Nusantara Bioscience vol. 5, no. 2, November 2013

| Nus Biosci | vol. 5 | no. 2 | pp. 51-107 | November 2013 || ISSN 2087-3948 | E-ISSN 2087-3956 |

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EDITORIAL BOARD:

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PUBLISHER:Society for Indonesian Biodiversity

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FIRST PUBLISHED: 2009

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Society for Indonesia Biodiversity Sebelas Maret University Surakarta


I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

ISSN: 2087-3948Vol. 5, No. 2, pp. 51-56 E-ISSN: 2087-3956November 2013

Effects of autoclaving on the proximate composition of stored castor(Ricinus communis) seeds

ANTHONY NEGEDU1,♥, JOSEPH B. AMEH2, VERONICA J. UMOH3, SUNDY E. ATAWODI3, MAHENDRA K. RAI4

1Raw Materials Research and Development Council, P.M.B. 232, Garki, Abuja, Nigeria. Tel.: +.234-9-4137416-7, Fax.:+234-9-4136034,♥email: [email protected]

2Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria3Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria

4Department of Biotechnology, SGB Amravati University, Maharashtra, India.

Manuscript received: 12 February 2013. Revision accepted: 24 May 2013.

Abstract. Negedu A, Ameh JB, Umoh VJ, Atawodi SE, Rai MK. 2013. Effects of autoclaving on the proximate composition of storedcastor (Ricinus communis) seeds. Nusantara Bioscience 5: 51-56. The effect of autoclaving on the proximate composition, free fattyacids and peroxide value of castor (Ricinus communis L.) seeds in storage were studied. Seeds of castor were surface sterilized, driedand divided into two equal sets of 300g each. One set was autoclaved at 15 1b pressure for 30 minutes at 121oC and the other set servedas control. Each set was prepared in triplicates and both sets were stored under same room temperature conditions for a period of 180days and agitated intermittently. Analysis of the proximate composition showed that autoclaving treatment caused an increased total fatcontent, reduced moisture, protein, nitrogen free extract (soluble sugar) and ash contents of the seeds in storage, as well as a non-significant increase in crude fiber (non-soluble sugar) content. It increased the free fatty acid content and decreased the peroxide valueof seed oil.

Key words: autoclaving, castor seeds, free fatty acids, peroxide value, proximate composition

Abstrak. Negedu A, Ameh JB, Umoh VJ, Atawodi SE, Rai MK. 2013. Pengaruh perlakuan autoklaf terhadap komposisi proksimat daribenih jarak kepyar yang disimpan (Ricinus communis). Nusantara Bioscience 5: 51-56. Pengaruh perlakuan autoklaf terhadapkomposisi proksimat, asam lemak bebas dan nilai peroksida benih jarak kepyar (Ricinus communis L.) dalam penyimpanan dipelajari.Biji jarak kepyar disterilkan permukaannya, dikeringkan dan dibagi menjadi dua kelompok yang sama, masing-masing sebanyak 300g.Salah satu kelompok itu diautoklaf pada tekanan 15 lb selama 30 menit pada suhu 121oC, sedangkan kelompok lainnya digunakansebagai kontrol. Setiap kelompok diperlakukan dalam tiga ulangan dan kedua kelompok disimpan dalam kondisi suhu ruangan yangsama dalam jangka waktu 180 hari serta beberapa kali dibalik-balik. Analisis komposisi proksimat menunjukkan bahwa perlakuanautoklaf menyebabkan peningkatan kadar lemak total, mengurangi kadar air, protein, ekstrak nitrogen bebas (gula larut) dan kandunganabu dari biji dalam penyimpanan, serta peningkatan secara tidak signifikan kandungan serat kasar (non larut gula). Perlakuan inimeningkatkan kadar asam lemak bebas dan menurunkan nilai peroksida minyak biji.

Kata kunci: perlakuan autoklaf, biji jarak kepyar, asam lemak bebas, angka peroksida, komposisi proksimat

INTRODUCTION

Vegetation belt influences dietary pattern in WestAfrica. For instance, in the Southern Nigeria, legumes,nuts, seeds, starchy roots or tubers dominate, while cerealsdominate the northern part (Ajayi et al. 2005). In the southeastern Nigeria, popular among the oil seeds used in soupsfor emulsification and stabilization are Irvingia gabonensis(Ataga and Ota-Ibe 2006); Brachystegia eurycoma andDetarium microcarpum (Ohegbu et al. 2002). But, ofparticular interest in this study is the castor (Ricinuscommunis) because of versatile industrial applications.

Castor (Ricinus communis L.) (Figure 1) bean plant is adicotyledonous and monoecious herb of the familyEuphorbiaceae and it is considered by most authorities tobe native to tropical Africa and may have originated inAbyssinia/Ethiopia (CSIR 1976). The castor is cultivated

for its seeds which yield versatile oil known as castor oil.The seed contains 45-50.6% oil, 12-16% protein, 23-27%fibre, 3-7% NFE, 5% moisture and 2% ash (CSIR 1976).The annual worldwide production stands at 1,311, 669metric tonnes. The demand for the castor oil is about453,590 metric tonnes and valued at more than US $500million (FAO 2005). The oil has about one thousandpatented industrial applications and has been used in theproduction of over four hundred industrial products such aspaints, dyes, soaps, cosmetics, polishes, lubricants, plastics,paper, hydraulic fluids, inks, lacquers, machining oils,pigments, sealants, electrical liquids etc. (Roetheli et al.1991; Gobin et al. 2001). Castor oil enjoys tremendousworld demand in the pharmaceutical, cosmetic, textile,paint, leather, lubricant, chemical, plastic, synthetic fibre,automobile and engineering industries (Roetheli et al.1991; Anjani et al. 2004).

5 (2): 51-56, November 201352

Figure 1. Ricinus communis. A. Habit, B. Development of flowers into fruit.

After the extraction of castor oil, the high nitrogencontaining pomace (meal) is suitable for fertilizer and whendetoxified, it can be employed in livestock feedformulation (Uzogara et al. 1990; Joshua 2000).

Castor oil is used as an ingredient in the folk medicinefor arthritis, cancer, cholera, convulsion, dogbite, guineaworm, oesteomelitis, rheumatism, veneral diseases,tuberculosis and considered an antidote, bactericide,emetic, emollient, insecticide, larvicidal, laxative, purgativeetc (Roetheli et al. 1991). It has been reported that theproximate composition of the seeds of some used as soupthickners, such as Mucuna flagellipes (Udensi et al. 2010);Mucuna utilis (Ukachukwu and Obioha 2000; Udensi et al.2008) have been improved by autoclaving. However, thereappears to be dearth of information on the effect thatautoclaving would have on the proximate composition ofcastor seeds.

This study was therefore, undertaken to evaluate theeffect that autoclaving treatment will have on the proximatecomposition of castor seeds with a view to recommendingit as a pre-storage treatment for the preservation of thenutritional values of seeds.

MATERIALS AND METHODS

Collection of seed samplesShortly after harvesting and sun-drying of castor seeds

by farmers, seed samples were purchased from localfarmers at Ankpa, Kogi State, Nigeria. Visibly mouldy as

well as necrotic lesioned seeds were handpicked and wholeseeds that failed to pass through ¾ x ¾ inch mesh wereused for treatments.

Sample preparationThe approximately uniform and clean seeds were

surface sterilized using 1% sodium hypochlorite solution(NaOCl) and rinsed consecutively in sterile de-mineralizedwater. The surface sterilized seeds were divided into twosets of 300g each and placed in 1 litre autoclavable plasticjars. Each set was prepared in triplicates and one set wasautoclaved at 15 1b pressure for 30 minutes at 121oC andcooled, while the second set served as control (raw seed).Both sets were stored under same normal room temperatureof 27±1oC for a period of 180 days (6 months). At intervalsof sixty days (2 months), samples were taken from each set(autoclaved and control) and analyzed for proximatecomposition using standard methods of AOAC (1995). Thebiochemical changes that occurred in both sets of seedsamples were compared.

Analysis of proximate compositionMoisture content of the samples was determined by

drying to a constant weight of 105oC in a forced draughtoven. Crude protein content was determined using themicro Kjeldahl digestion method described by AOAC(1995). The total ash content was determined using themethod of Kirk and Sawyer (1991). The total ash present in5g of the sample was determined by incinerating thesample in a muffle furnace at 550 oC for 3 hours. The

BA

NEGEDU et al. – Effects of autoclaving on Ricinus communis seeds 53

method described by Kirk and Swayer (1991) was used todetermine the crude fibre content of the samples. Theprotocol for the crude fibre content is briefly given. Twogrammes of defatted sample were boiled in 200cm3 of0.1275 M sulphuric acid solution for 30 minutes withconstant agitation. The boiling mixture was poured into aBuckner funnel and washed with boiling water twice. Thenthe residue was boiled in a 0.313 M sodium hydroxidesolution for 30 minutes with constant stirring. The residuewas then washed twice with boiling water followed by 1%HCl, then washed with boiling water until free from acid. Itwas then dried in an oven to a constant weight.Carbohydrate was determined by difference (100-(protein +fat + moisture + ash).

The nitrogen value, which is the precursor for protein ofa substance, was determined by micro-Kjeldahl method(Guebel et al. 1991). The nitrogen value was converted toprotein by multiplying with a factor of 6.25. The crudelipid content of the sample was determined using Soxhlettype of the direct solvent extraction method. The solventused was petroleum ether (boiling range 40-60 oC). Allproximate values were reported in percentage (AOCS2000; Okwu and Morah 2004).

Data analysisData were expressed as mean±standard error of M

(SEM). The data were subjected to one-way analysis ofvariance (ANOVA). SPSS soft ware was used to analysethe data and P< 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Effects of autoclaving on moisture content of castorseeds after 180 days of storage

In the control (un-autoclaved) and autoclaved, therewas a similar trend in moisture content levels (Figure 2.).From an initial level of 7.9±0.21%, the moisture declinedthrough 60 days to lower values (5.57±0.16% and6.45±0.16%) in the control and autoclaved respectively.Following this point, the seed moisture content increased to8.71±0.19% in the un-autoclaved. At the end of storageperiod of 180 days, the level of moisture in the control (un-autoclaved) was higher than in the autoclaved seeds.Statistical analysis shows that the difference in the valuesof moisture between the control and autoclaved wassignificant (P≥0.05).

The significant decrease in moisture content of theautoclaved seeds (Figure 2.) agreed with Ward and Diener(1961) on steamed peanuts and Ohegbu et al. (2009) onBrachystegia eurycoma . The decrease could have been asa result of physical damage to the structural integrity of theseeds and denaturation of protein structure leading toreduced water holding capacity of the seeds during storage.

Effect of autoclaving on crude protein (cp) content ofcastor seeds after 180 days of storage

Figure 3 presents the trend in the level of crude proteincontent of the castor seeds after 180 days storage. Within

0-60 days, in the control (un-autoclaved), there was asimilar trend in crude protein values. A rise in the initialvalue (21.28±0.03%) to a higher value (27.07±1.76%) wasobserved. A slight decline from the initial value(21.28±0.03%) to a lower level (20.80±1.76%) wasobserved in the autoclaved seeds. Between 120-180 days, adecline in the crude protein value was observed in the un-autoclaved and autoclaved (from 26.36±0.40% to24.47±0.55% and from 20.26±0.40% to 17.78±0.55%respectively). At the end of storage period, the level ofcrude protein in the control (un-autoclaved) was higherthan in the autoclaved seeds). Statistical analysis showsthat, the difference in the crude protein values between theun-autoclaved and the control was significantly different (P≤ 0.05).

The decline in the level of crude protein in theautoclaved seeds (Figure 3.) agreed with Ward and Diener(1961) who reported similar result on steamed peanuts andUdeni et al. (2004) who reported similar observation onautoclaved seeds of Mucuna utilis. The increase in proteincontent could have been due to reduction/destruction ofcertain protease inhibitors and anti-nutrients like phyticacid and tannins which form complexes with protein andmake it unavailable during hydrolysis.

Effect of autoclaving on total fat content of castor seedsafter 180 days of storage

Figure 4 presents the mean values of the total fatcontent with respect to autoclaving treatment. In the un-autoclaved seeds, it was observed that the total fat declinedfrom the initial value (47.55±0.42%) to a lower value(24.93±2.23%) at 60 days, 21.77±0.15% at 120 days and13.19±1.44% at 180 days. But in the autoclaved, after thedecline from the initial value (47.55±0.42%) to a lowervalue (32.84±2.23%),at 60 days, the value remained almostunchanged till end of storage (180 days). However, at theend of storage, the level of fat content in the autoclavedwas higher than in the control (un-autoclaved) and thedifference in the level, between the control and theautoclaved was statistically significant (P ≤ 0.05).

The higher total fat content of the autoclaved seeds(Figure 4.) agreed with the findings of Ezeokonkwo (2005)who observed increased total fat content of steamed seedsof an oil seed crop (African almond-Terminalia catappa).The significantly higher total fat value in the autoclavedseeds as compared to the control could be as a result of thepreservation of the total fat by the autoclaving treatmentwhich might have inactivated the endogenous enzymes(lipoxygenases) of the seeds. This reasoning is supportedby the findings of Majunder (2007) and Sreerama et al.(2008) who reported that post harvest practices acceleratemoisture migration, together with thermogenesis leading toenhanced deterioration of seed constituents such as fat, but,because endogenous enzymes were inactivated in theautoclaved seeds, there was lesser deterioration of the totalfat compared to the raw seeds in which the endogenousenzymes might have been reduced the total fat content.

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2 3 4 5

6 7 8 9

Figure 2. Effects autoclaving on the moisture content of castor seeds after 180 days of storageFigure 3. Effect of autoclaving on the crude protein content of castor seeds after 180 days of storageFigure 4. Effects of autoclaving on total fat content of castor seeds after 180 days of storageFigure 5. Effects of autoclaving on crude fiber content of castor seeds after 180 days of storageFigure 6. Effects of autoclaving on the ash content of castor seeds after 180 days of storageFigure 7. Effects of autoclaving on the nitrogen free extract content of castor seeds after 180 days of storageFigure 8. Effects of autoclaving on free fatty acid content of castor seeds after 180 days of storageFigure 9. Effects of autoclaving on the peroxide value of castor seeds after 180 days of storage

Effects of autoclaving on crude fiber content of castorseeds after 180 days of storage

The trend in the crude fiber content from 0-180 days ofstorage is presented in Figure 5. In both the un-autoclavedand autoclaved), a similar trend occurred. From the initialvalue (10.68±2.11%), the level of crude fiber content rosesteadily to a higher value (35.60±30.18%), while a gradualrise was recorded in the autoclaved till the end of storage(36.80a±30.18). However, at the end of storage, thedifference in the level of crude fiber between the controland the autoclaved was not statistically significant (P≥ 0.05).

The non-significantly higher level of crude fiber content(Figure 5.) observed in the autoclaved seeds during thisstudy agreed with the report of Apata (2008) who observedthat autoclaving did not cause appreciable changes in the

crude fiber content of groundnut meal. The non-significantdifference between autoclaved and un-autoclaved seedswith respect to crude fiber content could have been due tothe presence of some heat-stable factors in the seedscausing less hydrolysis of the structural carbohydrates. Thepresence of heat-stable factors in other oilseeds such asJatropha curcas has been reported (Martinez-Herrera et al.(2005).

Effects of autoclaving on the total ash content of castorseeds after 180 days of storage

The trend in the values of ash content from 0-180 daysof storage is presented in Figure 6. In un-autoclaved andautoclaved seeds, a similar trend was observed. A gradualrise from the initial value (3.61±1.01%) to higher values

NEGEDU et al. – Effects of autoclaving on Ricinus communis seeds 55

after 180 days (6.20±0.25% and 5.24±0.25% respectively)was observed. After 180 days, the value of the ash contentin the un-autoclaved was higher than that in the seeds.However, in both (control and autoclaved), at 180 days ofstorage, the difference in values between them, werestatistically significant (P ≤ 0.05) with respect to ashcontent.

The significantly higher level of ash content in theautoclaved seeds than that of un-autoclaved seeds(Figure5.) agreed with the findings of Ezeokonkwo (2005)who obtained similar result from steamed seeds of anotheroil seed, African almond (Terminalia catappa). Salunkheand Desai (1986) had also reported increased ash content ofsteamed groundnuts seeds. The higher level of ash in theun-autoclaved seeds could be attributed to non-leaching ofthe minerals from the seeds which might have occurred inthe autoclaved seeds during the autoclaving process. Thisreasoning is supported by Ataga and Ota-Ibe (2006) whoreported leaching of minerals from steamed seeds of WildMango (Irvingia gabonensis) leading to decreased total ashcontent in the autoclaved seeds than in the control.

Effects of autoclaving on the nitrogen free extract(NFE) content of castor seeds after 180 days of storage

From the trend in the values of the NFE content ofcastor seed presented in Figure 5.24, the un-autoclavedshowed a rise from the initial value (11.79±1.11%) to ahigher value (21.31±1.28%) at 60 days and followed by agradual decline to a lower level (16.29±1.82%). The valuegradually rose to a level higher (19.17±0.07%). In theautoclaved seeds, after the initial rise from the pre-storagevalue (11.79±1.11%) to a higher level (22.44±1.28%), asteady decline followed till end of storage to a lower value(12.55±0.70%). Statistical analysis reveals that at 180 days,the mean values in the level of the NFE, between theautoclaved and controls (un-autoclaved) were significantlydifferent (P ≤ 0.05).

The significant decrease in the soluble sugar (NFE-nitrogen free extract) of the autoclaved seeds (Figure 7.)disagreed with the findings of Ezeokonkwo (2005) whoreported that roasting or steaming increased the level ofsoluble carbohydrates (NFE) in another oil seed(Terminalia catappa). In addition, Apata (2008) reportedthat autoclaving did not induce appreciable changes in thecomposition of cellulose, non-cellulosic polysaccharidesand lignin of processed groundnut meal. It has beenreported that some seeds may possess heat– stable factorssuch as lectins and typsin inhibitors (Martinez–Herrera etal. 2005) which make such seeds more resistant tohydrolysis by heat. The decrease in the level of NFE couldbe as a result of more stability of some structuralcarbohydrates of the castor seeds that allowed lesshydrolysis of the insoluble sugars (crude fiber) into solublesugars (NFE).

Effects of autoclaving on free fatty acid (FFA) contentof castor seeds after 180 days of storage

The trend in the levels of free fatty acid content ofcastor seed due to autoclaving is presented in Figure 8. Inboth autoclaved and control, a similar trend was observed.

From the initial value (9.21±0.02%) of the free fatty acid, arise to higher values occurred (31.74±2.34% in theautoclaved and 62.90±2.34% in the control seeds). Thiswas followed by a decline to lower values (21.41±2.98%and 32.94±2.98% in the autoclaved and un-autoclavedrespectively) at the end of storage. At the end of the storageperiod of 180 days, the level of free fatty acid in theautoclaved seeds was lower compared to the un-autoclavedcontrol). Statistical analysis showed that difference in thelevels of free fatty acids between the control and theautoclaved was statistically significant (P ≤ 0.05).

The increase in the level of free fatty acid in theautoclaved seeds (Figure 8.) agreed with Oso (1974), Manjiet al. (2006) who reported increased free fatty acid insteamed oil palm fruits. The increase could be due togreater liberation of free fatty acid by the heat process orconversion of the oil into their constituent fatty acids. Thisis supported by Onyeka et al. (2005) on heated fruits ofanother oil seed, Black pear (Dacryodes edulis). Thedecline could be as a result of the transformation of the freefatty acid into fatty acid hydroxy peroxides at a rate fasterthan they were formed, since the peroxides themselves areunstable and decomposed into stable compounds such asaldehydes, ketones, epoxides (Sowunmi 1981).

Effects of autoclaving on the peroxide value of castorseeds after 180 days of storage

Figure 9, presents the trend in the levels of peroxidevalue of castor seed during storage. An initial rise in thelevel of peroxide was observed in the autoclaved and un-autoclaved seeds. After 120 days of storage, a decline inthe level of peroxide value occurred in the autoclaved andun-autoclaved. The level of peroxide was lower in theautoclaved compared to the un-autoclaved seeds after 180days of storage period. Statistical analysis showed thatbetween the autoclaved and un-autoclaved seeds, thedifference in the level of peroxide value was statisticallysignificant (p≤0.05).

The significantly higher level of peroxide value in theun-autoclaved seeds (Figure 9) compared to the autoclaveddisagreed with Bankole et al. (2005) who reported higherperoxide in the steamed melon seeds. This variance couldhave been due to the decrease in the peroxide valueresulting from the faster rate of decomposition of thehydroperoxy fatty acids (which are themselves unstable)into secondary products such as ketones, aldehydes,epoxides which are more stable and are largely responsiblefor the off flavours and objectionable odours in deterioratedseeds or oily products. This is supported by the findings of(Going 1968; Gaillard 1975; Arumughan et al. 1984; Amooand Asoore 2006), they reported faster rate of decompositionof hydroperoxyfatty acids into secondary products.

CONCLUSION

The study has shown that when castor seeds areautoclaved and stored, the total fat content of the seedsincreased, with increased free fatty acid level of the seedoil. The increase in the free fatty acid level of an oilseed is

5 (2): 51-56, November 201356

not healthy for the economic values of the seed, becauseincreased free fatty acid level will cause rise in cost ofprocessing and also attract reduction in seed price aspenalty. In addition, the decreased protein and solublesugars may not be economically advantageous for thoseinterested in using the seed protein and soluble sugars.Therefore, it is not advisable to autoclave seeds beforestorage. If storage of seeds is for the purpose of economicend products, such as the oil, protein and soluble sugars,then, autoclaving may not be recommended.

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Ward HS, Diener UL. 1961. Biochemical changes in shelled peanutscaused by storage fungi. 1. Effect of Aspergillus tamarii, four speciesof Aspergillus glaucus group and Penicillium citrinum. Phyptopathol51: 244-250.


Changes in growth, hormones levels and essential oil content ofAmmi visnaga plants treated with some bioregulators

IMAN M. TALAAT1, HEMMAT I. KHATTAB2, AISHA M. AHMED1,♥1Botany Department, National Research Centre, Cairo, Egypt. Tel.: +20-1140355848, Fax.: +20-233370931, ♥e-mail: [email protected]

2Botany Department, Faculty of Science, Ain-Shams University, Cairo, Egypt.

Manuscript received: 6 August 2013. Revision accepted: 10 September 2013.

Abstract. Talaat IM, Khattab HI, Ahmed AM. 2013. Changes in growth, hormones levels and essential oil content of Ammi visnagaplants treated with some bioregulators. Nusantara Bioscience 5: 57-64. The effects of foliar application of different concentrations ofamino acids (tyrosine and phenylalanine) and phenolic acids (trans-cinnamic acid, benzoic acid and salicylic acid) on growth, pigmentcontent, hormones levels and essential oil content of Ammi visnaga L were carried out during two successive seasons. It is clear thatfoliar application of either amino acids or phenolics significantly promoted the growth parameters in terms of shoot height, fresh and drybiomass, number of branches and number of umbels per plant. The increment of growth parameter was associated with elevated levelsof growth promoters (IAA, GA3, total cytokinins) and low level of ABA. The greatest increase in the previously mentioned parameterswas measured in plants exposed to different concentrations of phenols particularly in benzoic acid-treated plants. Such effect wasconcentration dependent. All treatments led to significant increments in seed yield and essential oil content. Moreover, Gas LiquidChromatographic analysis revealed that the main identified components of essential oil were 2,2-dimethyl butanoic acid, isobutylisobutyrate,α-isophorone, thymol, fenchyl acetate and linalool. Phenolics and amino acids treatments resulted in qualitative differencesin these components of oil.

Key words: Ammi visnaga, phenolic compounds, amino acids, hormones, growth criteria, essential oil

Abstrak. Talaat IM, Khattab HI, Ahmed AM. 2013. Perubahan dalam pertumbuhan, kadar hormon dan kandungan minyak atsiritanaman Ammi visnaga yang diperlakukan dengan beberapa bioregulator. Nusantara Bioscience 5: 57-64. Pengaruh aplikasi daunberbagai konsentrasi asam amino (tirosin dan fenilalanin) dan asam fenolat (asam trans-sinamat, asam benzoat dan asam salisilat)terhadap pertumbuhan, kandungan pigmen, kadar hormon dan kandungan minyak atsiri Ammi visnaga L. telah dilakukan selama duamusim berturut-turut. Hasilnya secara jelas menunjukkan bahwa aplikasi daun dari salah satu asam amino atau asam fenolat secarasignifikan meningkatkan parameter pertumbuhan dalam hal tinggi tunas, biomassa segar dan kering, jumlah cabang dan jumlah tangkaibunga per tanaman. Kenaikan parameter pertumbuhan terkait dengan meningkatnya kadar hormon promotor pertumbuhan (IAA, GA3,total sitokinin) dan rendahnya kadar ABA. Peningkatan terbesar parameter tersebut terukur pada tanaman yang terkena berbagai kadarfenol terutama tanaman yang diperlakukan dengan asam benzoat. Efek seperti itu tergantung kadarnya. Semua perlakuan menyebabkankenaikan signifikan dalam jumlah biji dan kandungan minyak atsiri. Selain itu, analisis Kromatografi Gas Cair mengungkapkan bahwakomponen utama yang teridentifikasi dari minyak atsiri adalah asam 2,2-dimetil butanoat, isobutil isobutirat, α-isoforon, timol, fensilasetat dan linalool. Perlakuan fenolat dan asam amino mengakibatkan perbedaan kualitatif komponen minyak atsiri ini.

Kata kunci: Ammi visnaga, senyawa fenolat, asam amino, hormon, kriteria pertumbuhan, minyak atsiri

INTRODUCTION

Ammi visnaga, known as Khella, is an annual orperennial herb belongs to family Apiaceae (Umbelliferae).Khella is native to the Mediterranean and is cultivated inEgypt. Ammi visnaga is antiasthmatic, diuretic, lithontripicand vasodilator. It is an effective muscle relaxant and hasbeen used for centuries to alleviate the excruciating pain ofkidney stones (Chevallier 1996). The seeds used as a folkmedicine for diuretic and lithontripic (Uphof 1959).Visnaga seeds contain oil that includes the substance'khellin', which is used in the treatment of asthma. Theyhave antispasmodic action on the smaller bronchialmuscles, dilate the bronchial, urinary and blood vesselswithout affecting blood pressure (Bown 1995). Essential oil

of A. visnaga is known for its proprieties against coronarydiseases and bronchial asthma (Rose and Hulburd 1992;Satrani et al. 2004). The major components were linalool,isoamyl 2-methyl butyrate, and isopentyl isovalerate(Khadhri et al. 2011).

Furthermore, phenolics are low molecular compoundsubiquitous in all tissues of higher plants with greatsignificance in plant development. Phenolic compounds aresome of the most widespread molecules among plantsecondary metabolites, and are of great significance inplant development (Curir et al. 1990). However, theirbiological, ecological and agronomical significance in therhizosphere is much less clear. Furthermore these bio-molecules may contribute in soil and water conservation,weed management, mineral element nutrition, as well as

5 (2): 57-64, November 201358

they impact as signal molecule in certain symbioticrelationships, and act as defense molecules against soilpests and pathogens (Makoi1 and Ndakidemi 2007).Additionally, they serve as flower pigments, act asconstitutive protection agents against biotic and abioticstress (Delalonde et al 1996), function as signal molecules,act as allelopathic compounds, and affect cell and plantgrowth (Dakora 1995; Dakora and Phillips 1996;Ndakidemi and Dakora 2003), are important natural animaltoxicants (Adams 1989) and some may function aspesticides (Vidhyasekaran 1988; Waterman and Mole1989; Beier 1990). They are also functional components ofthe rhizosphere and its soil organic matter (Haider et al.1975; Martin 1977). They have long been recognized asallelochemicals for weed control (Rice 1984; Putnam andTang 1986) phytoestrogens in animals (Adams 1989) andplant defense molecules (Vidhyasekaran 1988). In therhizosphere, they act as important precursors for thesynthesis of soil humic substances (Haider et al. 1975).Salicylic acid participates in the regulation of severalphysiological processes in plant such as stomatal closure,nutrient uptake, chlorophyll synthesis, protein synthesis,inhibition of ethylene biosynthesis, transpiration andphotosynthesis (Khan et al. 2003; Shakirova et al. 2003).SA increase cell metabolic rate (Amin et al. 2008). Thebiosynthesis of salicylic acid in plants starts fromphenylalanine and follows one of two known paths ofsynthesis which involves trans-cinnamic acid thenhydroxylation of benzoic acid which is a direct precursor ofsalicylic acid (Rask 1995).

Moreover, amino acids as organic nitrogenouscompounds are the building blocks in the synthesis ofproteins (Davies 1982). Amino acids are particularlyimportant for cell growth stimulation. They act as bufferswhich help to maintain favorable pH value within the plantcell. They protect the plants from ammonia toxicity. Theycan serve as a source of carbon and energy, as well asprotect the plants against pathogens. Amino acids alsofunction in the synthesis of other organic compounds, suchas protein, amines, purines and pyrimidines, alkaloids,vitamins, enzymes, terpenoids and others (Goss 1973;Abdel-Aziz and Balbaa 2007). Furthermore, Hass (1975)stated that the biosyntheses of cinnamic acids (which arethe starting materials for the synthesis of phenols arederived from phenylalanine and tyrosine.

The aim of this study is to investigate the role of somephenolic substances (salicylic acid, t-cinnamic acid andbenzoic acid) and amino acids (tyrosine and phenylalanine)on the growth, endogenous hormones, photosynthetic pigments,total, soluble and insoluble carbohydrates of A. visnagaplants as well as the essential oil content of the seeds.


ExperimentalTwo pot experiments were conducted in the greenhouse

of National Research Centre (NRC), Dokki, Cairo, Egypt,during two successive seasons of 2009/2010 and2010/2011. Ammi visnaga seeds were obtained from theDepartment of Medicinal and Aromatic Plants, Ministry of

Agriculture, Giza, Egypt. Ten sterilized seeds were sown ineach pot (30cm diameter) in the third week of October.Each pot was filled with 10 kg of air-dried clay soil.Physical and chemical properties of the soil used in thisstudy were determined according to Jackson (1973) andCottenie et al. (1982) and are presented in Table (1). Eightweeks after sowing, the seedlings were thinned and threeplants per pot were left. Pots were divided into three maingroups. The first group was exposed to different levels ofphenolic compounds (salicylic acid, trans-cinnamic acidand benzoic acid) at concentrations 5, 10 and 20 mg L-1.The second group was sprayed with different levels ofamino acids (phenylalanine and tyrosine) at concentrations50,100 and 200 mg L-1. Phenolic compounds and aminoacids were applied after 30 days from the sowing date. Thethird group was sprayed with H2O to serve as control. Theexperiments conducted under natural day conditions, withphotoperiod 11hr ± 2 and temperature about 27oC ± 2.

Table 1. Physical and chemical properties of the soil used

Soiltexture pH EC*

Organic C

Organicmatter

TotalN Total P Total K

(%)

Sandyloam

7.2 0.6 0.9 1.9 0.3 0.1 0.1

Note: EC * = Electric conductivity (salinity)

All agricultural practices were conducted according tothe recommendations by the Egyptian Ministry ofAgriculture as follows: fertilizers were added to all pots asfollows: cattle manure (2g pot−1), phosphorus (2g pot −1) ascalcium super phosphate (15.5% P2O5), nitrogen (2g pot −1)as ammonium sulphate (20.5% N) and potassium (1.5 g pot−1) as potassium sulphate (48% K2O). Weeds were removedby hand and only natural pesticides were used for any plantdiseases. The growth parameters of differently treatedAmmi plants were measured after 75, 119, 180 and 210days from sowing (stages A, B, C and D respectively).Stage A was at the vegetative growth while stage B at thebeginning of flowering and stages C and D were at earlyfruiting and harvest time.

Vegetative growth charactersPlant height (cm), fresh and dry weights of shoot (g

plant-1) were recorded during the vegetative stage. Plantheight (cm), number of branches and umbels (plant-1),fresh and dry weights of shoots (plant-1) were recorded atflowering, early fruiting and fruiting stages.

Endogenous hormonesThe endogenous hormone levels were determined

using the method described by Wasfy and Orrin (1975).Chlorophyll (chl) a, chl b and total carotenoids contentwas measured according to the method o f Associationof Official Agricultural Chemists (AOAC 1970).

Total and soluble carbohydrateTotal and soluble carbohydrate contents were deter-

mined according to the method described by Dubois et al.(1956). Then, the insoluble carbohydrates were calculated.

TALAAT et al. – Growth, hormones levels and essential oil content of Ammi visnaga 59

Essential oil isolationThe ripening fruits of A. visnaga were collected air dried

and weighed for extraction of the essential oil. Five gramsof dry fruits were crushed into smaller pieces and reducedto fine powder with the aid of a mechanical grinder. Thepowder sample was extracted with petroleum ether (PE 40-60ºC) for 48h at room temperature. The extract was evapo-rated to dryness using a rotary evaporation at reduced pressure.The essential oil was passed over dark anhydrous sodiumsulfate to remove moisture. The fraction obtained was storedin a refrigerator at 4ºC in dark to identify the chemicalconstituents of oil (Adams, 2007). GC-MS analysis werecarried out on a Varina 3400 system equipped with a DB-5fused silica column (30 m x 0.25 mm i.d.); Oventemperature was 40 to 240°C at a rate of 4°C min-1, transferline temperature 260°C, injector temperature 250°C, carriergas helium with a linear velocity of 31.5 cm s-1, split ratio1/60, flow rate 1.1 mL min-1, Ionization energy 70 eV; scantime 1 s ; mass range 40-350 amu.

Identification of componentsThe components of the oil were identified by comparison

of their mass-spectra with those of a computer library orwith authentic compounds and confirmed by comparison oftheir retention Indices with those of authentic compounds.Kovats, Indices (Kováts 1958) were determined by co-injection of the sample with a solution containing ahomologous series of n-hydrocarbons, at a temperature runidentical to that described above.

Statistical analysisIn this experiment, one factor was considered: different

concentrations of amino acids (50, 100 and 200 mg L-1),phenolic compounds treatments (5, 10 and 20 mg L-1) andcontrol. The experimental design followed a completerandom block design. According to Sendecor and Cochran(1990), the average of data were statistically analyzedusing 1-way analysis of variance (ANOVA-1). Significantvalues determined according to the Least SignificantDifference (LSD at 0.05 and at 0.01 p) by using the STAT-ITCF program (1982).


Effect of amino acids and phenolic compounds ongrowth parameters

Foliar application of different concentrations of eitherphenols or amino acids stimulate a gradual increases in growthparameters in terms of plant height, number of branches,number of umbels fresh and dry weights and water contentof A. visnaga shoots throughout the experimental periods.Results also, investigated that phenols stimulate all theprevious morphological parameters particularly at 20 mg L-1

compared with those of amino acids (tyrosine and phenyl-alanine) throughout the experimental period (Figures 1-6).The greatest increases in all investigated morphologicalcriteria was measured in A. visnaga plants exposed to 20 mgL-1 benzoic acid at all stages. Similar results were obtained by

Figure 1. Ammi visnaga L. (Khella, bisnaga or toothpickweed)

5 (2): 57-64, November 201360

Balbaa and Talaat (2007) who concluded that phenylalaninetreatments significantly promoted plant height, number ofbranches, fresh and dry weights of rosemary plants. AbdEl-Aziz et al. (2007) indicated also that foliar applicationof tyrosine significantly promoted plant height, number ofleaves and branches, fresh and dry weights of branches andshoots and stem diameter in both cuttings of Salvia farinaceaplants. It was recorded that application of certain aminoacids significantly increased the vegetative growth ofChrysanthemum (El-Fawakhry and El-Tayeb 2003),peppermint (Refaat and Naguib 1998), datura (Youssef etal. 2004) and Pelargonium graveolens (Mahgoub andTalaat 2005). Furthermore, salicylic acid caused significantincreases in most growth parameters of different plantspecies (Abd El-Wahed et al. 2006; El-Khallal et al. 2009;Delavari et al. 2010; Dawoode et al. 2012). The promotiveeffect of salicylic acid could be attributed to itsbioregulator effects on physiological and biochemicalprocesses in plants such as ion uptake, cell elongation, celldivision, cell differentiation, sink/source regulation,enzymatic activities, protein synthesis and photosyntheticactivity as well as increase the antioxidant capacity ofplants (Raskin 1992; Blokhina et al. 2003; El-Tayeb 2005).

Effect of amino acids and phenolic compounds onchemical composition

The changes of chlorophylls a and b as well as carote-noids content in response to amino acids and phenolicstreatments are shown in Figure 7. High pigments levels (chla, b, carotenoids) were measured in A. visnaga leavestreated with phenols compared with those of amino acids.The maximum increase in chlorophylls and carotenoids arerecorded in leaves treated with 20 mg L-1 benzoic acid. Theincrements in pigment level were attributed to thepromotion in its synthesis and/or retardation of pigmentdegradation. These results are similar to those obtained bySharma et al. (1995) who found that excised leaves ofTropaeolum majus, treated with t-cinnamic acid, retainedmore chlorophyll (60% higher at 10-3 M) compared tocontrol. Moreover, the potent effects of particularlysalicylic acid might be ascribed firstly to the reduction inchlorophyll loss due to its ability to increase the antioxidantcapacity of the plants (Kuorzer et al. 1999) or inducing thesynthesis of stabilizing substances (Nemeth et al. 2002).Salicylic acid caused significant increases in photosyntheticpigments (Figure 8). These results corrborate with those ofKhodary (2004) and Gunes et al. (2005) on maize, El-Tayeb(2005) on barley, and Dawood et al. ( 2012) on sunflower.

The enhancing effects of SA on photosynthetic capacitycould be attributed to its stimulatory effects on Rubiscoactivity and pigment contents (Khodary 2004) as well asincreased CO2 assimilation, photosynthetic rate andincreased mineral uptake by the plant (Szepesi et al. 2005).In addition, Arfan et al. (2007), pointed that application ofsalicylic acid improved the photosynthetic capacity andretain pigment content through increasing IAA andCytokinins therefore inhibits their senescence. Similarresults were obtained by Hassanein (2003) on Foeniculumvulgare plants and Abou Dahab (2006) on Philodendronerubescens plant. They reported that foliar application of

the amino acid (tryptophan) caused an increase inphotosynthetic pigments contents.

The increments of the photosynthetic pigments in thetreated A. visnaga leaves were concomitant with a gradualincrease in total, soluble and insoluble carbohydrates(Figure 8). The maximum increases in soluble andinsoluble carbohydrates were measured in the plantsexposed to foliar application of phenolic compoundscompared to those treated with amino acids. Moreover,such increments in the levels of total, soluble and insolublecarbohydrates were recorded in leaves exposed to 20 mg L-

1 benzoic acid. These results are in agreement with thoseobtained by Goss (1973), who indicated that amino acidscan serve as a source of carbon and energy whencarbohydrates become deficient in the plant; amino acidsare determinate, releasing the ammonia and organic acidfrom which the amino acid was originally formed. Theorganic acids then enter the Kreb's cycle, to be brokendown to release energy through respiration. These resultscould also be explained by the findings obtained by GamalEl-Din et al. (1997) who found that treatment of lemongrass plants with 100 ppm phenylalanine in the first cut andornithine in the second cut recorded the highest level ofcarbohydrate percentage compared with control. Refaat andNaguib (1998) reported that application of all amino acids(alanine, cytosine, guanine, thiamine and L-tyrosine)increased the total carbohydrates percentage in peppermintleaves. The effect of the amino acids on the totalcarbohydrates content may be due to their important roleon the biosynthesis of chlorophyll molecules which in turnaffected carbohydrate metabolism. In this respect, Talaatand Balbaa (2010) reported that chemical analysis of theleaves of sweet basil indicated that the contents of totalsoluble and total carbohydrates were significantly increasedas a result of foliar application of trans-cinnamic acid. Tariet al. (2002) and Dawood et al. (2012) reported thatsalicylic acid application resulted in a significant increasein total soluble carbohydrates content in leaves of tomatoand sunflower, thus maintaining the carbohydrates pool inthe chloroplasts at a high level.

Plant hormones play an important role in developmentprocesses; some of them have a key in the most plantmechanisms. Data represented in Figure 9 showedincrements in gibberellins (GA3), indole acetic acid (IAA)and cytokinins (Z & ZR) in plants treated with amino acidsand phenolic compounds. High concentrations ofgibberellins (GA3), Indole acetic acid (IAA) and cytokinins(Z & ZR) were measured in A. visnaga leaves treated withphenolic compounds compared with amino acids. Thehighest values of GA3, IAA and cytokinins were recordedin plants exposed to 20 mg L-1 benzoic acid. A reduction inabscisic acid (ABA) level was concomitant with suchincrements in growth promoters estimated in plantsexposed to either phenolic compounds or amino acids. Theincreases in the levels of endogenous growth promoterscould be attributed to the increase in their biosynthesisand/or decrease in their degradation and conjugation. Onthe other hand, the reduction in ABA level could be due tothe shift of the common precursor isopentenyl pyrophos-phate to biosynthesis of cytokinins and/or gibberellins


1 2

3 4

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9 10

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

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)


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Chl aChl bCaretnoidsChl a+Chl bChl a/Chl bChla+Chlb/Carotinoids


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t (g)

, oil(

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Fruit yelidOil %Oil yelid

Total carbohydrateInsoluble carbohydrateSoluble carbohydrate

CytokininsZRZABAIAAGA3

Chl a+Chl b/CarotenoidsChl a/Chl bChl a+Chl bCarotenoidsChl bChl a

FruitingEarly fruitingFloweringVegetative



FruitingEarly fruitingFlowering

Oil yieldOil %Fruit yield


FruitingEarly fruitingFlowering

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)

5 (2): 57-64, November 201362

Figure 1. Changes in the values of plant height of shoot system of A. visnaga plants (cm plant-1) treated with different concentrations ofamino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of tenreplicates ± SD

Figure 2. Changes in the values of branch number of shoot system of A. visnaga plants treated with different concentrations of aminoacids and phenolic compounds during the flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD

Figure 3. Changes in the values of umbels number of shoot system of A. visnaga plants treated with different concentrations of aminoacids and phenolic compounds during the flowering , early fruiting and fruiting stages, each value is mean of ten replicates ± SD

Figure 4. Changes in the values of fresh weight of shoot system of A. visnaga plants (g plant-1) treated with different concentrations ofamino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of tenreplicates ± SD

Figure 5. Changes in the values of dry weight of shoot system of A. visnaga plants (g plant-1) treated with different concentrations ofamino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of tenreplicates ± SD

Figure 6. Changes in the percentage of water content of A. visnaga shoots treated with different concentrations of amino acids andphenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD

Figure 7. Changes in the values photosynthetic pigments of A. visnaga plants (mg L-1) treated with different concentrations of aminoacids and phenolic compounds during the vegetative stage, each value is mean of ten replicates ± SD

Figure 8. Changes in the percentage of total, soluble and insoluble carbohydrates of A. visnaga plants (%) treated with differentconcentrations of amino acids and phenolic compounds during the vegetative stage; each value is mean of ten replicates ± SD

Figure 9. Changes in the values of phytohormone contents of A. visnaga Plants (μg g-1) treated with different concentrations of aminoacids and phenolic compounds during the vegetative stage

Figure 10. Changes in the values of fruit yield (g), oil percentage (%) and oil yield (ml plant-1) of A. visnaga plants treated withdifferent concentrations of amino acids and phenolic compounds, each value is mean of ten replicates ± SD

Table 2. The constituents of essential oil of A. visnaga plants

No. Components (%) KITreatments (ppm)

0 Tyrosine Phenylalanine Benzoic acid Tarns-cinnamic acid Salicylic acid50 100 200 50 100 200 5 10 20 5 10 20 5 10 20

1 α-Thujene 931 - 2.5 1.3 1.0 1.2 1.9 - 1.1 - 3.9 2.2 0.4 0.9 1.5 1.2 3.92 Myrcene 991 - 2.0 0.4 8.0 3.6 3.6 - 1.2 - 3.7 1.9 0.4 1.6 2.1 1.4 4.93 Isobutyl isobutyrate 1004 22.9 20.6 35.3 15.9 18.9 18.6 24.1 14.8 24.3 9.9 11.4 24.4 22.6 6.4 16.5 15.64 Linalool 1029 5.7 2.9 0.6 1.3 3.3 1.3 - 0.8 - 4.5 2.1 0.3 1.1 1.1 2.5 2.65 2,2-Dimethylbutanoic acid 1108 28.9 35.4 55.4 30.4 20.6 38.8 50.5 35.0 25.9 21.1 27.4 36.5 34.6 59.0 34.4 38.26 α-Isophorone 1121 13.4 17.9 0.9 3.0 2.7 1.2 9.2 11.9 16.7 9.6 13.8 19.3 21.1 6.4 11.3 13.87 Fenchyl acetate 1220 6.3 3.8 0.3 2.5 7.8 5.0 - 1.0 - 4.8 7.0 0.2 3.2 3.7 4.7 3.58 Bornyl acetate 1289 - 1.7 0.4 7.8 2.6 5.1 - 0.8 - 4.3 5.3 0.5 2.3 0.9 0.8 2.09 Thymol 1290 13.2 8.5 1.8 13.1 9.3 2.8 - 2.1 15.2 7.0 8.0 0.8 1.7 6.7 3.7 5.710 Geranyl acetate 1381 - - 0.3 1.4 4.9 2.6 9.1 11.5 - 5.2 3.8 11.2 2.7 0.9 6.9 4.511 Lavandulyl acetate 1439 - - 0.2 0.7 7.6 3.0 - 1.4 - 3.7 2.7 0.7 1.1 2.2 0.9 -12 Citronellyl propionate 1446 - - 0.6 5.6 7.9 3.3 - 1.0 - 5.3 1.6 - 1.2 3.1 2.4 -13 Croweacin 1460 9.6 4.7 1.5 6.7 8.1 11.0 7.1 10.4 15.0 5.9 7.2 2.8 3.3 6.0 8.7 5.314 α-Damascone 1689 - - 0.4 1.5 2.1 1.0 - 3.2 2.9 5.7 2.7 2.4 0.9 - 2.2 -15 (Z,E)-farnesal 1701 - - 0.6 1.1 1.4 0.8 - 3.8 - 5.4 2.9 0.1 1.7 - 2.4 -Total identified 100 100 100 82.6 100 100 100 100 100 100 100 100 100 100 100 100Monoterpene compounds 100 100 99.4 98.9 98.6 99.2 100 96.2 100 94.6 97.1 99.9 98.3 100 97.6 100Sesquiterpene compounds - - 0.6 1.1 1.4 0.8 - 3.8 - 5.4 2.9 0.1 1.7 - 2.4 -

instead of ABA (Hopkins and Huner 2004). These resultsare in accordance with those obtained by Shehata et al.(2000), Shehata et al. (2001) and Zaghlool (2002). Theincreases in IAA and GA3 in shoot tissues of sunflowerplant concurrently with the increase in growth rate due tothe role of these endogenous hormones in stimulating celldivision and/or the cell enlargement and subsequentlygrowth (Taiz and Zeiger 1998). It is well known thatsalicylic acid induces flowering, increases flower life,retard senescence and increases cell metabolic rate. Inaddition, salicylic acid may be a prerequisite for synthesis

of auxin and /or cytokinin. (Metwally et al. 2003, Gharib2006). Furthermore, these increments in growth regulatingsubstances might be a prerequisite for acceleration ofgrowth resumption of sunflower plant. In addition,salicylicacid effects on abscisic acid (Senaranta et al. 2000),gibberellins (Traw and Bergelson 2003) regulate manyphysiological process and plant growth. Moreover,Dawood et al. (2012) reported that SA caused markedincrements in IAA, GA3, zeatin and zeatin riboside, in themeantime decrease in ABA content comparing withuntreated controls.


Figure 10 indicated that the fruit yield, oil yieldpercentage and oil yield (ml plant-1) increased in plantstreated with phenolic compounds and amino acids. Themaximum levels of oil yield percentage (ml plant-1) wererecorded in seeds exposed to 20 mg L-1 benzoic acid. Theincrement in oil% and protein% might be due to theincrease in vegetative growth and nutrients uptake. Similarresults were reported by Gharib (2006) and Çag et al.(2009). In addition, Noreen and Ashraf (2010) mentionedthat high doses of salicylic acid caused marked increases insunflower achene oil content as well as some key fattyacids and significant decrease in stearic acid.

Table 2 represents the compounds of essential oilobtained from A. visnaga as detected by GC-MS. therelative levels of various constituents of oil yield wereincreased, decreased or disappeared in A. visnaga fruits ofplants treated with amino acids and phenolic compoundscompared with untreated control plants. 2,2-dimethylbutanoic acid, isobutyl isobutyrate, linalool,thymol and croweacin are the major constituents of A.visnaga fruits. These results are similar to those obtainedby Khalfallah et al. (2011) who found that the majorcomponent of essential oil in A. visnaga are 2, 2-dimethylbutanoic acid, isobutyl isobutyrate, croweacin,linalool and thymol. The effect of different treatments onessential oil and its constituents may be due to its effect onenzyme activity and metabolism of essential oil production(Burbott and Loomis 1969).

SA has a role in controlling gene expression (He et al.2005) reported that most of the genes regulated by SA aredefense related genes and many of them participate in plantresponses to biotic and abiotic stresses. Therefore SA maychange secondary metabolites and its pathway by effects onplastid, chlorophyll level and represent stress conditions.The SA like stress manipulated quality and quantity ofessential oil of Salvia macrosiphon. The yield of essentialoil was increased. The useful component such as Linaloolwas increased. Seventeen components were identified inSA-treated plants (Rowshan et al. 2010).

CONCLUSION

Finally, it is apparently clear that phenolics treatmentswere more effective in enhancing growth and productivityof A. visnaga. Moreover, the greatest increase in the growthparameters and chemical constituents obtained at 20 mg L-1

of benzoic acid. On the other hand, the major component ofessential oil gave the best percentage (59%) was assayed inseeds exposed to salicylic acid.

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Effect of water regime on the growth, flower yield, essential oil andproline contents of Calendula officinalis

SAMI ALI METWALLY1, KHALID ALI KHALID2,♥, BEDOUR H. ABOU-LEILA3

1Department of Ornamental Plants and Woody Trees, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt.2Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Tel. +202-3366-9948,

+202-33669955, Fax: +202-3337-0931, email: [email protected] of Water Relation and Field Irrigation, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt.

Manuscript received: 10 May 2013. Revision accepted: 16 July 2013.

Abstract. Metwally SA,Khalid KA, Abou-Leila BH. 2013. Effect of water regime on the growth, flower yield, essential oil and prolinecontents of Calendula officinalis. Nusantara Bioscience 5: 65-69. The effects of water regime on the growth, content of essential oil andproline of Calendula officinalis L. plants were investigated. Water regimes of 75% of field water capacity increased certain growthcharacters [i.e. plant height (cm), leaf area (cm2), flower diameter (cm) and spike stem diameter] and vase life (day). Water regimepromoted the accumulation of essential oil content and its main components as well as proline contents.

Key words: Calendula officinalis, essential oil, flower yield, growth, proline, vase life, water regime

Abstrak. Metwally SA,Khalid KA, Abou-Leila BH. 2013. Pengaruh tata air terhadap pertumbuhan, hasil panen bunga, kandunganminyak atsiri dan prolin pada Calendula officinalis. Nusantara Bioscience 5: 65-69. Pengaruh tata air terhadap pertumbuhan,kandungan minyak atsiri dan prolin dari tanaman Calendula officinalis L. diteliti. Pengaturan tata air sebanyak 75% dari kapasitas airlapangan meningkatkan karakter-karakter pertumbuhan tertentu [yaitu: tinggi tanaman (cm), luas daun (cm2), diameter bunga (cm) dandiameter tangkai bulir] dan masa hidup bunga (hari). Pengaturan tata air menyebabkan akumulasi kadar minyak atsiri dan komponenutamanya serta kandungan prolin.

Kata kunci: Calendula officinalis, minyak atsiri, hasil panen bunga, pertumbuhan, proline, tata air

INTRODUCTION

Calendula officinalis L. (English marigold, pot marigold;Figure 1) belongs to the Asteraceae (Compositae) family; itis an annual with bright or yellow orange daisy-like flowerswhich are used for ornamental and medicinal purposes(Bcerentrup and Robbelen 1987; Cromack and Smith1988). Calendula officinalis can be broadly applied as anantiseptic, antiinflammatory and cicatrizing (Correa Júnior1994) aswell as a light antibacterial (Chiej 1988) andantiviral (Bogdanova and Farmakol 1970) agent. ManyCalendula species have a characteristic scent or tastecaused by mono and sesquiterpenes within the essential oil,which in many cases are the reason for their application infolk medicine (Yoshikawa et al. 2001). Recently, manyattempts have been made to better characterize theirtherapeutic properties and to enhance the production ofthese useful compounds within their essential oils. SelectedCalendula chemo-types growing in soil or in vitro, forexample, flowers of the cadinol chemo-type, are veryimportant in European and western Asian folk medicinesand are used to treat inflammatory conditions (Yoshikawaet al. 2001). Distinct subspecies of C. officinalis have beenreported from various countries (Chalchat et al. 1991;Nicoletta et al. 2003), i.e. Herbaria, Mecsek, Melius,

Golden Dragon and Adamo (Bakó et al. 2002). Calendulaofficinalis can be used as a colorant because it primarilycontains two classes of pigments, the flavonoids andcarotenoids, which can be used as yellow and orangenatural colors, respectively. Natural colors are gainingconsiderable attention since several synthetic colorantshave given rise to allergic, toxic and carcinogenic effects(Lea 1988). Flavonoids have antioxidant activities whichplay an important role in food preservation and humanhealth by combating damage caused by oxidizing agents(Meda et al. 2005). Carotenoids are important to humansand other animals as precursors of vitamin A and retinoids.In addition, they act as antioxidants, immune-enhancers,inhibitors of mutagenesis and transformation, inhibitors ofpremalignant lesions, screening pigments in primate fovea,and non photochemical fluorescence quenchers(Castenmiller and West 1998).

In aromatic plants, growth and essential oil productionare influenced by various environmental factors, such aswater deficit (Burbott and Loomis 1969; Sabih et al. 1999).Solinas and Deiana (1996) reported that secondaryproducts of plants can be altered by environmental factorsand that water deficit is a major factor affecting thesynthesis of natural products. Water deficit resulted in asignificant reduction of fresh and dry matter, and essential

5 (2): 65-69, November 201366

oil yield of mint (Mentha sp.) plants (Mirsa and Strivastava2000). Fresh and dry weights of Ocimum basilicum L.decreased as plant water deficit increased while the linalooland methyl chavicol contents increased (Simon et al. 1992).The essential oil yield and proline contents of basil(Ocimum sp.) increased by subjecting plants to waterdeficit just before harvesting (Baeck et al. 2001). Khalid(2006) reported that fresh and dry weights of Ocimum sp.were significantly decreased by water deficit. Meanwhile,essential oil percentage, as well as the main constituents ofthe essential oil, proline content increased. Baher et al.(2002) showed that water deficit reduced the fresh and dryweights of Satureja hortensis L. plants, while severe waterdeficit increased essential oil content more than moderatewater deficit. The main constituents, such as carvacrol,increased under moderate water deficit, while -terpinenecontent decreased under moderate and severe water deficit.Hendawy and Khalid (2005) showed that essential oil, andproline contents showed a pronounced increased byincreasing the water stress levels of Salvia officinalis L.plants. On the other hand, Petropoulos et al. (2007) notedthat water deficit had relatively little effect on the essentialoil composition of parsley (Petroselinum crispum).

The Egyptian climate is mostly arid and semi-arid,where water availability is a major problem for cropproduction (Abou El-Fadl et al. 1990). In such conditionscultivation of resistant plants is one way to utilize theselands and therefore the selection of suitable crops, whichcould cope with these conditions, is a necessity. In arid andsemi-arid regions, where water availability is a majorlimitation in crop production, using alternative waterresources. The major challenge facing water managementis the availability of water. Its amount is fixed, but itsdemand will continue to increase steadily into the

foreseeable future. Reclamation of desertlands has been a top priority and challengefor the Egyptian government over the lastfew decades. In this study, we investigatethe possible effect of water defect on theflower yield, essential oil composition andproline content of C. officinalis flowers, aneconomically important medicinal andornamental plant in Egypt.


Experiments were carried out in agreenhouse at the National Research Centre,Egypt, during 2010/2011 and 2011/2012.Calendula officinalis seedlings wereobtained from the Medicinal and Aromaticdepartment, Agriculture Research Centre,Egypt. Uniform seedlings were transplantedinto plastic pots (30 cm diameter and 50 cmheight). In the first week of Novemberduring both seasons, the pots weretransferred to a greenhouse adjusted to thenatural conditions. Each pot was filled with10 kg of air-dried Typic Torrifluvents soil

(USDA 1999), with a field water capacity (FWC) of 62.5%based on the weight of the soil. Physical and chemicalproperties of the soil used in this study were determinedaccording to Jackson (1973) and Cottenie et al. (1982) andare presented in Table 1. Three weeks after transplanting,the seedlings were thinned to three plants per pot.Calendula officinalis plants were divided into four maingroups were subjected to different levels of water regime:25, 50, 75, or 100% (the control) corresponding to theFWC determined in the soil by weight. All agriculturalpractices, other than the experimental treatments were doneaccording to the recommendation of the Ministry ofAgriculture, Egypt.

Table 1. Physical and chemical properties of the soil used

Soiltexture

pH EC* Org-C OM TotalN

TotalP

TotalK

%Sandyloam

7.2 0.6 0.9 1.9 0.3 0.1 0.1

Note: EC* = electronic conductivity (salinity), Org-C = organicC, OM = organic matter

HarvestingFresh flowers were collected from each treatment at

three flowering stages, start flowering or flower budinitiation (25 days after bud formation), full flowering (86days after bud formation) and end of flowering (119 daysafter bud formation) in both seasons, all of which were airdried. Yield (dry weights of flower) was recorded (gplant−1). On the other hand the vegetative growth characters[Plant height (cm), leaf area (cm2), flower diameter (cm)and spike stem diameter] and vase life (day) were recordedduring the start flowering stage.

Figure 1. Calendula officinalis L. (English marigold, pot marigold)

METWALLY et al. – Effect of water regime on Calendula officinalis 67

Essential oil isolationFresh flowers were collected from each

treatment and from the three floweringstages in both seasons, air dried andweighed to extract the essential oil. Dryflowers (500 g) from each of thesetreatments were hydro-distilled for 3 h usinga Clevenger-type apparatus (Clevenger1928). The essential oil content wascalculated as a percentage. Also g essentialoil plant −1 was calculated according to thedry weight of flowers per plant.

Gas Chromatography-MassSpectrophotometric (GC-MS) analysis

The ADELSIGLC MS system, equippedwith a BPX5 capillary column (0.22 mm id× 25 m, film thickness 0.25 µm) was used.Analysis was carried out using He as thecarrier gas, with a flow rate of 1.0 mL/min.The column temperature was programmedfrom 60 to 240°C at 3°C/min. The samplesize was 2 µl, the split ratio 1: 20; injectortemperature was 250°C; ionization voltageapplied was 70 eV, mass range m/z 41-400amu. Kovat’s indices were determined byco-injection of the sample with a solutioncontaining a homologous series of n-hydrocarbons in a temperature run identicalto that described above.

Identification of essential oil componentsThe separated components of the

essential oil were identified by matchingwith the National Institute of Standards andTechnology (NIST) mass spectral librarydata, and by comparison with Kovat’sindices of authentic components and withpublished data (Adams 2001). Quantitativedetermination was carried out based on peakarea integration.

Proline determinationProline content was determined in fresh

leaves during three flowering stages usingthe method of Bates et al. (1973).

Statistical analysisIn this experiment, 2 factors were

considered: water deficit (100, 75, 50 and 25% FWC) and flowering stages. For eachtreatment there were 4 replicates, each ofwhich had 8 pots; in each pot 3 individualplants were planted. The experimentaldesign followed a complete random blockdesign. According to Snedecor and Cochran(1990) the averages of data for two seasonswere statistically analyzed using 2-wayanalysis of variance (ANOVA-2) for floweryield (g plant-1), Proline (µm mg-1) and

Table 2. Effect of water regime on the vegetative growth characters and vase life

Water stresstreatment

Vegetative growth charactersVase life(day)

Plantheight(cm)

Leaf area(cm2)

Flowerdiameter(cm)

Spike stemdiameter(cm)

100 36.3 79.3 5.0 0.3 8.375 39.3 95.0 5.7 0.5 10.050 38.3 85.3 5.5 0.4 9.325 35.7 52.7 4.5 0.2 6.7F. values 0.3 0.1 *** 0.1 0.1 ** 0.1 **LSD at 0.05 NS 6.4 NS 0.04 1.4Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of the 2-wayanalysis of variance (ANOVA-1).

Table 3. Effect of water regime, flowering stages and their interactions on floweryield, proline and essential oil content

Treatments Floweryield(g plant -1)

Proline(µm mg-1)

Essential oil

Stages Waterregime % mL plant -1

Start 100 9.3 2.1 0.1 0.00975 113.1 2.5 0.2 0.22650 50.4 3.8 0.2 0.10125 44.1 8.6 0.3 0.132Over all 54.2 4.3 0.2 0.117

Full 100 135.7 3.4 0.2 0.27175 233.6 3.9 0.2 0.46750 222.9 5.8 0.3 0.66925 100.3 10.9 0.4 0.401Over all 173.1 6.0 0.3 0.452

End 100 69.1 2.4 0.2 0.13875 152.6 2.9 0.3 0.45850 103.1 4.8 0.4 0.41225 58.8 9.8 0.5 0.294Over all 95.9 5.0 0.4 0.326

Over all water regime 100 71.4 2.6 0.2 0.13975 166.4 3.1 0.2 0.38450 125.5 4.8 0.3 0.39425 67.7 9.8 0.4 0.276

F valuesWater regime 2466.2*** 2555.9*** 9.727*** 31606.9***Stages 15248.6*** 247.4*** 7.364*** 140934.9***Water regime Ҳ stages 3796.1 *** 5.7 *** 0.455 5553.3***LSD at 0.05Water stress 38.8 0.6 0.04 0.04Stages 22.8 0.3 0.03 0.03Water stress Ҳ stages 21.7 0.2 NS 0.02

Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of the 2-wayanalysis of variance (ANOVA-2).

Table 4. Effect of water regime on the chemical constitution of essential oil

Constituents (%) RI Water regime treatments F. values LSD100 75 50 25γ-Cadinene 1514 2.5 2.6 2.7 2.8 1.3 NS∆ -Cadinene 1523 18.8 18.9 19.3 19.8 8.9** 0.2β-Calacorene 1564 4.4 4.5 4.7 4.9 1.3 NSNerolidol (E) 1565 6.9 7.1 7.8 7.9 11.9*** 0.2β-Acorenol 1635 4.9 5.2 5.3 5.5 1.4 NSα-Eudesmol 1653 10.8 10.9 11.8 12.9 36.9*** 0.1α-Cadinol 1663 33.9 34.8 34.9 35.2 1.6 NSPentacosane 2501 3.8 4.2 4.4 4.9 6.3* 0.2Total identified 86.0 88.2 90.9 93.9Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of the 2-wayanalysis of variance (ANOVA-2).

5 (2): 65-69, November 201368

essential oil (% and mL plant-1); while using 1-wayanalysis of variance (ANOVA-1) for plant height (cm), leafarea (cm2),flower diameter (cm),Spike stem diameter, Vaselife (day) and essential oil constituents. The applications ofthat technique were according to the STAT-ITCF program(Foucart 1982).


Effect of water regime on the vegetative growthcharacters and vase life

Vegetative growth characters [Plant height (cm), leafarea (cm2), flower diameter (cm) and spike stem diameter]and vase life (day) of Calendula plants were affected bychanges of soil moisture. The highest values of thesemeasurements were recorded when plants subjected to 75%of FWC with the values of 39.3, 95.0, 5.7, 0.5 and 10.0respectively. On the other hand the lowest values wererecorded when the plants were subjected to 25% of FWC.ANOVA indicated that the changes in plant height andflower diameter were insignificant but highly significantfor leaf area while more significant for pike stem diameterand Vase life (Table 2). The inhibition of plant growthcharacters and flower yield under water deficit treatment(25% of FWC) may be due to exposure to injurious levelsof drought causing a decrease of turgor which would resultin a decrease of growth and development of cells, especiallyin stems and leaves (Merrill and Eckard 1971). Cell growthis the most important process and is affected by water stress.Plant size is indicated by a decrease in height or smallersize of leaves when there is a decrease in the growth of cells(Hsiao 1973). When leaf size is smaller, the capacity to traplight decreases too and the capacity of total photosynthesisdecreases, i.e. photosynthesis is restricted in watershortage conditions, with a subsequent reduction in plantgrowth and performance (Hsiao 1973). Water stressresulted in significant reductions in CO2 exchange rate,total assimilatory area, fresh and dry matter andchlorophyll in Japanese mint (Mentha arvensis L. cv. MS77) (Misra and Srivastava 2000). The loss ofphotosynthesis in drought stress conditions results in a lossof dry matter production at the leaf level of mungbean, bean,topiary bean, Sesuvium portulacastrum (ambiguously) andPesquisa Agropeularia (embrapa) plants (Cox and Jolliff1987; Abdul-Hamid et al. 1990; Castonguay and Markhart1991; Nunez-Barrios 1991; Viera et al. 1991; Slama et al.2007). However the decrease in flowers vase life understress condition may be due to loss of turgidity (Hirt andShinozaki 2003).

Effect of water regime, flowering stages and theirinteractions on the flower yield

Water regime and/or flowering stage affected the totalflowers (g plant-1) (Table 3). Thus, various characteristicsof the flowers decreased under the various water regimelevels, especially at 25 % of FWC at the end of floweringstages. Greatest yields were obtained at 75 % of FWC,especially at full flowering stage (Table 3). The decrease inflower heads was highly significant for water regime

treatments and for flowering stages. In addition, thechanges in this variable were highly significant for thewater regime × flowering stage interaction (Table 3). Ourresults showed that growth and flower yield of C.officinalis plants was clearly affected by the different waterregime, where the growth parameters recorded the highestvalues when plants irrigated after adaptation of 75 % ofFWC. These superiority may be due to treatment providethe plant all time of growth with adequate supply of waterwhich accelerate physiological processes and plant growth.In this respect Tayel and Sabreen (2011) indicated that soilwater potential through the growing season is necessary tomaintain crop growth. Moreover, addition of adequatewater decreased abssic acid and increased cytokinins,gibrellin and indole acetic acid hormones, which reflectinggood plant growth and finally yield (Hayat 2007).

Effect of water regime, flowering stages and theirinteractions on proline content

The accumulation of proline in C. officinalis leaves waspromoted by applying various levels of soil moisture,flowering stages and their interaction (Table 3). Thehighest proline content resulted from 25 % FWC treatmentat full flowering stage (Table 3). The increase in prolinecontent was highly significant for water regime floweringstages and their interaction treatments (Table 3). Theresults of proline content agree with those of Salama et al.(2001) and Blum and Ebercon (1993) who indicated thatproline is regarded as a source of energy, carbon, andnitrogen for recovering tissues under stress conditions.

Effect of water regime, flowering stages and theirinteractions on essential oil content and its chemicalcomposition

Data in table indicates that the highest percentage of C.officinalis essential oil was obtained from flower heads atthe full flowering stage, i.e. full flowering stage, therefore,this stage was investigated to identify the essential oilcomponents. Treatment with 25% (FWC) caused the mostpronounced increase in the essential oil percentage at allflowering stages; however, this percentage decreased atcontrol treatments. The essential oil yield (g plant-1)increased in the most water regime treatments comparedwith the control treatment in. The essential oil% and yieldof flower heads was greatest at full flowering. Waterregime treatments and flowering stage, when assessedseparately, had a greater effect on essential % and yield offlowers than their interaction, i.e. water regime treatments× flowering stages. The changes in essential oil yield werehighly significant for water regime treatments andflowering stages (Table 3).

A qualitative and quantitative comparison of the mainconstituents present with the water regime treatments inhydro-distilled C. officinalis essential oil was studied(Table 4). A total of 8 compounds, accounting for 86.0-94.3% of the oil, were identified. The effects of soilmoisture levels on the chemical composition of essential oilextracted are shown in Table 4. The main components wereγ-cadinene, ∆-cadinene, β-calacorene, nerolidol, β-acoreno,α-eudesmol, α-cadinol and pentacosane. Moreover, the

METWALLY et al. – Effect of water regime on Calendula officinalis 69

highest percentages of the main components resulted fromthe treatment of 25% FWC. These percentages decreasedas soil moisture levels increased. The changes in nerolidoland α-eudesmol constituents were highly significant forwater regime treatments while the changes in ∆-cadineneconstituent were more significant. The changes inpentacosane were significant. On the other hand, changesin γ-cadinenem, β-calacorene, β-acorenol and α-cadinolwere insignificant for water regime treatments (Table 4).The effect of different treatments on essential oil and itsconstituents may be due to its effect on enzyme activity andmetabolism of essential oil production (Burbott andLoomis 1969).

CONCLUSION

It may be concluded that water stress affects on growth,flower yield, vase life. essential oil composition and prolinecontents of Calendula officinalis L. plants.

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Phenotypic recurrent selection on herb growth yield of citronella grass(Cymbopogon nardus) grown in Egypt

MOHAMED M. IBRAHIM1,♥, KHALID A. KHALID2

1Department of Genetics and Cytology, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Tel. +202-3371615.email: [email protected]

2Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt.

Manuscript received: 26 June 2013. Revision accepted: 16 July 2013.

Abstract. Ibrahim MM,Khalid KA. 2013. Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus)grown in Egypt. Nusantara Bioscience 5: 70-74. This investigation was conducted in four generations: base population (G0, G1, G2)and G3 (clone selection generation) to evaluate the genetic variability of citronella clones. Thirteen clones were selected from basepopulation to study the herb growth yield characters and oil production as well as genetic parameters, correlation and regression. Resultswere recorded for herb growth characters (i.e. plant high (PH), no. of tillers (NOT), dry yield (DY), viability percentage (VP) and oilproduction. Significant variation was observed among citronella clones in base population for most studied traits. Wide range of meanvalues was observed among the characters for generations and cuts in most of traits. High heritability values (0.95, 0.93, 0.89 and 0.72)were estimated in NOT, LG, HY and VP., respectively. Clone code no. 39/3, 17/4 and 8/1 gave highest values of dry weight, oil yieldand viability percentage. Selected clones showed significant positive regression and correlation between dry weight and each of numberof tillers and linear growth. On contrary, viability percentage had significant negative correlation and regression with other characters.These results raveled high yielding selected citronella clones will be utilized in medicinal plant breeding program.

Key words: citronella, essential oil, heritability, selection

Abstrak. Ibrahim MM,Khalid KA. 2013. Seleksi fenotipik berulang pada pertumbuhan herba rumput serai (Cymbopogon nardus) yangditanam di Mesir. Nusantara Bioscience 5: 70-74. Penelitian ini dilakukan dalam empat generasi: populasi dasar (G0, G1, G2) dan G3(generasi pilihan klon) untuk mengevaluasi keragaman genetik klon-klon serai (sereh wangi). Tiga belas klon dipilih dari populasi dasaruntuk mempelajari karakter hasil pertumbuhan herba dan produksi minyak serta parameter genetik, dengan korelasi dan regresi. Hasilyang dicatat berupa karakter hasil pertumbuhan herba (yaitu: tinggi tanaman (PH), jumlah anakan (NOT), hasil berat kering (DY),persentase viabilitas (VP) dan produksi minyak. Variasi yang signifikan teramati diantara klon-klon serai dalam populasi dasar untuksebagian besar sifat-sifat yang dipelajari. Nilai rata-rata yang berjangkauan luas teramati diantara sifat-sifat untuk generasi danpemotongan di sebagian besar sifat. Nilai heritabilitas tinggi (0,95, 0,93, 0,89 dan 0,72) diperkirakan secara berturut-turut pada NOT,LG, HY dan VP. Kode klon no. 39/3, 17/4 dan 8/1 memiliki nilai tertinggi dalam berat kering, hasil minyak dan persentase viabilitas.Klon terpilih menunjukkan regresi dan korelasi positif yang signifikan antara berat kering dan jumlah anakan serta pertumbuhan linear.Sebaliknya, persentase viabilitas memiliki korelasi dan regresi negatif yang signifikan dengan karakter lain. Hasil ini memberikan klon-klon serai terseleksi dengan hasil panen yang tinggi yang akan digunakan pada program pemuliaan tanaman obat.

Kata kunci: serai, minyak esensial, heritabilitas, seleksi

INTRODUCTION

Citronella, Cymbopogon nardus L. is a tufted perennialgrass with long narrow leaves and numerous stems arisingfrom short rhizome roots (Figure 1) which are indigenousto India (Weiss 1997). The importance of citronella grass isrelated to widely use in perfumes, soaps, insect repellent. Itis also used in Chinese medicine and traditional medicinefor the treatment of rheumatism, digestive problems, fever,and intestinal problems and in aromatherapy to treat colds,flu, and headaches (Akhila 2010).

Few efforts had been carried out on the cropimprovement through the clone selection of herb andessential oil yield. Plant breeders primarily estimatevariability in initial population of its importance in

choosing the most efficient breeding procedures. Kulkarni(1994) studied the herb growth characters, oil yield andvariability in lemon grass though phenotypic recurrentselection, found that realized gains from selection wereslightly smaller or smaller to predicted gains. Kole and Sen(1986) studied the selection strategy of yield and yieldcomponent in lemon grass clones in base population andselected clones. Selection is more efficient to improvementessential oil and yield component. The importance of cloneselection in genus Cymbopogon and genetic variabilitywere studied by many researchers (Patra et al. 1991; Raoand sobti 1991; Siugh and pathak 1994). They foundrecently a few clonal varieties have been developed toovercome wide fluctuation in quantity production of genusCymbopogon.

IBRAHIM & KHALID – Phenotypic recurrent selection on Cymbopogon nardus 71

Information on genetic variability, heritabilityestimates and interclass correlations for importantcharacters are essential for plant breeding program. Thepresent work aims to study genetic variability in populationof citronella grass and then attempt to select somecharacterized and improved clones in herb growth yieldand essential oil production.


Cultivation methodThis work was carried out in the Experimental farm of

Nation Research Centre (NRC) in El Nobaria, Egypt,during four successive seasons 2008 - 2011 seasons orgenerations. Clones of citronella grass (Cymbopogomnards L.) were obtained from the groups of genetics andbreeding of medicinal plants, Nation Research Centre(NRC), Egypt. All tented clones were cultivated usingtillers witch separated from mother plants. On the 1st ofMay in base population and selected clones over two trials.A randomized – complete block design with 3 replicationswas used. Each replicate had one line of 3m long and 60cm in between. Each line had six ridges with 50 cm space.All cultural practices were followed. The first cut wastaken after six months after planting, while the second cutwas taken three months later. The plants of base populationand selection experiment were carried out though fourgeneration. The plant records included linear growth,number of tillers, herb dry yield and viability percentagesof 13 clones were cultivated.

Statistical analysisStatistical analysis of data was computed

according to Steel and Torrie (1965).Heritability estimates were according toRobinson et al. (1951).Correlation andregression analysis were estimates withaccording to Mode and Robinson (1959).

Extraction of essential oilsThe essential oils were extracted by

harvesting the plants of selected clone'sbasis on dry weight. 30 grams of driedleaves from each of three replications foreach selected clone were hydro distilledusing Clevenger apparatus (Gunter 1962)for three hours. The obtained oil wasmeasured and then computed as percentagevalue.


Rang, mean values, coefficient ofvariation. (%) analysis of variance,heritability and L.S.D. of four herb growthcharacters (no. of tillers.linear growth, herbdry yield and viability percentage) at threegenerations (G0, G1 and G2) of citronella

clones population are presented in Table 1. Significantdifferences were showed in all studied characters. Widerang observed in all studied traits among three generations.Mean values ranged from(8.3±0.6) to (87.3±0.6),(43.4±0.9) to (108.5±5.1), (32.3±2.7) to (165.9±2.48),(77.0±1.9) to (98±0.07) % of numbers of tillers, lineargrowth, herb dry yield and viability percentagerespectively. Coefficient of variation (C.V. %) varied in allgenerations and all studied characters. Heritability estimaterevealed highest values (0.9528, 0.9303, 0.8935 and 0.725)in no. of tillers,linear growth, herb dry yield and viabilitypercentage., respectively,while the values 0.4253, 0.1589,0.372 and 0.1986 were the lowest in the same characters.,respectively. Moreover, the first cut had higher heritabilityvalues comparing with the second cut values for all studiedcharacters.

Evaluation of selected citronella clonesThirteen clones of citronella were selected from earlier

population to study clonal variation parameters andevaluate herb growth characters and yield components

Analysis of varianceHighly significant differences were shown among

generations in all studied characters and only related tolinear growth among clones

Covariance analysisAverage values, coefficient of variation and parent-

progeny regression of 13 selected clones over four differentgenerations presented in Table (3). clonal variations werefound for herb growth yield characters in the four

Figure 1. Cymbopogon nardus L. (citronella grass)

5 (2): 70-74, November 201372

generations. Average values ofclones cod no. 39/3, 15/4, 17/4,1/2and 14/3 had the highest values ofherb dry character.Linear growth hadthe highest spatial regression (bij) incase of clone 25/1 followed by clone39/3 in dry weight and no. of tillers

Interclass correlation andregression

Interclass correlation andregression among four growth herbyield in the mean of 13 citronellaclones was presented in Table 4.Viability percentage showed highnegative correlation with each oflinear growth, no. of tillers and herbdry yield. One the other hand herbdry yield was high positivecorrelation with linear growth andno. of tillers. Viability percentagewith each of no. of tillers, lineargrowth and herb dry yield revealednegative regression values (-0.52, -1.01, -13.25) respectively. Herb dryyield with each of linear growth andno. of tillers had high (25.23, 18.66)and positive values of regressionrespectively.

Variation in oil production inselected citronella clones

Data presented in Table 5 showedvariation in the oil content variationand oil yield of the thirteen selectedclones basis on dry weight..Oilpercentage ranged from 2.00 to 2.66for clones (24/4, 14/3) and 36/3respectively. Clones no. 15/4, 17/4and 1/2 had highest values of oilyield. Oil yield ranged from 2.96 inclone 34/3 to 11.99 in case of clone15/4 respectively.

DISCUSSION

Cymbopogon species displaywide variation in morphologicalattributes and essential oilcomposition at inter- and intra -specific levels. Germplasm diversityis important for plant conservationand improvement, therefore there isinterest in determining the geneticdiversity in Cymbopogon germplasm.Although, morphological traits canbe used to assess genetic diversitythey are strongly influenced byenvironment conditions and show

Table 1. Biometrical genetic parameters of four major herb yield component in citronellagrass under clone selection

Items Range Mean C.V.%

ANOVA h2b LSD noơ2g ơ2e 0.05 0.01

Number of tillers1st gen (G0) 5.5-11.3 8.3±0.6 25 24.43** 33.64 0.4253 4.36 5.74 412nd gen (G1) 11.4-17.7 14.9±0.6 14 226.38** 11.33 0.9528 1.71 2.25 371st Cut 5.1-13.0 8.3±0.6 25 77.15** 10.15 0.8828 2.29 3.01 372nd Cut 16.0-28.5 21.1±0.8 14 143.48** 39.48 0.7837 4.6 6.06 373rd gen(G2) 42.5-119.3 75.7±1.2 24 1289.07** 105.44 0.5508 44.94 59.16 16

Linear growth (cm)1st gen (G0) 39.6-49.0 43.4±0.9 7 29.78* 157.69 0.1589 8.99 0.0 412nd gen (G1) 51.0-63.1 57.0±0.6 8 216.92** 52.6 0.7574 3.7 4.9 371st Cut 37.5-52.5 46.4±1.7 12 147.22** 105.35 0.5829 7.6 10 372nd Cut 62.3-74.5 63.6±1.3 6 46.04* 121.92 0.2741 7.9 10.4 373rd gen (G2) 77.5-127.5 108.5±5.1 15 435.26** 32.63 0.9303 7.9 10.4 16

Herb dry yield (g/plant)1st gen (G0) 18.4-95.0 35.3±1.2 56 1328.92** 678.41 0.662 20 26.4 412nd gen (G1) 30.0-78.1 56.0±3.6 23 2021.91* 1197.83 0.372 17.9 23.5 371st Cut 18.9-52.7 32.3±2.7 30 1083.82** 596.35 0.6451 17.8 23.4 372nd Cut 32.5-121.7 80.0±1.3 28 1048.57** 1799.3 0.3682 30.9 40.7 373rd gen (G2) 420.344 1659±248 54 8.79** 0.26 0.8935 70.7 930 16

Parentage viability1st gen (G0) 75-100 98±0.7 72ndgen (G1) 42-100 79±4.2 19 429.59** 164.99 0.725 20.6 27.1 413rd gen (G2) 68-89 77±1.9 9 75.23** 315.57 0.1986 22 29 41

Table 2. Analysis of variance of 13 citronella clones grown in four generations underclone selection

S.O.V.A DF No. of tillers DF Linear growth DF Herb dry yield Viability %

Clones 12 117.93 10 140.57** 12 203566.21 82.57Generations 3 13435.49*** 3 9822.34 *** 3 8423104.3** 1187.16***Error 36 101.13 36 51.07 36 199424.21 123.467

Table 3. Average values, coefficient of variation and regression on the median index of13 citronella clones grown in four generations

Viability % Herb dry yield (gm.) Linear growth (cm.) Number of tillers Codeno.bij C.V.

% Average bij C.V.% Average bij C.V.

% Average bij C.V.% Average

1.02 16 87 2.10 18.7 905 1.26 52 75 1.62 13.1 40 39/31.22 20 83 0.98 18.7 424 1.28 57 68 0.79 5.6 27 54/40.98 16 85 1.84 18.7 794 00 00 000 0.92 12.1 25 15/41.72 40 74 1.54 18.4 673 1.09 46 72 1.04 10.4 32 17/41.16 19 86 0.63 16.4 309 1.27 49 74 1.09 12.2 29 24/11.16 25 73 0.94 17.0 443 0.75 35 64 0.92 12.1 25 22/10.91 14 88 0.75 17.8 341 5.85 32 56 1.04 12.6 27 25/11.01 19 88 1.01 17.9 455 1.26 51 74 0.61 8,9 23 14/30.69 11 92 0.24 13.9 137 0.83 43 59 0.47 7.4 21 34/30.74 11 90 0.58 15.4 293 0.73 33 66 0.05 11.5 32 8/11.30 12 88 0.42 15.1 245 00 00 00 0.87 10.1 24 36/31.20 17 84 1.29 18.6 560 1.03 52 64 1.36 12.7 35 1/20.04 12 77 0.63 17.7 287 1.01 49 62 1.05 12.4 28 18/2

84.23±1.667.42

451±62.5349.97

66.59±149444.57

28.0±15.9411.3.85

MeanCV%

IBRAHIM & KHALID – Phenotypic recurrent selection on Cymbopogon nardus 73

little variation at the intra-specific level (Dhar et al. 1981;Kulkarni and Rajagopol 1986; Kulkarni 1997). Thefluctuation of viability percentage of citronella clonespopulation from generation to other due to compensationamong yield component, fitness characters and genotype –environmental interaction Adams and Gutfuls (1971). Theanalysis of variance and covariance in the actual andpredicted selection in base population and selected top 30%of citronella clones were studied in herb growth charactersand oil yield production. Highly significant differenceswere shown among generations in three characters only(number of tillers, linear growth and viability percentage).This finding revealed that clones carried genes withdifferent additive effects. Meanwhile, in citronella clonesreveled highly significant differences between clones incase of linear growth character, these results were reflectedthe role of gene type–environment interaction and seasonalvariations (Western and Lawrence 1970). The result ofclonal variation for herb growth yield characters in thethree generations are in agreement with the investigation ofOmkhafe and Alika (2004) and Jezowski (2008).

Table 4. Estimates of interclass correlation (above) andregression (down) coefficients among four herb growth yieldcomponents.

Characters X1 X2 X3 X4

No. of tillers (X1) 1.000 0.3368* 0.5911** -0.5202**Linear growth ( X2) 0.360 1.000 0.4699** -0.85414**Herb dry yield ( X3) 25.23 18.66 1.0000 -03706**Viability% ( X4) -0.52 -1.01 -13.25 1.0000

Table 5. Average values, coefficient of variation in oil productionfor selected citronella clones at two generations.

Code no. Oil percent % Oil yield ml./plantAverage C.V.% Average C.V.%

39/3 2.05 17.02 6.33 24.2224/4 2.00 13.20 8.02 67.2115/4 2.05 4.80 11.99 61.1217/4 2.10 7.20 10.43 50.9324/1 2.46 14.30 7.43 39.0422/1 2.03 28.70 8.43 56.5025/1 2.08 16.23 7.09 40.3014/3 2.00 16.06 9.12 60.1234/3 2.16 11.03 2.96 53.618/1 2.14 10.25 6.22 24.3036/3 2.66 16.22 7.33 40.151/2 2.12 12.80 10.40 61.3018/2 2.16 10.30 6.22 25.26MeanC.V.%

2.16 ±0.05313.70

7.85 ±0.64546.47

In citronella studied clones the correlation analysisconfirms the role of clones x environment interactionViability percentage showed high negative correlation witheach of linear growth, no. of tillers and herb dry yield. Onethe other hand herb dry yield was high positive correlationwith linear growth and no. of tillers. These results reflectedcompensation in yield components and variation inexcretion. That is confirmed clear homeostasis of genetic

background and differential expression in these charactersexpression (El- Balal et al. 1983).

Comparison of oil content and yield of the selectedclones in the four seasons revealed apparent interaction(Harridy et al. 2001). Herb dry yield express of growthactivity and essential oil production in grasses (Barristerand GutBurg 1965). Variations in oil production andaccumulation (oil/yield ml/plant) due to many factors, suchas growth stage, elongation of tillers and dry matterproduction were influenced. These finding is in agreementwith Misra and Srivastava (1991) and Behura et al. (1991).

CONCLUSION

The results from this study leads to the conclusion thatthere are significant genetic variability among citronellaclones has been made through phenotypic recurrentselection for studied traits. Wide rang were showed in allstudied characters among three generations. Heritabilityestimates revealed highest values in no. of tillers, lineargrowth, and herb dry yield and viability percentage.Coefficient of variation (C.V. %) varied in all generationsand all studied characters. One the other hand herb dryyield was high positive correlation with linear growth andno. of tillers. Highly significant differences were shownamong generations in three characters only (number oftillers, linear growth and viability percentage). This findingrevealed that clones carried genes with different additiveeffects. Meanwhile, in citronella clones reveled highlysignificant differences between clones in case of lineargrowth character, these results were reflected the role ofgene type–environment interaction and seasonal variations

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Burmeister VA, Gutenberg A. 1960 On the formation of essential oils inplants. Planta Medica 8: 1-30.

Dhar AK, Thappa RK, Tel CKA. 1981. Variabililty in yield andcomposition of essential oil in Cymbopogon jawarancusa. PlantaMedica 41: 366-388

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El-balal AA, Mandour MS, Nofel M, Tawfik MSH. 1983. Physiologicalhomestasis of essential oil production in lemon grass (Cymbopogoncitratus L). The IX Inter. Cong. Ess. Oils Singapore 147-151.

Guenther E. 1962. Essential Oils vol.1.4D Van Nostrand Co., New Jersey.Harridy IM, Gabr AS, Shalan MN. 2001. Comparative study on some

species of the genus Cymbopogon grown in Egypt. Egypt J Res 79 (4)48-1469.

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Kole CR. 1986. Path- coefficient analysis in citronella. Indian Journal ofAgric Sci 56 (4) 241-244.

Kole CR, Sen S. 1986. Selection strategy for improvement of oil yield ofcitronella Cymbopogon winterianus. Environ Ecol 4 (4) 613-618.

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Kulkarni RN. 1994. Phenotypic recurrent selection for oil content in EastIndian Lemongrass. Euphytica 78 (1-2): 103-107.

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Patra NK, Sharma S, Ram RS. 1991. correlation and several plant traits tocitral content in lemongrass (Cymbopogon spp.). Exp Genet 7: 102-106

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Sing RS, Pathak MC. 1994. Variability in herb yield and volatileconstituents of Cymbpobogon jawarancusa (Jones) Schull cultivars.Ind Crops Prod 2 (3) 197-199.

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Weiss EA. 1997. Essential oil Crops. CAB International, New YorkU.S.A

Westerman JM., Lawrance G. 1970. Genotype- environmental interactiondevelopmental regulation in Aabidapsis thaliana L. Heredity 25:1457-1465


Experimental effect of temperature and sedimentation on bleaching ofthe two Red Sea corals Stylophora pistillata and Acropora humilis

MOHAMMED S.A. AMMAR1,♥, AHMED H. OBUID-ALLAH2, MONTASER A.M. AL-HAMMADY3

1Department of Hydrobiology, National Institute of Oceanography and Fisheries, P.O. Box 182, Suez, Egypt. Tel. +20 111 1072982,Fax. +20 623360016, E-mail: [email protected]

2Department of Zoology, Faculty of Science, Assiut University, Assiut, Egypt3National Institute of Oceanography and Fisheries, Hurghada, Egypt

Manuscript received: 30 July 2013. Revision accepted: 15 August 2013.

Abstract. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2013. Experimental effect of temperature and sedimentation onbleaching of the two Red Sea corals Stylophora pistillata and Acropora humilis. Nusantara Bioscience 5: 75-85. At 26°C (the controlsample), the loss of zooxanthellae by each of the two studied corals Stylophora pistillata and Acropora humilis was very low. Cellviability of the two studied corals was similar at 26 and 29°C, but depicted a sharp decline of zooxanthellae lost at 31°C through time.As the temperature increased to 35°C, the loss of zooxanthellae from each host increased both with time and temperature elevation. Thecoral A. humilis had a higher decrease in its zooxanthellae densities than S. pistillata at the same treatment. Bleaching temperaturethreshold was 33°C or less for the two species S. pistillata and A. humilis where 51% of their zooxanthellae were lost after 24 h ofexposure. In samples exposed to sediment concentration of 0.1 mg/cm2/L, zooxanthellae densities of A. humilis and S. pistillata did notshow any decrease after 1 day. However, after 1 days of exposure to 0.5 mg/cm2/L, zooxanthellae densities were significantly differentfrom those of the controls. Increases in sediment concentration to 1 mg/cm2/L caused a decrease in zooxanthellae densities that varygreatly over time. Measurements of zooxanthellae densities of A. humilis and S. pistillata at this stage revealed a highly significantdifference between exposed and control sample. At 1 g/cm2/L, the number of zooxanthellae lost from A. humilis was higher than thoselost from S. pistillata at same time. It is suggested that, the normal sedimentation rate for A. humilis and S. pistillata to be in an order of1 mg/cm2/L or less.

Key words: Acropora humilis, bleaching, Red Sea corals, sedimentation, Stylophora pistillata, temperature

Abstrak. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2013. Pengaruh perlakuan suhu dan sedimentasi terhadap pemutihan duajenis karang dari Laut Merah Stylophora pistillata dan Acropora humilis. Nusantara Bioscience 5: 75-85. Pada suhu 26°C (kontrol),hilangnya zooxanthellae dari dua jenis karang yang diteliti Stylophora pistillata dan Acropora humilis sangat rendah. Viabilitas selkedua jenis karang tersebut serupa pada suhu 26 dan 29°C, tetapi pada suhu 31°C terjadi penurunan tajam zooxanthellae sejalan denganbertambahnya waktu. Pada saat suhu meningkat menjadi 35°C, hilangnya zooxanthellae dari masing-masing inang meningkat sejalandengan bertambahnya waktu maupun suhu. Karang A. humilis mengalami penurunan kepadatan zooxanthellae yang lebih tinggidaripada S. pistillata pada perlakuan yang sama. Batas suhu pemutihan karang adalah 33°C atau kurang untuk kedua jenis karang, S.pistillata dan A. Humilis, dimana 51% dari zooxanthellae-nya hilang setelah 24 jam paparan. Pada sampel yang terpapar sedimendengan konsentrasi 0,1 mg/cm2/L, kepadatan zooxanthellae A. humilis dan S. pistillata tidak menunjukkan penurunan apapun setelah 1hari. Namun, setelah 1 hari paparan sedimen 0,5 mg/cm2/L, kepadatan zooxanthellae secara signifikan berbeda dari kontrol. Peningkatankonsentrasi sedimen 1 mg/cm2/L menyebabkan penurunan kepadatan zooxanthellae yang sangat bervariasi dari waktu ke waktu. Padatahap ini, pengukuran kepadatan zooxanthellae A. humilis dan S. pistillata menunjukkan perbedaan yang sangat signifikan antara yangterpapar dan kontrol. Pada konsentrasi sedimen 1 g/cm2/L, jumlah zooxanthellae yang hilang dari A. humilis lebih tinggi daripada yanghilang dari S. pistillata pada waktu yang sama. Hal ini menunjukkan bahwa, tingkat sedimentasi normal untuk A. humilis dan S.pistillata berada pada kisaran 1 mg/cm2/L atau kurang.

Kata kunci: Acropora humilis, pemutihan, karang Laut Merah, sedimentasi, Stylophora pistillata, suhu

INTRODUCTION

Bleaching (loss of pigmentation by corals) is awidespread phenomenon in coral reef ecosystems. Despitethis, the underlying of some forms of bleaching are poorlyunderstood. This study explores the conditions that inducedbleaching in two zooxanthellate, reef coral speciesAcropora humilis and Stylophora pistillata collected fromthe middle-reef in front of National institute ofoceanography and fisheries (NIOF), Hurghada Branch.

Environmental extremes, such as high temperature orirradiance, damage the symbionts’ photosyntheticmachinery, resulting in the over production of oxygenradicals. This leads to eventual cellular damage in thesymbionts and/or their hosts, and can lead to the expulsionof symbionts and the eventual break down of the symbiosis(Lesser 2006). The loss of zooxanthellae (and/or areduction in their pigment concentrations) as a result of thisprocess is referred to as‘‘bleaching’’ (Brown et al. 1999;Fitt et al. 2000).

5 (2): 75-85, November 201376

An important factor affecting coral communities is thewidespread, periodic bleaching of coral colonies (Celliersand Schleyer 2002). Coral bleaching results in thebreakdown of a mutualistic symbiosis that is essential forthe survival of corals, since the polyp receives a substantialpart of its energy from the zooxanthellae (Muller-Parkerand D’Elia 1997), and any disruption of this relationship willaffect photosynthetic potential, coral growth, reproductiveoutput and may eventually kill the coral (Richmond 1997).

Because of the intensity of recent bleaching events andassociated mortalities, bleaching is considered by most reefscientists to be a serious and relatively new challenge to thehealth of world's coral reef (Celliers and Schleyer 2002;Nesa and Hidaka 2009; Obura 2009; Miller et al. 2011).The widespread bleaching of corals on individual reefs haslargely been correlated with elevated sea temperature(Celliers and Schleyer 2002; Mc Clanahan et al. 2005).

Seasonal cycles in the quantum yields of chlorophyllfluorescence of corals have also been observed (Warner etal. 2002), revealing seasonal acclimatization in solarirradiance and seawater temperature. Moreover, Nesa andHidaka (2009) detected a negative correlation betweensurvival time and the zooxanthella density of tissue balls at31°C in both Fungia sp. and P. divaricata. Elevatedtemperature was found to significantly reduce the amountof zooxanthellae in primary polyps (Anlauf et al. 2010).Exposure to elevated temperatures reduces the photo-synthetic rate of zooxanthellae and predisposes theirphotosynthetic apparatus to further damage (Bhagooli andHidaka 2004).

Reef building corals and their zooxanthellae had showntwo broad ways that may be able to cope with elevatedtemperature (Clark 1983). Firstly, by micro-adaptivecombinations of symbiotic algae (Rowan 1997), secondlyby biochemical defense mechanisms, such as the inductionof heat shock proteins (Sharp et al. 1997; Ammar andMueller 2001). Despite their ability to acclimatize to theirthermal limits, reef building corals do not appear to haveacclimatized to the rapid increase in sea temperature overthe past 20 years.

Sedimentation is among the factors that leads to escapeof symbiotic zooxanthellae from the host coral (Dubinskyand Stambler 1996). Fabricius (2005) regarded sedimentationas an increasing threat to coral reefs. The impactsassociated with sedimentation and sediment burial includereduced photosynthesis and increased respiration (Philippand Fabricius 2003; Weber et al. 2006), tissue mortality(Lirman and Manzello 2009), reduced growth (Lirman andManzello 2009), and reduced fertilization, larvalsurvivorship, and recruitment (Babcock and Smith 2000).

The degree of coral mortality and bleaching depends onthe amount of sediment in the coral communities followinga tropical storm in the tropical Atlantic (Nowlis et al.1997). Burial of corals by sediments for 20 hours resultedin increased discoloration of coral tissue, after 68 hours ofburial, up to 98% of the tissue bleached in the first days,about 50% of this tissue disappeared subsequently and barecoral skeleton became exposed or were covered with algae(Wesseling et al. 1999). Cruz-Pinio et al. (2003) found that,high sedimentation rates, low light availability and

anthropogenic influence lead to cellular damage anddeteriorated coral skeletal density.

Turbidity reduces light levels, photosynthetic potentialand possibly coral growth rates (Yentsch et al. 2002;Anthony and Hoegh-Guldberg 2003) while elevated netsedimentation rates increase abrasion and smothering(Fabricius 2005), inhibiting coral growth and reefaccretion. In contrast, Palmer et al. (2010) found that, nearshore environments directly influenced by fluvialsediments and dominated by terrigenoclastic sedimentationare generally considered marginal for coral reef growth.The energetic costs of sediment clearing can beconsiderable (Riegl and Branch 1995), and the inability toclear sediments exposes corals to further stress as anoxicconditions under sediments can cause tissue bleaching andsubsequent mortality (Weber et al. 2006).

For the purposes of this study, the release of algalsymbionts at various temperatures and sedimentconcentrations (“bleaching response”) was studied on thetwo reef corals S. pistillata and A. humilis using theprotocols of Hoegh-Guldberg and Smith (1989) and Anlaufet al. (2010). The focus here on increasing seawatertemperature reflects the choice of an environmental factorthat is an integral component of global climate changeeffects and is tractable to experimental manipulation.Moreover both temperature and sedimentation have a well-established effect on coral bleaching.


Two experiments were carried out in the NIOFlaboratories using the hermatypic coral S. pistillata and A.humilis in which corals were exposed to elevated watertemperatures and different concentrations of sediments.Corals were collected from the middle reef in front of theNational institute of Oceanography and Fisheries (NIOF),Hurghada Branch, Egypt.

Experiments were conducted in open topped glassaquaria, under controlled conditions. Seawater was aeratedusing air pumps and heated and recirculated usingsubmersible pumps and aquarium heaters. Refrigeratedcoolers and refrigerated water-baths were used to controlthe temperatures to be within ±0.3°C of the desired level.Temperature readings were made every 5-15 min between8.00 a.m. and 6.00 p.m. During the night, the readings weremade only every 2 hours since the temperature was morestable. The temperatures were increased and decreasedfollowing the diurnal variation in water temperature in thefield by approximately 6°C. Temperature in the field wasbetween 29°C and 30°C at midday (at 3 m depth).

Small colonies of the two studied species werecollected from individual colonies located at 3 m depthfrom the Middle reef, which is located 200 m offshorebetween the northern reef and the crescent reef, directly infront of NIOF, Hurghada, Red Sea. Samples were thentransferred directly into glass aquaria supplied with airpumps for aeration.

Two corals for studying the effect of temperature wereincubated at each temperature test (24, 29, 31, 33 and

AMMAR et al. – Effect of temperature and sedimentation on corals bleaching 77

35˚C). Control samples were placed at room temperature(26˚C). An air stone was placed in each aquarium for watercirculation and the sample was put in a good light conditionduring the day for photosynthesis. Three colonies fromeach species were then taken after 6, 12 and 24 hours andtheir tissues were removed using sea water jet (Water PikTeledyne) for counting the zooxanthellae and measuringthe chlorophyll concentration. Zooxanthellae were countedusing count Rafter cell and the chlorophyll concentrationwas measured by spectrophotometer method of Jeffrey andHumphrey (1975).

Branches for studying the effect of sediments were putin glass aquaria, exposed to 0.1, 0.5, 1 mg/cm2/L and1g/cm2/L different concentrations of sediments, an air stonewas placed in each aquarium for aeration in a good lightingconditions, then three branches of each colony were takenafter 1, 5 and 10 days for counting zooxanthellae andmeasuring chlorophyll concentration.

Biomass measurementsFor zooxanthellae density, tissues were striped from the

skeletons (Plate 15) with a jet of recirculated 0.45 µmmembrane filtered sea water using a water pikTM (Johannesand Wiebe 1970). The slurry produced from the tissue-stripping process was homogenated in a blender for 30s andthe volume of homogenate was recorded. The number ofzooxanthellae in 10 ml aliquotes of homogenate wasmeasured in triplicate by light microscope (X 400) usingCount Rafter Cell. The total number of zooxanthellae percoral was measured after correcting the volume ofhomogenate. Zooxanthellae density was calculated as anumber per unit surface area.

Zooxanthellae number / cm2 = counted cells / cellsurface area x cell depth x dilution

For chlorophyll analysis, 10-20 ml sub samples of thehomogenate were filtered through Whatman GF/C 0.45filters, which were then homogenized for 30s using a tissuehomogenizer. Chlorophyll was extracted twice with 10 mlof acetone 90% for 24 h in darkness at-4 C°. Extracts werecentrifuged at 6000 g rev/min for 20 minutes to remove filterfiber from suspension and the supernatants read on aspectrophotometer at 630, 645, 665 and 750 wave lengths.Chlorophyll concentrations were calculated according tothe equation of Jeffery and Humphrey (1975), as follow:

Ch.a (mg m3) = 11.85D663-665-1.54 D647-0.08D630 ) v I-1 V-1

D = absorbance at wave length incubated by subscript,after correction by the cell to cell bank and subtraction ofthe cell-to-cell blank corrected absorbance at 750 nm

V = volume of acetone (ml)I = cell (cuvette) length (cm)V = volume of filtered water (L)

Spectrophotometric readingWhen possible, the absorption spectra in the range 630-

750 nm were collected. Our spectrophotometer SPECORDM40 was connected with pc computer. Some of the earlier

data were registered on the paper. The length of cuvetteswas chosen according to chlorophyll range (usually L2cm). Surface area of the bare skeletons remaining afterremoval of tissue was measured independently using theparaffin wax technique (Stambler et al. 1991), by immersingthe skeleton bar in hot wax; the mass of wax added to theskeleton bare was determined by weighing the skeletonbare before and after immersion. A relationship betweenchange in mass and surface area was obtained byimmersing a known surface area cubes in the wax.


Experimental effect of elevated temperature on thepopulation density of zooxanthellae of A. humilis

The changes observed in zooxanthellae densities lostfrom A. humilis indicate clearly increasing susceptibility toboth elevated temperature and prolonged exposure. Thecontrol samples maintained at 26°C exhibited no variationin symbiont density at both the beginning and the end of theexperiment. In samples exposed to 24°C, zooxanthellaedensities were highly significantly different from those atthe control sample (P < 0.01, HSD = 0.324), wherezooxanthellae densities were 0.78±0.015x106 cells/cm2

after 6 hours, 0.77±0.014x106 cells/cm2 after 12 hours and0.64±0.015x106 cells/cm2 after 24 hours. A. humilis at 29°Chad lower counts of symbiotic algae (0.71±0.023 x106

cells/cm2 after 6 hours, 0.6±0.015x106 cells/cm2 after 12hours and 0.55±0.015x106 cells/cm2 after 24 hours) withinhost tissue compared to control sample. Moreover,zooxanthellae densities of A. humilis were significantlydifferent from those at the control sample (P < 0.01, HSD =0.18). Increases in temperature to 31°C caused a decreasein zooxanthellae densities and vary greatly over time.Measurements of zooxanthellae densities at this stagerevealed a highly significant difference between exposedand control sample (ANOVA, P < 0.01, HSD = 0.296).Where, zooxanthellae densities were 0.62±0.015 x106

cells/cm2, 0.5±0.015x106 cells/cm2, 0.43±0.015 x106

cells/cm2 after 6 hours, 12 hours and 24 hours respectively.At 33°C, the number of zooxanthellae lost from A. humiliswas decreased to 38% (content after loss= 0.5±0.015x106

cells/cm2) after 6 hours, 48% (content after loss=0.42±0.015x106 cells/cm2) after 12 hours and to 51%(content after loss= 0.4±0.015x106 cells/cm2) after 24 hourscompared to control sample. This indicates that, thephysiological state of zooxanthellae is clearly influencedby elevated temperatures and the duration of heat exposure.In samples exposed to 35°C, the density of zooxanthellaewas (0.41±0.015x106 cells/cm2). This represents a 50%decrease compared to controls after 6 hours. Samplesexposed to 35°C and analyzed after 12 hours, showed azooxanthellae density of (0.3±0.015x106 cells/cm2) (63%decrease) compared to (0.81±0.0125x106 cells/cm2) forcontrols. While after 24 hours the loss of zooxanthellae wasabout 75% (content after loss= 0.2±0.015x106 cells/cm2)compared to the control sample (ANOVA, P < 0.01, HSD=0.503).

5 (2): 75-85, November 201378

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof zooxanthellae at the different time of exposure (Table 1).It revealed that, the mean value of zooxanthellae densitiesafter 6 hours of exposure was significantly different withthose after 12 hours . However, the difference was highlysignificant between zooxanthellae densities after 12 hoursof exposure and 24 hours. This was driven from the datathat zooxanthellae densities after 6 hours exposure(0.638x106 cells/cm2) were higher than those after 12 hours(0.616 106 cell/cm2), which in turn were higher than thoseafter 24 hours (0.503x106 cells/cm2).

Table 1. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in temperature (°C) onzooxthancellae density (10 6 cells/cm2) of A. humilis by using thesampling frequency as dependent variables.

After 6 h.(0.638)

After 12 h.(0.766)

After 24 h.(0.503)

After 6 h.(0.638)After 12 h.(0.766)

0.128 (Sig)

After 24 h.(0.503)

0.135 (Sig) 0.263 (H. Sig)

Note: Number in parentheses = Zooxthancellae density (10 6

cells/cm2). Minimum significant difference 0.0102. H. = Highlysignificant differences. Sig. = Significant difference

Experimental effect of elevated temperature onchlorophyll contents of A. humilis

The amount of chlorophyll per zooxanthellae wasinversely related to increased temperature. All chlorophyllconcentrations showed a significant decrease with increasedtemperature (P < 0.01 for all cases). Control samples (26°C)had higher content of chlorophyll (2.1±0.11, 2.1±0.07 and1.99±0.04 µg/cm2 after 6, 12 and 24 hours, respectively)within host tissue compared to test samples. In samplesexposed to 24°C, the content of chlorophyll was (2±0.09,1.97±0.04 and 1.91±0.06 µg/cm2 after 6, 12 and 24 hoursrespectively). Although the difference was statisticallysignificant (ANOVA, P < 0.01, HSD = 0.108). Coloniesimmediately sampled after 6 hours exposure to 29°C,showed a slight decrease in chlorophyll contents(1.98±0.0106 µg/cm2). While, chlorophyll contents incolonies collected after 12 hours were 1.9±0.04 µg/cm2,and after 24 hours were 1.87±0.106 µg/cm2. Coloniesexposed to 29°C showed a significant difference relative tothe control samples (ANOVA, P < 0.01, HSD = 0.15).

Measurements of chlorophyll contents at 31°C ofexposure revealed a significant difference between exposedand control sample (ANOVA, P < 0.01, HSD = 0.42).However, chlorophyll contents were 1.82±0.0104 µg/cm2,1.6±0.08 µg/cm2, 1.5±0.02 µg/cm2 after 6 hours, 12 hoursand 24 hours respectively. An increase in temperature to33°C caused an increase in the amount of chlorophyll lostfrom A. humilis. Chlorophyll content decreased from2.1±0.11 µg/cm2 in controls to 1.43±0.039 µg/cm2 inexposed samples after 6 hours (P < 0.01, HSD = 0.73). Ananalyzed A. humilis, after 12 hours of exposure to 33°C,showed a chlorophyll content of 1.37±0.03 µg/cm2

compared to 2.1±0.07 µg/cm2 in controls. While after 24hours it was 1.21±0.08 µg/cm2. Exposure of A. humilis to35°C reduced the content of chlorophyll sharply comparedto the control sample. However, the difference wasstatistically highly significant (P < 0.0001, HSD = 1.166).About 48% of chlorophyll content was lost from A. humilisafter 6 hours when incubated at 35°C (1.1±0.13 µg/cm2),after 12 hours about 57% of chlorophyll content (0.9±0.03µg/cm2) was lost. While after 24 hours the loss ofchlorophyll content was about 65% (0.7±0.019 µg/cm2)relative to the control sample.

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof chlorophyll contents at different times of exposure(Table 2). It was revealed that, the mean value ofchlorophyll contents after 6 hours of exposure wassignificantly different with those after 12 hours and 24hours Also the difference was significant betweenchlorophyll contents after 12 hours of exposure and 24hours where the contents of chlorophyll after 6 hoursexposure (1.74 µg/cm2) were higher than those after 12hours (1.65 µg/cm2), which in turn were higher than thoseafter 24 hours (1.53 µg/cm2).

In general, the physiological state of Symbiodinium isclearly influenced by elevated temperatures and duration ofheat exposure.

Table 2. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in temperature (°C) onchlorophyll concentration (µg/cm2) of A. humilis by using thesampling frequency as dependent variables.

After 6 h.(1.74)

After 12 h.(1.65)

After 24 h.(1.53)

After 6 h.(1.74)After 12 h.(1.65)

0.087 (Sig)

After 24 h.(1.53)

0.206 ( H. Sig) 0.11 (Sig)

Note: Number in parentheses = Chlorophyll concentration(µg/cm2). Minimum significant difference 0.043 .H. = Highlysignificant differences. Sig. = Significant difference

Experimental effect of elevated temperature on thepopulation density of zooxanthellae of S. pistillata

The number of zooxanthellae showed a significantdecrease with increasing temperature (ANOVA, P < 0.01)and exposure time (P < 0.01), additionally there wasinteraction between temperatures and exposure time (P <0.01). To detect the different effect of changes intemperatures on zooxanthellae densities after differentexposure times, Turkey’s Studentized Rang StatisticalAnalysis (HSD) was applied. It was shown that the meanvalue of zooxanthellae densities exposed to 24°C wassignificantly different from those in controls (HSD =0.083). However, the control samples (26°C) had higherdensities of zooxanthellae (0.83±0.015 after 6 and 12 hoursand 0.82±0.018x106 cells/cm2 after 24 hours) within hosttissue compared to test samples. In samples exposed to24°C, the densities of zooxanthellae were (0.81±0.012,


0.73±0.015 and 0.69±0.018x106 cells/cm2 after 6, 12 and24 hours, respectively).

Zooxanthellae densities of S. pistillata at 29°C werealso significantly different from those of the control sample(P < 0.01, HSD = 0.118). Stylophora pistillata had lowercounts of symbiotic algae cells (0.76±0.015x106 cells/cm2

after 6 hours, 0.75±0.015 x106 cells/cm2 after 12 hours and0.62±0.018x106 cells/cm2 after 24 hours) within host tissuecompared to control sample. While, the difference betweenzooxanthellae densities at 31°C and those in controlsamples were highly significant (P < 0.01, HSD = 0.273),zooxanthellae densities at this stage were 0.66±0.015x106

cells/cm2, 0.53±0.015x106 cells/cm2, 0.47±0.015x106

cells/cm2 after 6 hours, 12 hours and 24 hours respectively.Measurements of zooxanthellae densities at 33°C

revealed a highly significant difference between theexposed and the control sample (ANOVA, P < 0.01, HSD= 0.373). Increases in temperature to 33°C caused adecrease in zooxanthellae densities and vary greatly overtime. Zooxanthellae densities at this stage (33°C) were0.51±0.015x106 cells/cm2, 0.44±0.015x106 cells/cm2,0.41±0.015x106 cells/cm2 after 6 hours, 12 hours and 24hours respectively. In samples exposed to 35°C, the densityof zooxanthellae was (0.43±0.015x106 cells/cm2). Thisrepresents a 48% decrease compared to the control sampleafter 6 hours. Exposure to 35°C for 12 hours, showed azooxanthellae density of (0.35±0.015x106 cells/cm2)compared to (0.83±0.015x106 cells/cm2) in controlsrecording a decrease of 58%. While after 24 hours the lossof zooxanthellae was about 67% (content after loss=0.27±0.015x106 cells/cm2) relative to the control sample(ANOVA, P < 0.01, HSD= 0.476).

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof zooxanthellae at the time of exposure (Table 3). It wasdetected that, the mean value of zooxanthellae densitiesafter 6 hours of exposure was significantly different fromthose after 12 hours. However, the difference was highlysignificant between zooxanthellae densities after 12 hoursand 24 hours of exposure. This revealed that zooxanthellaedensities after 6 hour exposure (0.67x106 cells/cm2) werehigher than those after 12 hours (0.605x106 cells/cm2)while zooxanthellae densities after 12 hours were higherthan those after 24 hours (0.546x106 cells/cm2).

Table 3. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in temperature (°C) onzooxthancellae density (10 6 cells/cm2) of S. pistillata by usingthe sampling frequency as dependent variables.

After 6 h.(0.666)

After 12 h.(0.605)

After 24 h.(0.546)

After 6 h.(0.666)After 12 h.(0.605)

0.061 (Sig)

After 24 h.(0.546)

0.12 (Sig) 0.342 (H. Sig)

Note: Number in parentheses = Zooxthancellae density (10 6cells/cm2). Minimum significant difference 0.01; H. = Highlysignificant differences . Sig. = Significant difference.

Experimental effect of elevated temperature onchlorophyll contents of S. pistillata

The amount of chlorophyll concentration showed asignificant decrease with increasing temperature (ANOVA,P < 0.01) and prolonged exposure (P < 0.01). Additionally,there was interaction between the treatments. Control samples(26°C) had chlorophyll contents of (2.2±0.11, 2.1±0.088and 2±0.015 µg/cm2 after 6, 12 and 24 hours, respectively).The contents of chlorophyll in samples exposed to 24°Cwas (1.98±0.082, 1.94±0.051 and 1.9±0.018 µg/cm2 after 6,12 and 24 hours, respectively). Also, the differencebetween theses samples was statistically significant(ANOVA, P < 0.01, HSD = 0.163). Six hours exposure to29°C showed a slight decrease in chlorophyll contents(1.84±0.056 µg/cm2). While, chlorophyll content incolonies exposed for 12 hours was 1.83±0.046 µg/cm2,while it was 1.8±0.015 µg/cm2 after 24 hours. Coloniesexposed to 29°C showed a significant difference from thecontrol samples (ANOVA, P < 0.01, HSD = 0.28).

Measurements of chlorophyll content at 31°C ofexposure revealed also a significant difference betweenexposed and control sample (ANOVA, P < 0.0001 andHSD = 0.43). Also, chlorophyll contents were 1.7±0.016µg/cm2, 1.67±0.045 µg/cm2, 1.62±0.018 µg/cm2 after 6hours, 12 hours and 24 hours respectively. An increase intemperature to 33°C caused an increase in chlorophyllcontent loss from S. pistillata. Chlorophyll contentdecreased from 2.2±0.11 µg/cm2 in controls to 1.5±0.028 insamples exposed for 6 hours (P < 0.01, HSD = 0.73).

An analyzed S. pistillata after 12 hours of exposure to33°C showed a chlorophyll content of 1.1±0.035 µg/cm2

compared to 2.1±0.088 µg/cm2 in controls. While after 24hours it was 0.9±0.015 µg/cm2 compared to 2±0.015 for thecontrols. Exposure of S. pistillata to 35°C sharply reducedthe content of chlorophyll relative to the control sample. Inaddition, the difference was statistically highly significantbetween both samples (P < 0.0001, HSD = 1.36). About63% of chlorophyll content was lost from S. pistillata after6 hours when incubated at 35°C (content after loss=0.81±0.023 µg/cm2), while it was 65% after 12 hours(content after loss= 0.74±0.007 µg/cm2). While after 24hours, the loss of chlorophyll content was about 66%(content after loss= 0.68±0.015 µg/cm2) relative to thecontrol sample.

Table 4. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in temperature (°C) onchlorophyll concentration (µg/cm2) of S. pistillata by using thesampling frequency as dependent variables.

After 6 h.(1.67)

After 12 h.(1.56)

After 24 h.(1.48)

After 6 h.(1.67)After 12 h.(1.56)

0.11 (Sig)

After 24 h.(1.48)

0.19 (Sig) 0.08 (Sig)

Note: Number in parentheses = Chlorophyll concentration(µg/cm2). Minimum significant difference 0.0304. Sig. = Significantdifference

5 (2): 75-85, November 201380

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof chlorophyll contents at different times of exposure(Table 4). It was revealed that, the mean value ofchlorophyll contents after 6 hours of exposure wassignificantly different from those after 12 hours (ANOVA,P < 0.0001, HSD = 0.11) and 24 hours. Also the differencewas significant between chlorophyll contents after 12 hoursof exposure and 24 hours, this revealed the recorded datathat the contents of chlorophyll after 6 hours of exposure(1.67 µg/cm2) were higher than those after 12 hours (1.56µg/cm2), and chlorophyll contents after 12 hours werehigher than those after 24 hours (1.48 µg/cm2).

Experimental effect of increased sediment concentrationson the population density of zooxanthellae density of A.humilis

The changes observed in zooxanthellae densities lostfrom A. humilis indicated an increasing susceptibility toboth increased sediment concentrations and prolongedexposure. The control samples exhibited slight variations inalgal density at the beginning and the end of theexperiment. In samples exposed to 0.1 mg/cm2/L,zooxanthellae densities did not show any decrease after 1day (0.8±0.025x106 cells/cm2 in controls and exposedsamples). However, after 5 days of exposure to 0.1mg/cm2/L, zooxanthellae densities decreased from0.79±0.015x106 cells/cm2 (controls) to 0.76±0.007 x106

cells/cm2, while after 10 days zooxanthellae densities were0.73±0.014x106 cells/cm2 compared to 0.77±0.01 x106

cells/cm2 for the controls. The mean of zooxanthellaedensities in samples exposed to 0.1 mg2/cm/L weresignificantly different from those of the controls (P < 0.01,HSD = 0.023). Acropora humilis at 0.5 mg/cm2/L hadlower counts of symbiotic alga cells (0.75±0.015x106

cells/cm2 after 1 day, 0.7±0.015x106 cells/cm2 after 5 daysand 0.65±0.01x106 cells/cm2 after 24 hours) within hosttissue compared to the control sample. Moreover,zooxanthellae densities were significantly different fromthose of the control sample (P < 0.01, HSD = 0.086).

Increases in sediment concentration to 1 mg2/cm/Lcaused a decrease in zooxanthellae densities and varygreatly over time. Measurements of zooxanthellae densitiesat this stage revealed a highly significant differencebetween exposed and control sample (ANOVA, P < 0.0001and HSD = 0.173). Where, zooxanthellae densities were0.72±0.015 x106 cells/cm2, 0.68±0.015x106 cells/cm2,0.44±0.01 x106 cells/cm2 after 1 day, 5 days and 10 days,respectively. At 1g/cm2/L, the number of zooxanthellae lostfrom A. humilis was decreased to 11% (content after loss=0.71±0.012x106 cells/cm2) compared to control sampleafter 1 day, 24% (content after loss= 0.6±0.015 x106

cells/cm2) after 5 days and to 60% (content after loss=0.31±0.012x106 cells/cm2) after 10 days, indicating that thephysiological state of zooxanthellae is clearly influencedby elevated sedimentation rate and the duration ofexposure. In addition, the differences in zooxanthellaedensities between samples exposed to 1 g/cm2/L andcontrols were highly significant (ANOVA, P < 0.01, HSD=0.246).

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof zooxanthellae densities after different times of exposure(Table 5). It was revealed that, the mean value ofzooxanthellae densities after 1 day of exposure wassignificantly different from those after 5 days. However,zooxanthellae densities after 10 days of exposure werehighly significantly different from the sample of 1 day ofexposure and 5 days. This revealed the result thatzooxanthellae densities after 1 day exposure (0.755x106

cells/cm2) were higher than those after 5 days (0.706x106

cells/cm2), also those after 5 days were higher than thoseafter 10 days (0.58x106 cells/cm2).

Table 5. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in sedimentation onzooxthancellae density (106 cells/cm2) of A. humilis by using thesampling frequency as dependent variables.

After 1 day(0.755)

After 5 days(0.706)

After 10 days(0.58)

After 1 day(0.755)After 5 days(0.706)

0.049 (Sig)

After 10 days(0.58)

0.175 ( H. Sig) 0.126 (H. Sig)


cells/cm2). Minimum significant difference 0.01. H. = highlysignificant differences. Sig. = Significant difference

Experimental effect of increased sedimentconcentrations on chlorophyll contents of A. humilis

Chlorophyll contents within the host tissue showed asignificant decrease with increased sedimentation rate (P <0.01 for all cases). Control samples had higher content ofchlorophyll (1.99±0.15, 1.73±0.015 and 1.31±0.018 µg/cm2

after 1, 5 and 10 days, respectively) compared to the testsamples. In samples exposed to 0.1 mg/cm2/L the contentof chlorophyll was (1.99±0.02, 1.71±0.015 and 1.27±0.01µg/cm2 after 1, 5 and 10 days, respectively). In addition, thedifference between 1, 5 and 10 days samples wasstatistically significant (ANOVA, P < 0.01, HSD = 0.02).Colonies immediately sampled after 1 days exposure to 0.5mg/cm2/L showed a slight decrease in chlorophyll contents(1.91±0.01 µg/cm2). While chlorophyll contents were1.66±0.015 µg/cm2 in colonies collected after 5 days and1.1±0.05 µg/cm2 after 10 days. Colonies exposed to 0.5mg/cm2/L showed a significant difference in chlorophyllcontents compared to the control samples (ANOVA, P <0.001, HSD = 0.121).

Chlorophyll contents of samples exposed to 1 mg/cm2/Lwas highly significantly different from the control sample(ANOVA, P < 0.01, HSD = 0.242). Chlorophyll contentswere 1.84±0.015 µg/cm2, 1.58±0.015 µg/cm2 and0.89±0.01 µg/cm2 after 1 day, 5 days and 10 daysrespectively. Exposure of A. humilis to 1 g/cm2/L sharplyreduced the content of chlorophyll in relative to the controlsample, giving a high significance of difference betweenboth treated and control sample (P < 0.01, HSD = 0.374).About 10% of chlorophyll content was lost from A. humilisafter 1 day when exposed to 1 g/cm2/L (content after loss =


1.79±0.02 µg/cm2), after 5 days for about 18% ofchlorophyll content (content after loss = 1.42±0.012µg/cm2) was lost. While after 10 days the loss ofchlorophyll content was about 47% (content after loss =0.7±0.019 µg/cm2) relative to the control sample.

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof chlorophyll contents at the different times of exposure(Table 6). It was revealed that, the mean value ofchlorophyll contents after 1 day of exposure to differentconcentrations of sediments was significantly differentfrom those after 5 days. However, the difference washighly significant between chlorophyll contents of 10 dayssamples and controls. In addition, the difference was highlysignificant between chlorophyll contents of 10 days and 5days of exposure. Contents of chlorophyll after 1 day ofexposure (1.904 µg/cm2) were higher than those after 5days (1.62 µg/cm2), which in turn were higher than thoseafter 10 days (1.056 µg/cm2).

Table 6. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in sedimentation rates(mg/cm2/L) on chlorophyll concentration (µg/cm2) of A. humilisby using the sampling frequency as dependent variables.

After 1 day(1.904)

After 5 days(1.62)

After 10 days(1.056)


0.284 (Sig)


0.848 ( H. Sig) 0.564 (H. Sig)

Note: Number in parentheses = Chlorophyll concentration(µg/cm2). Minimum significant difference 0.015. H. = highlysignificant differences. Sig. = Significant difference

Experimental effect of increased sediment concentrationson the population densities of zooxanthellae of S. pistillata

The number of zooxanthellae showed a significantdecrease with increasing sediment concentrations(ANOVA, P < 0.01) and exposure time (P < 0.01).Additionally, there was an interaction between temperatureand exposure time (P < 0.01). To detect the distinctdifferent effects of changes in sediment concentrations onzooxanthellae densities after different exposure times,Turkey’s Studentized Rang Statistical Analysis (HSD) wasapplied. It was shown that the mean value of zooxanthellaedensities exposed to 0.1 mg/cm2/L was significantlydifferent from those in the controls (HSD = 0.02).However, control samples had higher densities ofzooxanthellae (0.81±0.015, 0.8±0.015 and 0.78±0.015x106

cells/cm2 after 1, 5 and 10 days respectively) compared totest samples. While, in samples exposed to 0.1 mg/cm2/L,the densities of zooxanthellae were (0.8±0.015 after 1 day,0.78±0.007 after 5 days and 0.75±0.01x106 cells/cm2 after10 days, respectively). Zooxanthellae densities of S.pistillata exposed to 0.5 mg/cm2/L had also a significantdifference from those of the control sample (P < 0.01, HSD= 0.073). Recorded data revealed that, S. pistillata hadlower counts of symbiotic algae cells (0.77±0.015x106

cells/cm2, 0.72±0.015x106 cells/cm2 and 0.68±0.01x106

cells/cm2 after 1, 5 and 10 days respectively) within hosttissue compared to control sample. The difference washighly significant between zooxanthellae densities at 1mg/cm2/L and those in control samples (P < 0.01, HSD =0.156). At this stage zooxanthellae densities were0.74±0.015x106 cells/cm2, 0.69±0.018x106 cells/cm2, and0.49±0.01x106 cells/cm2 after 1 day, 5 days and 10 daysrespectively. An increases in sediment loading to 1 g/cm2/Lcaused a decrease in zooxanthellae densities which varygreatly over time.

Measurements of zooxanthellae densities after exposureto 1 g/cm2/L revealed a high significant difference betweenexposed and control samples (ANOVA, P < 0.01, HSD =0.226). Zooxanthellae densities at this stage were0.71±0.014x106 cells/cm2 after 1 day, 0.63±0.015 x106

cells/cm2 after 5 days and 0.37±0.015x106 cells/cm2 after 510 days. This represents a 13.4% decrease in zooxanthellaedensity after 1 day and 31% after 5 days compared tocontrols. While after 24 hours the loss of zooxanthellae wasabout 53.6% relative to the control sample.

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof zooxanthellae at the time of exposure (Table 7). It wasrevealed that, the mean value of zooxanthellae densitiesafter 5 days of exposure was significantly different fromthose after 1 day. However, the difference was highlysignificant between zooxanthellae densities after 10 days ofexposure and those after 1 and 5 days. However,zooxanthellae densities after 1 day of exposure were 0.766x106 cells/cm2, being higher than those after 5 days (0.724x106 cells/cm2). However, which in turn were higher thanthose after 10 days (0.614x106 cells/cm2).

Table 7. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in sedimentation rates(mg/cm2/L) on zooxthancellae density (10 6 cells/cm2) of S.pistillata by using the sampling frequency as dependent variables.

After 1 day(0.766)

After 5 days(0.724)



0.042 (Sig)


0.152 ( H. Sig) 0.11 (H. Sig)


cells/cm2). Minimum significant difference 0.0096. H. = highlysignificant differences. Sig. = Significant difference.

Experimental effect of increased sedimentconcentrations on chlorophyll contents of S. pistillata

The amount of chlorophyll showed a significantdecrease with increasing sediment concentration (ANOVA,P < 0.01) and prolonged exposure (P < 0.01). Additionally,there was interaction between the treatments. Controlsamples had chlorophyll contents 2±0.015, 1.84±0.018 and1.34±0.015 µg/cm2 after 1, 5 and 10 days respectively. Thecontents of chlorophyll in samples exposed to 0.1mg/cm2/L was (1.98±0.015 µg/cm2 after 1 day, 1.74±0.013µg/cm2 after 5 days and 1.32±0.013 µg/cm2 after 10 days).The difference between exposed and controls was

5 (2): 75-85, November 201382

statistically significant (ANOVA, P < 0.01, HSD = 0.044).One day exposure to 0.5 mg/cm2/L showed a slightdecrease in chlorophyll contents (1.93±0.01 µg/cm2).However, chlorophyll contents in colonies analyzed after 5and 10 days were 1.69±0.013 µg/cm2 and 1.21±0.013µg/cm2 respectively. Colonies exposed to 0.5 mg/cm2/Lshowed a significant difference relative to the controlsamples (ANOVA, P < 0.0001, HSD = 0.114). While,measurements of chlorophyll content in samples exposed to1 mg/cm2/L expressed a high significant differencebetween exposed and control sample (ANOVA, P < 0.01,HSD = 0.242). Where is chlorophyll contents were1.87±0.013 µg/cm2, 1.61±0.013 µg/cm2, 0.97±0.013µg/cm2 after 1 day, 5 days and 10 days respectively.

An increase in sediment concentration to 1 g/cm2/Lcaused a sharp loss in chlorophyll content from S.pistillata. Chlorophyll content decreased from 2±0.15µg/cm2 in controls to 1.83±0.016 µg/cm2 in exposedsamples after 1 day (8.5% decrease). An analyzed A.humilis after 5 days of exposure to 1 g/cm2/L, showed achlorophyll content of 1.47±0.007 µg/cm2 compared to1.84±0.088 µg/cm2 in controls (20% decreased). After 10days it was 0.81±0.016 µg/cm2 compared to 1.34±0.013µg/cm2 in controls (40% decrease). The difference between1 g/cm2/L exposure and controls was statistically highlysignificant (P < 0.01, HSD = 0.354).

Turkey’s Studentized Rang Statistical Analysis (HSD)was applied to detect the distinct variance between meansof chlorophyll contents at different times of exposure. Itwas revealed that, the mean value of chlorophyll contentsafter1 day of exposure was significantly different fromthose after 5 days but it was highly significantly differentfrom those after 10 days In addition, the difference betweenchlorophyll contents after 5 and 10 days exposure washighly significant. However, the measured chlorophyllcontent after 1 day of exposure (1.92 µg/cm2) was higherthan those after 5 days (1.66 µg/cm2) which in turn washigher than those after 10 days (1.129 µg/cm2).

Table 8. Turkey’s studentized rang statistical analysis (HSD) forthe experimental effect of changes in sedimentation rates(mg/cm2/L) on chlorophyll concentration (µg/cm2) of S. pistillataby using the sampling frequency as dependent variables.

After 1 day(1.921)

After 5 days(1.669)



0.252 (Sig)


0.792 ( H. Sig) 0.54 (H. Sig)

Note: Number in parentheses = Chlorophyll concentration(µg/cm2). Minimum significant difference 0.01. H. = highlysignificant differences. Sig. = Significant difference.

Experimental effect of changes in temperature onbleaching

The changes observed in zooxanthellae lost from thetwo corals A. humilis and S. pistillata clearly indicateincreasing susceptibility to both elevated temperature andprolonged exposure. At 26°C (the control sample), the loss

of zooxanthellae by each of these corals was very low. Cellviability of these corals was similar at 26 and 29°C, butdepicted a sharp decline of zooxanthellae lost from thesecorals at 31°C through time. This result confirms the resultof Strychar et al. (2004) that zooxanthellae lost fromAcropora hyacinthus, Favites complanata, and Poritessolida at 32°C was greater than that at 28°C. However,Berkelmans and Willis (1999) found a temperature increaseof 2-4°C is to have caused coral bleaching within dayswhile a temperature increase of 1-2°C caused bleachingwithin weeks. Moreover, Nesa and Hidaka (2009) detecteda negative correlation between survival time and thezooxanthella density of tissue balls at 31°C in both Fungiasp. and Porites divaricata. This relationship was clearlyobserved in the Caribbean basin during the 1980s and1990s, when annual coral bleaching increasedlogarithmically with sea surface temperature anomalies(McWilliams et al. 2005). A 0.1°C rise in regional seasurface temperature resulted in a 35% increase in the numberof areas that reported bleaching, and mass bleaching eventsoccurred at regional sea surface temperature anomalies of0.2°C and above (Baker et al. 2008).

As the temperature increased to 35°C in the presentexperiment, the loss of zooxanthellae from each hostincreased both with time and temperature elevation. Thisresult agrees with the finding of Riegl (2002) that bleachingmortalities were reported in Abu Dhabi at 1996 whentemperatures remained above 35°C for 3 weeks. Elevatedtemperature was found to significantly reduce the amountof zooxanthellae in primary polyps (Anlauf et al. 2010).

The result reported in the present experiment indicatedthat, A. humilis had a higher decrease in its zooxanthellaedensities than S. pistillata at the same treatment; where50% of zooxanthellae in the host tissue of A. humilis werelost after 6 hours while S. pistillata lost 48% ofzooxanthellae after equal time of exposure. Differences inthe response of these species of coral to thermal stress mayresult from difference in hospite irradiances driven by thecombination of skeletal architecture and light scatteringproperties (Enríquez et al. 2005). An additional source ofvariation in the response to thermal stress in these coralsmay originate from differences in tissue thickness that isassociated with difference in the initial protein content(Warner et al. 2002). Fitt et al. (2009) found physiologicaland biochemical differences of both symbiont and hostorigin in the response to high-temperature stress of Poritescylindrica and S. pistillata. Ferrier-Pagès et al. (2010)studied the changes in feeding rates of three scleractiniancoral species between normal and short-term stressconditions, and assessed the effect of feeding on thephotosynthetic capacity of corals exposed to a thermalstress. He found that, S. pistillata significantly decreased itsfeeding rates at 31°C, while rates of Turbinaria reniformisand Galaxea fascicularis were increased between 26 and31°C. Exposure to elevated temperatures reduces thephotosynthetic rate of zooxanthellae and predisposes theirphotosynthetic apparatus to further damage (Jones et al.1998; Bhagooli and Hidaka 2004).

In the present experiment, the bleaching temperaturethreshold was 33°C or less for the two species S. pistillata


and A. humilis where 51% of their zooxanthellae were lostafter 24 h of exposure. This result confirms that of Leletkin(2002) that water temperature of 32°C and above inevitablycaused coral bleaching. However, the thermal bleachingthreshold for primary polyps might be below that reported(30-31°C) from most adult coral species in the easternPacific (D'Croz et al. 2001; Hueerkamp et al. 2001).Bleaching temperature thresholds vary locally, andconditions that result in coral mortality in some regions canhave no effect on corals in others. For example, while30.5°C and 30.8°C represent bleaching thresholds for atleast some regions of the Caribbean and Great Barrier Reef,respectively (Berkelmans et al. 2004; Manzello et al.2007), temperatures as high as 35.5°C do not affect coralsin the Arabian Gulf or in the Samoan Manu’a Islands(Craig et al. 2001; Riegl 2002; Birkeland et al. 2008).Indeed, individual corals have been reported surviving inAbu Dhabi at temperatures up to 40°C (Kinsman 1964),although mortality did occur in this region in 1996 whentemperatures remained above 35°C for 3 weeks (Riegl 2002).

Experimental effect of changes in sedimentation rate onbleaching

In samples exposed to 0.1 mg/cm2/L, zooxanthellaedensities of A. humilis and S. pistillata did not show anydecrease after 1 day. However, after 1 days of exposure to0.5 mg/cm2/L, zooxanthellae densities were significantlydifferent from those of the controls. This result agrees withthe laboratory finding of Peters and Pilson (1985) whoexamined the effect of heavy sedimentation rate oncorals using both symbiotic and asymbiotic colonies ofAstrangia danae at a rate of 200 mg. cm.−2 day-1 for 4weeks. He found no difference from controls while slightadverse effects relative to controls were noted afterincreasing the sand applications to three times per day. Theeffects of varying rates of sedimentation (0.5 to 325 mgcm-2d-1) on settlement rates of Acropora millepora larvaewere examined experimentally, in aquaria. Highersedimentation rates reduced the number of larvae settlingon upper surfaces, but total numbers of settled larvae werenot significantly affected by sedimentary regime (Babcockand Davies 1990). Increases in sediment concentration to 1mg/cm2/L caused a decrease in zooxanthellae densities andvary greatly over time. Measurements of zooxanthellaedensities of A. humilis and S. pistillata at this stagerevealed a highly significant difference between exposedand control sample. However, Sofonia and Anthony (2008)found no effect of sediment loads greater than 110 mg cm−2

on any of the physiological variables of Turbinariamesenterina, that tolerant to sediment loads an order ofmagnitude higher than most severe sediment conditions insitu. Likely mechanisms for such tolerance are that: (1)colonies covered in sediment in low-flow were able to clearthemselves rapidly (within 4-5 h) and (2) sedimentprovides a source of food. These results suggest thatintensified sediment regimes on coastal reefs mayshift coral communities towards dominance by a few well-adapted species (Weber et al. 2006; Palmer et al. 2010).

Cruz-Pinion et al. (2003) found that, high sedimentationrates, low light availability and anthropogenic influence

lead to cellular damage and deteriorated coral skeletaldensity. At 1 g/cm2/L, the number of zooxanthellae lostfrom A. humilis was higher than that was lost from S.pistillata at same time. This result agrees with the findingof Fabricius et al. (2007). Who reported a contrast betweenspecies susceptibility at 39.6 mg cm-2 day-1 sedimentationrate in Ngardmau, Palau, Micronesia, that small polypcorals such as Porites rus suffered greatest mortality whiledamage in Galaxea fascicularis was less severe assediments were shifted and removed by the large polyps.However, Acropora spp. Appeared partially bleachedalthough little time sediment remained on branches. Withreference to the present experiment, we can suggest that thenormal sedimentation rate for A. humilis and S. pistillata tobe in an order of 1 mg/cm2/L or less. Chronic rates andconcentrations above these values are high. This result isconflicted with the finding of Rogers (1990), whoconcluded that the mean sediment concentration was < 10mg/L at reefs subject to stresses from human activities.Sedimentation is regarded as an increasing threat to coralreefs (reviewed by Fabricius 2005). The impacts associatedwith sedimentation and sediment burial include reducedphotosynthesis and increased respiration (Philipp andFabricius 2003; Weber et al. 2006), tissue mortality,reduced growth (Rice and Hunter 1992; Lirman and Manzello2009), and reduced fertilization, larval survivorship, andrecruitment (Babcock and Smith 2000). Turbidity reduceslight levels, photosynthetic potential and possibly coralgrowth rates (Yentsch et al. 2002; Anthony and Hoegh-Guldberg 2003); however, elevated net sedimentation ratesincreases abrasion and smothering (Rogers 1990; Fabricius2005). On the other hand, Palmer et al. (2010) found that,near shore environments directly influenced by fluvialsediments and dominated by terrigenoclastic sedimentationare generally considered marginal for coral reef growth.The same author did not mention if there is some way ofwashing or cleaning of these sediments. The energetic costsof sediment clearing can be considerable (Riegl and Branch1995), and the inability to clear sediments will exposecorals to further stress because anoxic conditions due tosediments can cause tissue bleaching and subsequentmortality (Weber et al. 2006).

Increased temperature and sedimentation rate are bothknown to cause physiological stress in corals (Weber et al.2006; Lirman and Manzello 2009; Obura 2009). Corals arenot homoiothermic (Hoegh-Guldberg and Smith 1989) andshort-term temperature and sediment stress can causechanges in basal metabolism, such as respiration andzooxanthellae photosynthesis (Nyström et al. 2001; Philippand Fabricius 2003; Weber et al. 2006).

Some key questions on the present work and their answersQ: How does the fact that “the temperature treatments

were just 6, 12 or 24 hours long” relate to the aims and howdoes this design advance our understanding of temperaturechange as a result of climate change effects? A: Theseexperimental choices should not be construed to mean thatthe response of reef corals to global climate change can befully understood without addressing other facets of globalclimate change and through a comprehensive analysis of

5 (2): 75-85, November 201384

the coral holobiont. This study addresses mainly the earlyeffects of heat stress on symbiotic dinoflagellates(zooxanthellae) within the tissues of a common reef-coral.

Q: Why were the experiments conducted under constanttemperature conditions and how does this design advanceour understanding of temperature change as a result ofclimate change effects? A: The experiments wereconducted under constant temperature conditions as theywere conducted in an aquarium, and to maintain a stablereef tank ecosystem, a constant temperature is required.Also, as the reef tank is a closed system and most types ofcoral can thrive a few degrees above or below the ideallevel in such a closed system, the study examined the earlyeffects of heat stress on symbiotic dinoflagellates(zooxanthellae) within the tissues of a common reef-coral.

Q: How are results from a constant temperatureexperiment reconciled with the normal cyclic conditionsexperienced by the corals in their natural habitat? A: Thetemperatures in the present experiment were increased anddecreased following the diurnal variation in water temperaturein the field by approximately 6 ˚C. Temperature in the fieldwas between 29 ˚C and 30 ˚C at midday (at 3 m depth).Therefore, corals for studying the effect of temperaturewere incubated at each temperature test (24, 29, 31, 33 and35 ˚C). Control samples were placed at room temperature(26˚C). Since coral bleaching occurs when the thermaltolerance of corals and their photosynthetic symbionts(zooxanthellae) is exceeded, corals in the presentexperiment, beside being subjected to the normal cyclictemperature, they were subjected to some few dgrees belowand above the normal cyclic temperature.

Q: How quickly was ambient temperature changedduring experimentation and how does this affectinterpretation of the outcome? A: Bleaching can be inducedby short-term exposure (i.e. 1 days) at temperatureelevations of 3°C to 4°C above normal summer ambient orby long-term exposure (i.e. several weeks) at elevations of1°C to 2°C. Temperature elevations above summer ambient,but still below the bleaching threshold (as in the presentwork), could impair growth and reproduction by the effectof increased zooxanthellae expulsion. In 1998 Red Seacorals were perilously close to their bleaching thresholdduring the summer months, and localized bleachings didoccur. In some cases, local warming of surface water onshallow reef flats exceeded this threshold temperature andcaused localized coral bleaching.

Q: What is briefly the design of the sedimentationexperiments and how do they allow the conclusions that arepostulated. A: The present experiment examined the effectof different sedimentation rates on corals. For detection ofthe short-term sediment, branches for studying the effect ofsediments were put in glass aquaria, exposed to 0.1, 0.5, 1mg/cm2/L and 1g/cm2/L different concentrations ofsediments. The present experiment postulated that, thenormal, safe sedimentation rate for A. humilis and S.pistillata to be in an order of 1mg/cm2/L or less.

Q: Exactly how do the outcomes of this work advanceour understanding? A: Threshold temperatures as well asnormal safe sedimentation rate determined in the presentwork can be applied directly to reef aquaria where they are

closed systems exactly like those used in our experiments.These experimental choices should not be construed tomean that the response of reef corals to global climatechange or to natural or human-made sedimentation in thesea can be fully understood without a comprehensiveanalysis of the coral holobiont and the surroundingenvironmental conditions.

CONCLUSIONS

The result reported in the present experiment indicatedthat, Acropora humilis had a higher decrease in itszooxanthellae densities than Stylophora pistillata at thesame treatment after equal time of exposure to temperature.In the present experiment, the bleaching temperaturethreshold was 33°C or less for the two species S. pistillataand A. humilis where 51% of their zooxanthellae were lostafter 24 h of exposure. On exposure to sedimentation,number of zooxanthellae lost from A. humilis was higherthan those lost from S. pistillata at same time. The normalsedimentation rate for A. humilis and S. pistillata wasfound to to be in an order of 1 mg/cm2/L or less.

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Review: Physical, physical chemistries, chemical and sensorialcharacteristics of the several fruits and vegetables chips produced by

low-temperature of vacuum frying machine

AHMAD DWI SETYAWAN♥, SUGIYARTO, SOLICHATUN, ARI SUSILOWATIDepartment of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java,

Indonesia. Tel./Fax.: +92-271-663375. ♥email: [email protected]

Manuscript received: 12 November 2012. Revision accepted: 4 May 2013.

Abstract. Setyawan AD, Sugiyarto, Solichatun, Susilowati A. 2013. Review: Physical, physical chemistries, chemical and sensorialcharacteristics of the several fruits and vegetables chips by produced low-temperature of vacuum frying machine. Nusantara Bioscience5: 86-103. Frying process is one of the oldest cooking methods and most widely practiced in the world. Frying process is considered as adry cooking method because it does not involve water. In the frying process, oil conduction occured at high temperature presses waterout of food in the form of bubbles. Fried foods last longer due to reduced water contents leading to less decomposition by microbes,even fried foods can enhance nutritional value and beautify appearance. Food frying technology can extend the shelf life of fruits andvegetables, while the frying oil are used to enhance the flavor of the products, but the use of improper frying oil can have harmfuleffects on human health. Vacuum frying is a promising technology and may become an option for the production of snacks such as fruitand vegetable crisps that present the desired quality and respond to the new health trends. This technique of frying food at a lowtemperature and pressure makes the nutritional quality of the food is maintained and the quality of the used oil is not quickly declined tobecome saturated oils that are harmful for human health. This technique produces chips that have physical, physico-chemical, chemical,and sensorial properties generally better than chips produced by conventional deep-fat frying methods.

Key words: chips, food, frying, preservation, vacuum frying

Abstrak. Setyawan AD, Sugiyarto, Solichatun, Susilowati A. 2013. Review: Karakteristik fisik, kimia fisik, kimia dan sensoris beberapakeripik buah-buahan dan sayuran yang dihasilkan dengan mesin vacuum frying bersuhu rendah. Nusantara Bioscience 5: 86-103.Proses penggorengan merupakan salah satu metode memasak yang paling tua dan paling banyak dilakukan di dunia. Prosespenggorengan dianggap sebagai metode memasak kering karena proses ini tidak memerlukan air. Dalam proses penggorengan, terjadikonduksi minyak bersuhu tinggi yang mendesak air keluar dari bahan makanan dalam bentuk gelembung-gelembung. Makanan yangdigoreng tahan lebih lama karena berkurangnya kadar air yang menyebabkan tidak terjadinya pembusukan oleh mikroba, bahkanmakanan yang digoreng dapat ditingkatkan nilai gizi dan kualitas penampakannya. Teknologi penggorengan makanan dapatmemperpanjang umur simpan buah-buahan dan sayuran, sementara itu minyak goreng yang digunakan meningkatkan cita rasa produk,namun penggunaan minyak goreng yang tidak tepat dapat merugikan kesehatan. Penggorengan hampa udara (vacuum frying) adalahteknologi penggorengan yang menjanjikan dan dapat menjadi pilihan untuk produksi makanan ringan seperti keripik buah dan sayurandengan kualitas yang diinginkan dan memenuhi kecenderungan kesehatan saat ini. Teknik ini menggoreng makanan pada suhu dantekanan rendah sehingga kualitas gizi makanan terjaga dan minyak yang digunakan tidak cepat rusak dan menjadi minyak jenuh yangberbahaya bagi kesehatan manusia. Teknik ini menghasilkan keripik yang memiliki sifat-sifat fisik, fisika-kimia, kimia, dan sensorisyang umumnya lebih baik daripada keripik yang dihasilkan dengan metode penggorengan konvensional.

Kata kunci: keripik, makanan, menggoreng, pengawetan, penggorengan hampa udara, vacuum frying

INTRODUCTION

All foods require preservation for several reasons, suchas to prevent spoilage, to maintain the availabilitythroughout the year, to retain the nutritional value and tomake value-added products (higher prices). Food spoilageor damage may occur during handling process due to theinfluence of physical, physiological, chemical or microbialdamage. Chemical and microbial factors are the maincauses of food spoilage. Several chemical and enzymaticreactions can occur during processing and storage of food(Mujumdar and Jangam 2012). Food preservation is usually

done by preventing the growth of bacteria, fungi (e.g.yeast), and other microbes (although in some method,benign bacteria or fungi has been used to make certainfoods, such as tempeh, oncom and tape), as well asretarding the oxidation of fats which cause rancidity.Preservation of food can also include inhibition of visualdeterioration during food preparation, such as theenzymatic browning reaction in salaks, apples and potatoesafter peeling. Maintaining or creating nutritional value,texture and flavor are important aspects of foodpreservation, although, historically, some methodsdrastically change the character of the food which is

SETYAWAN et al. – Chips characteristic by vacuum frying process 87

preserved. In many cases, these changes have come to beseen as a desirable quality, including cheese, yoghurt andpickled onions. To preserve food, some methods aresometimes used together. Preserving fruit by turning intojam, for example, involves boiling (to reduce the watercontent of fruit and to kill microbes), the provision of sugar(to prevent their re-growth) and sealing in an airtight jar (toprevent recontamination) (Vivante 2009). There are variousways to preserve food, including canning, freezing,pickling, salting, sugaring (providing sugar syrup),irradiation, vacuum packaging, etc. The pre and/or post-processing steps are critical to reduce the drying load aswell as to make better quality product. The commonlymethods used for pre-treatment are osmotic dehydration,blanching, salting and soaking. While post-processing suchas coating, blending, packaging, etc. are also importantafter drying of food (Mujumdar and Jangam 2012).

Water content is a major cause of food spoilage,therefore the drying process is often done to reduce levelsof water and extend the shelf life of food (Potter 1973).Drying or dehydration process by thermal is one of themost ancient food preservation and the most frequentlyused, which reduces water activity sufficiently to preventbacterial growth; although some loss of quality occursduring dehydration. Drying has been applied to grains,seafood, and meat products as well as fruits, tubers andvegetables. Food products can have wide ranges of watercontent; as low as 35% in grains and as high as 90% ormore in some fruits (e.g. 93% in water melon) which needsto be reduced to an acceptable value so as to avoidmicrobial growth. There is reported that microbes havedifferent water activity (which means free water availablefor microbial growth in solids) (Mujumdar and Devahastin2008). In addition, each food product must be dried usingvarious types of suitable dryers and also using appropriatepre- and post-processing to obtain a satisfactory valueaddition to the dried product (Chen and Mujumdar 2008;Mujumdar and Devahastin 2008).

Traditionally, food products were commonly dried byopen sun drying method. Recently various advanced dryingmethods have been practised for food application as aresult of the increased demand for high quality productsand to reduce energy consumption which is one of thehighest costs in the food processing industries (Kudra andMujumdar 2009). The use of some techniques, such assolar cabinet dryers, tray dryers, fluid bed dryers, vacuumdryers, freeze dryers, etc has resulted in a better productquality (Potter 1973; Chen and Mujumdar 2008; Jangam etal. 2010). These processes can also be made efficient costsin terms of energy consumption (Mujumdar and Jangam2012).

Dehydration is one of the main processes in foodpreservation. Frying is the most widely practiced cookingmethod and the most cost-effective techniques for foodpreservation as well as for the production of traditional andinnovative products such as processed snacks with desiredquality (Mujumdar and Devahastin 2008). Food fryingtechnology can extend the shelf life of fruits and vegetablesand frying oil can increase the flavors of the products,however, improper frying oil can have harmful effects on the

consumer health (Inprasit 2011). Fruit into chips processingrequires technological support so that the qualities of theresulting chips are acceptable for consumers. One way toproduce healthy food without changing its original form isby using the vacuum frying technology (Siregar et al.2004).

This article begins by reviewing some of theconventional drying techniques used in food preservation.Later, it will focus on recent advances in vacuum fryingtechnique for food production; as well as physical, physicalchemistries, chemical and sensory characteristics of somechips of fruits and vegetables (incl. tubers) processed bylow-temperature vacuum frying machine.

FOOD FRYING PRESERVATION

Fresh fruits and vegetables are highly perishable, shelflife is so short. If not handled properly, fruits andvegetables that have been harvested will undergophysiological, physical, chemical, and microbiologicalchanges that become damaged or rotten. Meanwhile, thetubers usually have a longer shelf life, although some willsoon rot in storage, such as cassava. One effort to maintainquality and shelf life of fruit and have a pretty good marketis processed into chips. Chips are more durable than thestored fresh fruits or vegetables (incl. tubers) because of thelow water contents and no longer occurring physiologicalprocesses such as fresh crops (Antarlina and Rina 2005;Shidqiana 2012).

Deep-fat (ordinary, atmospheric) frying is one of theoldest food preparation processes and is widely used in thefood industry. Frying is a complex operation process whichis basically the immersion of food pieces in hot vegetableoil, at a temperature of above the boiling point of water(Amany et al. 2009). This condition causes high rates ofheat transfer, so that water evaporates and an oil layercovering the product surface (Hubbard and Farkas 1999;Bouchon et al. 2003). Several models have been developedto describe the moisture evaporation and oil absorption indeep-fat frying (Moreira and Bakker-Arkema 1989; Riceand Gamble 1989; Kozempel et al. 1991). Oil temperatureand frying time are the main frying operation variablescontrolling mass transfer in deep-fat frying (Mittelman etal. 1984). The deep-fat frying seals the food by immersingin hot oil so that all the flavors and juices are retained by acrisp crust (Moreira et al. 1995; Troncoso et al. 2009).Fried is generally processed under atmospheric pressure athigh temperatures. During the process, food is rapidlycooked, browned, and the texture and flavor is developed(Farkas et al. 1996a). Therefore, deep-fat frying is oftenselected as a method for creating unique flavors, colors,and textures in processed foods. Due to the higher heattreatment, surface darkening and many other adversereactions may occur before the food is fully cooked(Blumenthal and Stier 1991).

Frying temperature can range from 130-190°C, but themost common temperatures are 170-190°C. The hightemperature of the frying fat typically leads to theappreciated surface color and mechanical characteristics of

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fried foods; and besides that, heating the reducing sugaraffects a complex group of reactions, called caramelization,which leading to browning development that defines thecolor of the final product (Arabhosseini et al. 2009). Somefat and oil decomposition products have also been involvedin producing adverse health effects when frying oilsdegraded with continued use (Taylor et al. 1983; Hagemanet al. 1989). In addition, heat toxic compound acrylamidecan be formed during this process (Gokmen and Palazoğlu2008; Pedreschi et al. 2004).

Frying is considered a dry cooking method because itdoes not require water. In the frying process, the food isimmersed in a container of oil at a temperature of above theboiling point of water. High temperature and high heatconduction of oil, causing the cooked food preserved andeven enhanced nutritional value and reduced degradation.The high temperature causes partial evaporation of water,which is moving away from foods and through thesurrounding oil; and a certain amount of oil absorbed bythe food replaces water lost (Inprasit 2011). Frying is oftenchosen as a method to create a unique flavor and texture inprocessed foods that can improve their overall tasty andpalatability (Moreira et al. 1999). High heat transfer rateslead to the development of desirable sensory properties infried foods (Farkas et al. 1996). During the frying process,physicochemical, physical, chemical and sensory propertiesof food will be modified. Texture, color and oil content arethe main quality parameters of fried foods (Aguilera 1997;Moreira et al. 1999).

Texture is important for prominent sensory attributes offood preferences (Thygesen et al. 2001), and is a criticalparameter for the quality of fried chips (Ross and Scanlon2004). The texture of fried chips is known to be directlyassociated with a specific gravity, total solids, starchcontent, cell size, surface area and pectin (Moyano et al.2007). Textural changes during frying are the result ofmany physical, chemical and structural changes resulting ina complex process operation unit, which includes heat andmass transfer together with chemical reactions. Goodquality chips should have a crispy crust about 1-2 mm,whereas most of the oil is located and awed, a soft center,like a cooked potato. For potato chips, a very crunchytexture is expected all the way through since crispness is anindicator of freshness and high quality (Troncoso andPedresch 2007). It is a well known fact that texture of thisproduct depends on the quality of raw potato andtechnological parameters used in the production process(Kita 2002). Crisp texture is associated with the dry matterof raw potato tubers (Thygesen et al. 2001). Crispsobtained from potatoes which is rich in dry matter (above25%) can exhibit hard texture, whereas crisps of too low aspecific gravity (low in dry matter), containing too muchoil, are characterized by greasy and sticky texture. The drymatter of potato tubers is composed of various substances,i.e.: starch (15%), sugar, nitrogen compounds, lipids,organic acids, phenolic compound, mineral substances andnon-starch polysaccharides (Amany et al. 2009).

Color development begins when a sufficient amount ofdrying has occurred in the chips and depends also on thedrying rate and heat transfer coefficients during the various

stages of frying. Color is visually regarded as one of themost important parameters in determining the quality offried chips (Scanlon et al. 1994) and is the result of theMaillard reaction that depends on the content of reducingsugars and amino acids or proteins on the surface, as wellas the temperature and frying time (Marquez and Anon1986). The reduction in weight and size of dehydratedproduct and the increase in shelf stability can reduceproduct storage and distribution costs (Toledo et al. 1991).Da Silva and Moreira (2008) shows that the vacuum friedsnacks (blue potato, green bean, mango and sweet potatochips) retain more of their natural colors and flavors due tothe less oxidation and lower frying temperature.

Oil absorption is one of the most important qualityparameters of fried foods, but this is incompatible withconsumer trend recently towards healthier foods and low-fat products (Bouchon and Pyle 2004). However, the oilconsumption derived from salty snack products is very high(Kuchler et al. 2004). Consumption of oils and saturatedfats are specifically related to significant health problems,including coronary heart disease, cancer, diabetes, andhypertension (Saguy and Dana 2003). Other undesirableeffects due to high temperatures in frying process andexposure to oxygen are the degradation of essential nutrientcompounds and the formation of toxic molecules in thefoodstuff or the frying oil itself (Fillion and Henry 1998).This information has raised a red flag on the humanconsumption of fried foods and has a significant impact onthe snack food industry (Dueik et al. 2010). As a result,healthy low-fat snack products have acquired a new levelof importance in the snack food industry (Moreira et al.1999). However, even the health-conscious consumers arenot willing to sacrifice organoleptic properties, an intensefull-flavor snacks continue to play an important role in thesalty snacks market (Mariscal and Bouchon 2008).

Traditional deep-fat frying and vacuum frying are twocommon types of applied frying processes (Garayo andMoreira 2002). Vacuum frying is an alternative techniqueto improve the quality of dehydrated food (Song et al.2007). It operates at relatively lower temperatures (e.g.130oC), thus the texture, color, flavor, and nutritional valueis more preserved and naturally. Apart from high qualityretention in the final product obtained by vacuum frying,the main difference of these two techniques is theinvestment cost and operational cost. For vacuum frying,specially designed machinery and equipment are required.Both frying techniques have different benefits anddisadvantages, therefore, it should carefully consider therequired properties of raw materials and desiredcharacteristics of the final product to avoid wastefulinvestment (Inprasit 2011).

DISADVANTAGES OF FRIED FOODS

Snack foods and especially fried chips, are popularforms of refreshment among consumers, because frying inoil helps to create a great flavor and texture. Frying is oneof the oldest and most popular cooking methods in theworld. Deep-fat frying is a method to produce dried food


where an edible fat heated above the boiling water servesas a heat transfer medium, fats also migrate into food,providing nutrients and flavor (Fan et al. 2005; Tarmizi andNiranjan 2013). Unfortunately, deep-frying foods havesome shortage. This process causes the foods contain a lotof oils and saturated fats that are related to coronary heartdisease, cancer, diabetes, and hypertension, as well as leadsto formation of acrylamide on foods containing amino acidand reducing sugars (starch) that is carcinogenic andneurotoxic for human health.

Oil contentOil absorption is one of the most important quality

parameters of fried food, which is incompatible with recentconsumer trends towards healthier food and low-fatproducts (Bouchon and Pyle 2004). Repeated use of oil athigh temperature causes quality degradation throughchemical reactions, such as oxidation, hydrolysis andpolymerization. The resulting decomposition products affectthe flavor and color of the frying oil and fried products.These reactions impair the oil quality by increasing theamount of free fatty acids and polar compounds that affectconsumers’ health by causing a higher risk of developingcancer, hypertension and coronary heart disease. Inaddition, food labors are also in danger, as they maybreathe the oil vapor that can cause lung cancer (Hein et al.1998; Goburdhun et al. 2000).

Consumer’s preference for low-fat products havebecome the driving force of the food industry to producelower oil content fried potatoes that still retain the desiredtexture and flavor. In order to obtain a low-fat product, it iscritical to understand when, how and where the oilabsorption occurs, so that the oil migration into thestructure can be minimized. Several studies have revealedthat most of the oil is confined to the surface area of friedproduct and is restricted to a depth of a few cells (Keller etal. 1986; Lamberg et al. 1990). Potato chips have becomepopular salty snacks for 150 years and retail sales in manycountries are around 6 billion/year, representing 33% oftotal sales in the market fried foods (Garayo and Moreira2002). However, potato chips have an oil content rangingfrom 35 to 45g/100g (wet basis), which is a major factoraffecting consumer acceptance for oil-fried products today(Dueik and Bouchon 2011).

Rimac-Brncis et al. (2004) reported that the osmoticdehydration pretreatment can be an effective operation toproduce low-fat chips. Pre-drying of potatoes is a commonway to reduce fat uptake in the final fried product (Moreiraet al. 1999, Krokida et al. 2001, Moyano et al. 2002).Drying step following the blanching step reduces the absorbedoil on potato chips (Pedreschi and Mayano 2005). Theapplication of a proper coating is promising to reduce theoil content (Mellema 2003). The major oil fraction issuctioned by the microstructure of the potato piece whenthis is removed from the fryer during the cooling period,indicating that the tight oil absorption associated with the lossof moisture (Erdogdu and Dejemek 2010).

Oil absorption is mainly a surface phenomena and mostof the oil is absorbed by the fried product in the post-fryingperiod (Doran 2007; Dueik et al. 2011). Oil absorption will

result from the competition between drainage and suctioninto the porous crust once the food is removed from the oil(Bouchon et al. 2003). However, oil absorption duringvacuum frying follows transport mechanisms are morecomplexly than those elucidated in conventional frying andis currently the subject of intensive study (Amany et al.2009).

Oil absorption is a surface phenomenon that happens asthe product is removed from the fryer due to temperaturedifference between the product and ambient temperature.Changes in temperature cause an increase in capillarypressure in the product pores, which causes the oil to flowinto the opened pore spaces. The de-oiling processbecomes more important during vacuum frying because ofthe pressurization process. Chip has increased the oilcontent following vacuum frying and depressurization dueto rapid change in pressure (vacuum to atmospheric)(Moreira et al. 1997). De-oiling is one of the mostimportant unit operation steps in vacuum deep-fat frying toensure best quality products (Da Silva et al. 2009). Vacuumfrying at 120˚C under a pressure of 5.37kpa produce potatochips with acceptable quality and improved the quality offrying oil. De-oiling mechanisms are generally centrifuges,installed in a special vacuum dome attached to the vacuumfrying (Amany et al. 2012a,b,c,d). However, it is sometimesmanufactured separately.

Vacuum fried products of apple and potato chipssignificantly absorb lower oil than atmospheric fried(56.7% and 18% less oil in vacuum fried potatoes andapples, respectively). There are large differences in thedrainage capacity of the two products. Apples significantlydrained more oil from their surface than potato chips; andhad smoother surfaces with a higher drainage. Surfaceroughness and drainage capacity are inversely related (r2=0.949). For potato products, vacuum fried chips have arougher surface than the atmospheric fried ones (29%rougher), which may explain the lower drainage of the firstones, along with the higher oil viscosity at a lowertemperatures. The higher roughness of vacuum friedpotatoes can be a result of pressure changes that suffer thetissue during depressurization and pressurization steps.Certainly, the surface roughness is a key factor determiningits capacity to drain oil during the post frying period, butthere might be other factors such as crust micro structuralparameters that affect the final oil content of fried products(Dueik et al. 2011). Moreno et al. (2010) determined thatproducts with higher surface roughness absorbed more oil.However, this relationship is restricted to products ofsimilar nature (either gluten or potato-flake based productscategories) and cannot be extended when comparingdifferent product categories.

Vacuum frying is a promising technology that ispotential to produce low-fat chips (Dueik et al. 2010; Dueikand Bouchon 2011; Mariscal and Bouchon 2008). It is anefficient method to reduce the oil content in fried chips,maintain nutritional quality of products, and reduce damage tothe oil deterioration. It is a technology that can be used toproduce fruits and vegetables chips with the necessarydegree of dehydration without excessive darkening orscorching of the product. Vacuum frying is a deep-frying

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process, which is carried out in a closed system, underpressures well below atmospheric levels (preferably lowerthan 7-8 KPa), making possible to substantially reduce theboiling-point of water, and therefore, the fryingtemperature. The low temperatures employed and minimalexposure to oxygen account for most of its benefits, whichinclude nutrient preservation (Da Silva and Moreira 2008),oil quality protection (Shyu et al. 1998) and a reduction inthe generation of toxic compounds (Granda et al. 2004).Compared with other dehydration methods for fruits andvegetables, vacuum frying is a viable option to obtain highquality dried foods in a much shorter process. Vacuumfried products can absorb between 25 and 55% less oil thanatmospheric fried products (Garayo and Moreira 2002;Dueik and Bouchon, 2010). Vacuum frying (driving from60°C) can reduce the oil content of carrot crisps by nearly50% (d.b.) compared with atmospheric fried crisps producedusing the same driving force (Passos and Ribeiro 2009; DaSilva and Moreira 2008; Dueik et al. (2009).

Acrylamide formationDeep-fat frying is one of the oldest and most common

unit operations used in the preparation of foods. However,consumer fears have started to arise as acrylamide, apossible carcinogen, has been detected in foods exposed tohigh temperatures, including fried and baked foods (Tarekeet al. 2002). Acrylamide is classified as possiblycarcinogenic and neurotoxic to humans. It has been foundin starch-rich foods cooked at high temperatures (Grandaand Moreira 2005). Acrylamide was accidentallydiscovered in foods in April 2002 by scientists in Swedenwhen they found the chemical in starchy foods, such aspotato chips, french fries, and bread that had been heated.Boiled foods and raw or unheated foods did not exhibit anyformation of acrylamide (Mottram et al. 2002; Stadler et al.2002; Tareke et al. 2002).

Although the researchers are still unsure of the precisemechanisms by which acrylamide formed in foods, manybelieve it is a by-product of the Maillard reaction.Development of acrylamide as a by-product of the Maillardbrowning is currently the most accepted theory (Stadler etal. 2002; Yaylayan et al. 2003). In fried foods, acrylamidecan be produced by the reaction between asparagine andreducing sugars (fructose, glucose, etc.) or reactivecarbonyls at temperatures above 120°C (Mottram et al.(2002).

Acrylamide formation in fried foods found to dependon the composition of raw materials as well as frying timeand temperature. In potato chips, acrylamide is rapidlyformed at more than 160°C, with the amount proportionalto the heating duration and temperature. Free amino acidsare used to reduce acrylamide, with lysine, glycine, andcysteine having the greatest effects in aqueous system.Lysine and glycine are effective at inhibiting the formationof acrylamide in wheat-flour snacks. In potato chips, theaddition of 0.5% glycine to pallets reduced acrylamide bymore than 70%. Soaking potato slices in a 3% solution ofeither lysine or glycine reduces acrylamide formation morethan 80% in potato chips fried for 1.5 minutes at 185°C.These results indicate that the addition of certain amino

acids by soaking the raw products in appropriate solutionsis an effective way to reduce acrylamide in processed foods(Kim et al. 2005). Granda and Moreira (2005) shows thatduring traditional frying potato, higher temperatures areused (150 to 180°C) and acrylamide produced after sometime but started to degrade, resulting in a constant rate onthe acrylamide content at longer times. In addition, duringvacuum frying (10 Torr), acrylamide increasesexponentially (but at lower levels) for all frying times.

Decreasing in pH is a way to reduce the Maillardreaction when it is undesirable (Schwartzberg and Hartel1992). Jung et al. (2003) proposed a theory of acrylamidereduction by lowering the pH of the raw product prior tocooking. Stadler et al. (2002) observed that when pyrolyzedat 180°C in the presence of glucose, asparagine formedsignificant amounts of acrylamide (368 ppm). Appropriatereactants enhanced interaction when water is added to thereaction mixture, and there was an increase of the product(acrylamide) in reaction (960 ± 210 ppm).

Schwartzberg and Hartel (1992) suggested that one wayto inhibit the Maillard reaction in cases where it isundesirable is the maintenance of lowest possibletemperatures. Tareke et al. (2002) showed that acrylamideformation depends on temperature; it increases as theincreasing of temperature. Mottram et al. (2002) indicatedthat acrylamide formation increases with temperature fromabout 120-170oC and then decreases. Surdyk et al. (2004)found that not only the temperature (above 200oC) but alsoheating time increased acrylamide content in yeast-leavened wheat bread crust. When the bread is baked at270oC for 18 and 32 minutes, the acrylamide contentincreased from about 300 ppb to 1200 ppb, respectively.

VACUUM FRYING TECHNOLOGY

Due to the increasing health concern and the trendstoward healthier food snacks, vacuum fried foods havebecome a common product that can be found in the localmarkets. Various kinds of fried chips are now offered onthe local market shelves, such as bananas, jackfruits,pineapples, salaks, mangoes, cassavas, potatoes, sweetpotatoes, etc. Nonetheless, the vacuum frying process,similar to the atmospheric frying process, is quitecomplicated, involving coupled heat and mass transferthrough a porous media, crust formation, product shrinkageand expansion, and so forth. These mechanisms allcontribute to the difficulties in predicting the physical andstructural appearance of the final product. Therefore, abetter understanding of the frying mechanism and the heatand mass transport phenomena would be useful for foodprocessors to produce and develop new fried and vacuumfried snack foods for growing allegiance of healthyconsumers (Yamsaengsung et al. 2008).

Vacuum frying is a promising technology that could bean option for the production of novel snacks such as fruitand vegetable crisps that present the desired qualityattributes and respond to new health trends (Dueik et al.2010). Fruits and vegetables are important sources ofvitamins and antioxidants. However, average consumption


of fruits and vegetables in modern societies is low due toearly decay and rather high price (Da Silva and Moreira2008). Fruits and vegetables are high in sugar content andheat sensitive, thus they are usually burned in temperatureof usual frying process and lose their natural colors andflavors, unless the frying process takes place at lowtemperature (Shyu and Hwang 2001). One of the modernmethods for fruits and vegetables processing in the world isthe vacuum frying that can be performed at lowtemperatures and minimal exposure to oxygen (Maadyradet al. 2011). This allows us to create products with thedesired crispy texture and high nutritional value(Escaladapla et al. 2007).

In vacuum-frying, food is heated under a reducepressure that lowered the boiling points of frying oil andwater in food (Troncoso et al. 2009). Water can beremoved from the fried food rapidly once the oiltemperature reaches the boiling point of water. Colors andflavors can be better preserved in vacuum-fried food,because the food is heated at a lower temperature andoxygen content (Hidaka et al. 1991; Shyu et al. 1998). Theabsence of air during frying can inhibit oxidation includinglipid oxidation and enzymatic browning; therefore, thecolor and nutrients of food can be largely preserved (Xu1996; Gao and Liang 1999; Tarzi et al. 2011). Dehydratedfood produced by vacuum frying can have crunchy texture,good color and flavor and good retention of nutrients.Vacuum frying also has less adverse effects on the qualityof oil (Kato and Sato 1991).

During frying, the heat from the oil is confected to theproduct surface and then conducted to the product’s center,thus increasing its temperature. Water evaporates as theproduct reaches the boiling-point temperature. This processis generally regarded a Stephan type of heat transferproblem, which is characterized by the presence of amoving interface that divides two areas of physical andthermal properties (Farkas et al. 1996a). Farkas et al.(1996a, b) gives separate equations for the two regions: thecrust and the core, with a moving boundary. The study ofthese transport mechanism have led to the investigation onthe effect of vacuum frying on the transport processes. Forinstance, several studies have shown that less oil isabsorbed during the vacuum frying process (Garayo andMoreira 2002; Krupanyamat and Bhumiratana 1994;Choodum and Rojwatcharapibarn 2002; Yamsaengsungand Rungsee 2003). It has been suggested that the pressuredifference between the internal pressure of the product andthe vacuum pressure of the fryer help to reduce the amountof surface oil present at the end of the frying process,which in turn limits the total amount of oil absorbed.

Another important advantage of the vacuum frying isthe reduced temperature which helps to maintain thenatural color of the product while minimizing the loss ofvitamins and minerals. In atmospheric frying, the productsare generally fried at 160-190 oC, and the water inside theproduct evaporates at approximately 100oC depending onthe presence of dissolved components. On the other hand,under vacuum frying, the boiling point of water can bereduced to as low as 35-40oC, thus the frying temperaturecan be as low as 90-100oC. Shyu and Hwang (2001) found

that the optimum conditions for frying apple chips are at apressure of 3.115 kPa, a frying temperature of 100-110oC, afrying time of 20-25 minutes, and a concentration ofimmersing fructose solution of 30-40%. Garayo andMoreira (2002) found that potato chips fried under vacuumconditions (3.115 kPa and 144oC) have more shrinkagevolume and slightly softer, and lighter in color than potatochips fried under atmospheric conditions (165oC). Vacuumfrying can reduce levels of fat fries to 26.63% (which isnormally 35.3-44.5% by deep-fat frying). Yamsaengsungand Rungsee (2003) also found that compared toatmospheric frying, vacuum fried potato chips retained in amore intense flavor and color.

Fruits and vegetables are generally dehydrated byfreeze drying, a process that can maintain their originalflavor and color (Luh et al. 1975), but it is energy and timeconsuming (Flink et al. 1977). Many fruits and vegetableswith high nutritional value, such as cauliflower, carrots,mangoes, and pineapples, cannot be processed by ordinaryfrying methods. However, they can be processed by avacuum frying due to low temperature (Mariscal andBouchon 2008). This technology is expected to improvethe nutrition and health by producing products that tastegood, keep most of their nutrition values, have lower fatcontents than conventional fried chips, they also safer withlittle or no acrylamide formation, and they can be keptlonger (Kemp et al. 2009). Compared with otherdehydration technologies for fruits and vegetables, vacuumfrying is a viable option to obtain high quality driedproducts within a much shorter process (Laura and Claudio2009). It has been shown that vacuum fried chips (bluepotato, green bean, mango and sweet potato chips) retainmore of their natural colors and flavors because of lessoxidation and lower frying temperature (Da Silva andMoreira 2008). Vacuum fried potato chips and sliced guavahave lower oil content and more natural colorations thanthe conventional frying process (Yamsaengsung andRungsee 2003). Instead, Diamante et al. (2010) reportedthat hot air drying of green and gold kiwifruits at increasingtemperatures (60 to 100oC) leads to increased browningand ascorbic acid loss. Vacuum frying may be a goodalternative for the production of fruit and vegetabledehydrated slices (Shyu et al. 1998, 2005).

DESCRIPTION OF VACUUM FRYING

The main factors influencing the fried products arecombinations of time and temperature of the cookingprocess; the correct combination is required in producingfood product with acceptable physical attributes (such ascolor, appearance, texture and flavor) as well as preservingnutritional, but not stable, compounds such as vitamin C(Inprasit 2011). Several processes have been developed tomanufacture low-fat products that possess the desiredquality attributes of deep-fat fried food whilst preservingtheir nutritional and better sensory properties; such asextrusion, drying, and baking, which can be applied to rawfood or formulated products. Unfortunately, none of themhave been as successful as expected because they are still

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unable to impart the desired quality attributes of deep-fatfried food, such as amour, texture, appearance and mouthfeel (Dueik et al. 2010).

Vacuum frying is a promising technology that could bean option for the production of novel snacks such as fruitand vegetable crisps that present the desired qualityattributes and respond to new health trends. This process iscarried out in a closed system under pressures well belowatmospheric levels, which makes it possible to substantiallyreduce the boiling point of water and thus the fryingtemperature (Garayo and Moreira 2002). In fact, most ofthe benefits of this technology are the result of the lowtemperatures employed and minimal exposure to oxygen.Said the benefits include: (i) reduction of adverse effects onoil quality (Shyu et al. 1998), (ii) preservation of naturalcolor and amours (Shyu and Hwang 2001), (iii) reducedlevels of acrylamide (Granda et al. 2004), and (iv)preservation of nutritional compounds (Da Silva andMoreira 2008).

Vacuum frying is the technique of deep-fat frying foodsunder pressures well below atmospheric levels, preferablybelow 6.65 kPa, which serves to reduce oil content,discoloration and loss of vitamins and other nutrients thatare usually associated with oxidation and high temperatureprocessing (Garayo and Moreira 2002). One of its firstapplications was to reduce the formation of acrylamide inpotato crisps, as this tends to occur during high-temperature processing of high carbohydrate foods (Grandaet al. 2004). It has also shown some success in producingvacuum fried products with other foods, includingpineapple, apples, carrots, blue potato, sweet potato, beans,mangoes and jackfruit (Da Silva and Moreira 2008;Diamante 2009; Mariscal and Bouchon 2008; Perez-Tinocoet al. 2008; Fan et al. 2005). Figure 1 shows the schematicdiagram of vacuum frying system.

Figure 1. Schematic of the vacuum frying system (Garayo andMoreira 2002)

When the frying is carried out under atmosphericpressure, boiling point of water reduces; hence, higher

temperatures are not required to remove moisture from thefood. Deteriorating effect on the food due to heat will beless. The following is a brief explanation of governingtheories: (i) Water evaporation under vacuum. Boilingpoint of water is 100oC at atmospheric pressure.Evaporation of water at this temperature occurred togetherwith the loss of some food nutrients. Under vacuum wateris boiled and evaporated at lower temperature even at 0 oCso that nutrients loss is reduced especially for heat sensitivenutrients. (ii) Heat transfer. For hot air dryers, heat istransferred by convection using hot air as the medium. Airhas relatively lower heat transfer coefficient. Contrast tothe air, in a vacuum frying, vegetable oil has higher heattransfer coefficient. Therefore shorter time is required toreduce water content. (iii) Frying temperature. Automatictemperature control system, when used, provides amechanism by which moisture is reduced while foodtemperature is controlled by the system. Constanttemperature of the food results in uniformity of productquality (Inprasit 2011).

Stages using vacuum frying machine is as follows: Thematerial to be fried is prepared (peeled and sliced with athickness of 0.50-1 cm). If the water content is high, thespinner machine can be used to reduce the water content.Fryer tube is filled with frying oil. To 4 kg of fresh fruit, itis required 40 liters of frying oil. The raw materials are putinto the frying basket; the basket position is appointed (notsubmerged in oil). The frying machine and gas stove isturned on and the temperature is set. Furthermore, fryertube is closed to get the vacuum condition. After thepressure needle pointing at-680 mm Hg, the basket islowered down into the submerged position. Raw material isfried until drying. After completed frying, the position ofthe basket is moved up (not submerged in oil), and theelectric instalation and stove is turned off. Tap of the fryertube is opened until the pressure needle pointing at 0 mmHg. Than, the fryer tube is opened; and chips is removedand dried by using spinner machine. Chips is cooled andpackaged in plastic (PP 0.80 mm) or alumunium foil bagsand then sealed (Kamsiati 2010).

The oil uptake mechanism of vacuum frying is still notfully understood. During normal operation, the product isplaced inside the frying basket once the oil reaches thetarget temperature. The lid is then closed and the chamberis depressurized. Subsequently, the basket is immersed inthe oil bath, where it remains for the required amount oftime. It is then lifted out and the vessel is pressurized usinga pressure release valve. This results in a sudden increasein the surrounding pressure, which may force the vaporinside of the pores to condense, which means that oilabsorption may precede cooling. The low pressure mayallow air to diffuse faster into the porous structure,obstructing oil passage and leading to lower oil absorptionthan is observed in atmospheric frying. Vacuum friedpotato crisps absorb less than half the oil of crisps friedunder atmospheric conditions (Garayo and Moreira 2002).Mariscal and Bouchon (2008) states vacuum fried appleslices absorbed slightly less oil, and presented better resultsfor color preservation than atmospheric fried samples.


BENEFIT OF VACUUM FRYING

Vacuum frying may be an option for the production offruits and vegetables with low oil content and the desiredtexture and flavor characteristics. It is defined as the fryingprocess that is carried out under pressures belowatmospheric levels, preferably below 50 Torr (6.65 kPa).Due to the lowering pressure, boiling points both of oil andmoisture in the foods lowered (Garayo and Moreira 2002).Vacuum frying technology has the advantage of lowoxygen existed in the system resulted in the low rates offrying oil oxidation and lower boiling point of below100ºC. Vacuum fried products have better aroma and flavorsimilar to that of fresh fruit and tubers (vegetables). In factmost of the benefits of this technology are the results oflow temperatures used and minimal exposure to oxygen.The benefits include: (i) reduction of adverse effects on theoil quality (Shyu and Hwang 1998; Shyu et al. 1998), (ii)the preservation of natural colors and flavors (Shyu andHwang 2001), (iii) decreased of the acrylamide content(Granda et al. 2004), and (iv) the preservation of nutritionalcompounds (Da Silva and Moreira 2008).

Vacuum frying was tested as an alternative technique todevelop low oil content of potato chips. During vacuumfrying, oil temperature and vacuum pressure had asignificant effect on the drying rate and oil absorption rateof potato chips. Fried potato chips at low vacuum pressureand higher temperature had less volume shrinkage. Colorwas not significantly affected by oil temperature andvacuum pressure. Hardness values increased withincreasing oil temperature and decreasing vacuum levels.Potato chips fried under vacuum (3.115 kPa, 144ºC) hadmore volume shrinkage, slightly softer, and lighter in colorthan the potato chips fried under atmospheric conditions(165ºC). Vacuum frying is a process that could be afeasible alternative to produce potato chips with lower oilcontent and the desirable color and texture (Garayo andMoreira 2002).

Vacuum fried products have low water content (<6%)and low water activity (aw<0.3) (Tawong 2000; Piamkhla2004; Wongsuwan and Laosuksuwan 2006), so it has along shelf life. Under vacuum condition, fryingtemperature is constant and not higher than 100oC andfrying time is not longer than 2 hours. Obviously, vacuumfrying is an energy efficient process. The products arecrispy and retain its original color, taste and odor as of thenatural foods (Granda et al. 2004).

Vacuum frying can process heat-sensitive commoditiessuch as fruits being processed in the form of crisps (chips),such as jackfruit chips, apple chips, banana chips,pineapple chips, melon chips, and papaya chips, etc.Compared with conventional fryers, vacuum systemsproduce a much better product in terms of colorappearance, aroma, and taste like a fruit because it isrelatively original (Siregar et al. 2004).

In vacuum frying, vegetable oil is used as a heattransfer medium. Oil may be absorbed by the foods,therefore, there will be oil remaining in fried productsmaking them undesirable and may raise health concernamong consumers. There are many research works which

has been described techniques to reduce oil absorptionduring vacuum frying. Pre-coating with guar gum is one ofthe recommended techniques. Banana coated with guargum before frying under vacuum has lower oil content of8% compared to vacuum fried banana without guar gumpre-coating, which has 12% oil content. Oil absorption,however, varies with the chemical properties of rawmaterials. Different pre-treatment techniques and processeswill be applied to produce a good quality of vacuum friedproducts (Inprasit 2011).

In financial terms, the investment cost of vacuum fryingprocess is much higher than that of deep frying. This isbecause the vacuum frying technique is basically designedfor large-scale industry. There is a lack of vacuum fryerdesign for small scale production. Small scale producerssuch as farmers' group, small community enterprise, andcooperatives are difficult to buy vacuum frying machineryand equipment without financial support from thegovernment. High investment costs are substantialdisadvantage in applying vacuum frying in small-scaleproduction (Inprasit 2011). Although, it is the cost-investment, among several deep-fat frying technologies,vacuum frying has significant strategic importance for thefuture fried food manufacturing. This technology offerssignificant benefits such as improved product safety andquality cooking oil and oxidation is reduced due to lowtemperature processing (Granda et al. 2004).

RAW MATERIALS FOR VACUUM FRYING

Raw materials that are not eligible for freshconsumption and are not eligible to be sold as fresh fruitsand vegetables such as too big or too small, not smooth onthe surface or have defects, are selected for vacuum frying.Unqualified fruits and vegetables are cheap. They shouldbe washed, peeled, and trimmed to remove defects anduneatable parts before vacuum frying. Defects are notdetected in fried fruit products. Therefore vacuum fryingshould be applied to reduce waste because they can useunqualified raw material for processing. Ripe fruits, whichhave high sugar content, are popularly fried under vacuumbecause it is not possibly fried at atmospheric pressure. Inthe fruit and vegetable industry, much kind of fruits andvegetables are wasted from pruning. These parts are ingood quality but their sizes and shapes do not meet theprocessing standards. Vacuum frying help improve thecommercial value of the waste (Inprasit 2011). Figure 2shows the flow diagram for the processing of vacuum friedpineapple.

The selection of fruits suitable for vacuum frying isbased on the followings (Inprasit 2011):

Variety. Chemical and physical properties of fruits andvegetables vary depending on the variety. These propertiesaffect the quality of fried products. Thin flesh jackfruits canbe fried as a lump of pulp whereas thick flesh jackfruitsshould be chopped into small pieces before frying. Thethickness of raw materials affects the vacuum fryingprocess. Crispy and soft texture of vacuum fried jackfruitsis obtained together with uniformity of the water content

5 (2): 86-103, November 201394

when raw material thickness is efficiently controlled.Freezing is a recommended pre-treatment to obtain crispyvacuum fried products.

Figure 2. Flow diagram for processing of vacuum-fried pineapple(Inprasit 2011)

Ripeness. Vacuum frying is effectively used for fryinghigh sugar fruits that has sweet taste fried products. Fruitsubjected to vacuum frying should not have any astringenttaste. Fruits should be ripe but not be too ripe because thehigh sugar content induces high oil absorption duringfrying. Likewise, ripe fruits should not have too soft texturedue to breaking of fruits to small pieces occurs from rapidevaporation of water during vacuum frying. Vacuum fryingof too ripe durian produce small pieces of fried products.

Taste. Vacuum frying is a process of evaporating ofwater at low temperature to retain natural flavor andminimize nutrient losses. Original fruit flavors should beconsidered for the selection of raw material. Stronger tasteof fried products is observed when compared to the taste ofraw materials. This is due to very little excreting salivaduring eating fried products, therefore, a strong taste of thehigh concentration of flavor components is observed.Vacuum fried pineapple has very sour taste when madefrom sour pineapple. Also too sweet ripe fruits should notbe selected as raw materials for vacuum frying due to itshigh content of sugar cause caramelisation.

Water content. It is usually difficult to fry high moisturefruits. Plenty of water must be removed during frying.Fruits will be burned before they dry and shrinkage willoccur during water evaporation. Freezing of high moistureraw materials before vacuum frying such as longan andlychee is recommended to keep the water evaporation at alower temperature.

Seasonal fruits. Some fruit are seasonally grown andharvested. Domestic and export markets cannot alwaysabsorb the entire production. Excess fruit during the seasonreduce the price. This is an important issue in thedeveloping agricultural countries. Frying can be applied toextend the shelf life of seasonal fruits.

PROCEDURE FOR HANDLING

Vacuum fried product is much susceptible to spoilageduring storage especially in tropical and humid conditions.It can easily absorb moisture from storage environments.Therefore, manufacturers should separate package of friedproducts that have different water content because, in thesame packaging, moisture can move out from highmoisture product and be absorbed by low moisture product.Then product crispiness decreases and consumer will rejectthe products. Fried product quality changes when storing athigh temperature such as shop standing in open space or incars and containers parking in the sun. This also causes lossof product crispiness (Inprasit 2011).

Among the important properties of deep and vacuumfried products are nutrition values, consumer acceptabilityand safety of frying oil. These qualities can be controlledby proper selection of raw materials. One of properties thataffect consumer acceptance is crispiness of fried products.Crispiness of deep fried product changes rapidly afterfrying because it mainly consists of sugar and easy toabsorb moisture. Whereas crispiness of vacuum friedproduct does not change much because it consists of starch,which absorbs less moisture after frying. In addition, thetype of raw material thickness affects product crispiness.Thin slices required to obtain soft crispy and dried textureafter frying. Manufacturers have to change frying oil whenoxidized and unsafe for consumers. During the fryingprocess, quality of frying oil should continue to bemonitored. Frying oil should be changed frequently due tochanges in the physicochemical properties of the oil thatwill affect the quality of the product and oil uptake duringfrying. In addition, toxic compounds can be generated indeteriorated frying oil, which is harmful to human health(Inprasit 2011).

Evaluation of frying oils can perform the followingmethods: (i) Sensory evaluation: Used frying oils aregenerally considered as deteriorated when they clearlyindicate an objection smell or taste, for example strongmildew, strongly gritty, rancid, vanish, or bitter; andshowed intense smoke and foam formation during frying.These sensory impressions are objectified through furtheranalytical criteria such as polar compounds and polymertriglycerides. Intensify the dark, however, is not a measureof deterioration. This color change is caused by the reaction

Pineapple

Washing

Peeling and core removing

Trimming

Washing

Slicing Tidbiting

Freezing (if any)

Vacuum frying

Cooling

Nitrogen packing in gas barrier plastic bags

Storing in cold room


of proteins with fat or sugars components. (ii) Quick tests:Colorimetric procedures to determine the amount ofdegradation products of fatty acids (carbonyl compounds).Color reaction aims to determine portion of the polarcompounds or acid value. Redox reaction determines theamount of oxidized fatty acids. It also needs to measure theheight of the foam, viscosity and dielectric properties. (iii)Analytical methods: Physical methods include determiningthe smoking point, viscosity, conductivity, constantdielectricity and the Lovibond color index. Chemicalmethods include the determination of free fatty acids (acidvalue) by acid-base titration, polar compounds bychromatographic procedures, and also triglyceridepolymers and oxidized fatty acids (Inprasit 2011).

Manufacturers should follow the following guidelinesto control the quality of fried chips products (Inprasit2011): (i) Manufacturers should use control sheet presentedto control frying operation and also responsible operators.(ii) After frying, manufacturers should remove highmoisture fried product by pressing and observing the softtexture and then bring that product to fry once more to getthe low water content and desire crispiness. (iii) For storingfried products prior to packaging, manufacturers should usecontainers that can be sealed to prevent moisture transferand store products in the shade at low temperatures. Thebest storing method is packaged in a double polypropylene(PP) plastic bag and then placed in a sealed plastic bucket.(iv) For retail packaging, manufacturers should usecontainers that can be sealed to prevent moisture transfersuch as aluminum foil bag, aluminum foil bag in a paperbox, paper can coated inside with aluminum foil and metalcan. Fried product shelf life is not less than six monthswhen the manufacturer follows the above guidelines. Longshelf life can be obtained when using nitrogen gaspackaging. According to Piamkhla (2004), the shelf life ofvacuum fried ripe banana is six months when packaged in aplastic bag that can be flushed with nitrogen or puttingmoisture absorbent.

LESSON LEARNED FROM SEVERAL CHIPS

Many vacuum fried products are introduced in themarkets. Fruits, tubers and other vegetables are the mostwidely processed food with vacuum frying method, butsome types of meat and fish are also treated with thismethod, such as shrimp, squid, green shell mussels(Taryana 2012), sepat-siam (Suwanchongsatit et al. 2004),lemuru (Manurung 2011), tongkol (Nufzatussalimah 2012),beefs (Shofiyatun 2012) and others. Some types of fruitsand vegetables that have been processed into chips withvacuum frying method are: banana (Garcia and Barette2002), banana peel (Dewantara 2012), jackfruit (Alamsyahet al. 2002), durian, mango, pineapple, taro, yam, baby corn(Inprasit 2011), carrot (Fan et al. 2005), okra (Arlai 2009),garlic, sapodilla (Paramita 1999), gembili (Dioscoreaaculeata) (Wibowo 2012), chickpea (Widaningrum et al.2008), salaks (Maulana 2012), apple (Shidqiana 2012),tapioca (Binti Zahroni2012), cassava (Aprillia 2007),eggplant (Nur-Aeny 2012), potato (Granda et al. 2004),

kiwi fruits (Diamante et al. 2011), pumpkin (Mehrjardi etal. 2012), melon (Arum 2012), sweet potato (Abdullatif2012), tempeh (Kato and Sato 1991), etc.

Potato (Solanun tuberosum)Potato (Solanum tuberosum L.) is one of the world’s

major agricultural crops and it is consumed daily bymillions of people from diverse cultural backgrounds(NPC1988). The potato is best known for its carbohydratecontent (approximately 26 grams in a medium potato). Thepotato contains vitamins and minerals, as well as anassortment of phytochemicals, such as carotenoids andnatural phenols. Large variation in suitability of potato forprocessing of crisp and French fries have special qualitydemands compared to ware potatoes. Unfortunately, potatochips fried conventionally produce acrylamide that harmfulto human health.

Potatoes and other foods that have a high content of theamino acid asparagine and a high accumulation of reducingsugars are subject to the formation of acrylamide duringfrying (Granda et al. 2004). Acrylamide has been classifiedas probably carcinogenic in humans (Rosen and Hellenas2002; Tareke et al. 2002). Reducing acrylamide in foodindustry can only help the public perception about safety,which has suffered in recent years. Acrylamide formationcan be diminished by adding amino acids such as lysine,glycine and cysteine (Kim et al. 2005). Lowering the pHwith citric acid before frying was effective in diminishingacrylamide formation (by about 73%) in French fries whenfried for 6 minutes at 190 °C in an atmospheric fryer (Junget al. 2003). However, according to Pedreschi et al. (2004),the effect of citric acid immersion on acrylamide reductionwas not obvious in their experiment with potato chips friedat 170°C and 190°C. On the other hand, the blanchingprocess led to a significant reduction in acrylamide contentof their chips. Haase et al. (2003) reported that by loweringthe frying temperature of potato chips from 185°C to165°C, it was possible to reduce the acrylamide formationby half.

Among several deep-fat frying technologies, vacuumfrying has a significant strategic importance for future friedmanufacturing and in reducing acrylamide formation(Garayo and Moreira 2002; Granda et al. 2004). Vacuumfrying reduced acrylamide formation in fried potatoes by94%. As the frying temperature decreased from 180°C to165°C, acrylamide content in potato chips reduced by 51%during traditional frying and by 63% as the temperaturedecreased from 140°C to 125°C in vacuum frying.Increased frying time increased acrylamide formationduring frying for all temperatures and frying methodsanalyzed. However, the effect on acrylamide concentrationwas greater for the traditional frying than the vacuumfrying (Granda et al. 2004). Acrylamide formationdecreased dramatically as the frying temperature decreasedfrom 190 to 150°C for all the pre-treatments tested. Colorrepresented by the parameters L* and a* showed highcorrelations (r2 of 0.79 and 0.83, respectively) with Frenchfry acrylamide content (Pedreschi et al. 2006).

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Carrot (Daucus carota)Carrot (Daucus carota L. var. sativa D.C.) has the

highest carotene content of any human foods (Desobry etal. 1998). Carotene, a source of pro-vitamin A, may play arole in protecting the body from numerous diseases that areassociated with oxidative stress and damage (Handelman2001), and it also has many non-antioxidant properties thataffect cellular signaling pathways, modify the expression ofsome genes and can act as inhibitors of regulatory enzymes(Stahl and Ale-Agha 2002). To maximize the use of carrotas a source of pro-vitamin A, it is important to find anappropriate processing method to manufacture productsthat are not only highly preferred by consumers but also aregood nutritional sources of pro-vitamin A.

Vacuum-fried carrots may be a promising snackcategory due to the fact that this technology makes itpossible to overcome major carotenoids degradationpathways due to isomerization and oxidation and thuspreserve biological activity. Vacuum fried crisps (drivingforce of 60°C) may reduce the oil content of carrot crispsby nearly 50% (d.b.) compared to atmospheric fried crispsproduced using the same driving force. Furthermore, theypreserve around 90% of trans a-carotene and 86% trans b-carotene, which leads to the preservation of the color ofraw carrots. This is reflected by L*, a*, b* color coordinateanalyses, excellent linear correlations between a* and transb-carotene content (r2 = 0.95), b* and trans a-carotenecontent (r2 = 0.78), and hue and total carotenoids content(r2 = 0.91), when comparing values of fried crisps atbubble-end point. As a result, vacuum frying may be auseful process in the production of novel snacks thatpresent desired quality attributes and respond to new healthtrends (Dueik et al. 2010).

Bananas (Musa paradisiaca)Bananas (Musa x paradisiaca L.) are one of the world’s

most traded fruit in both fresh and processed forms.Bananas are an excellent source of vitamin B6, solublefiber, and contain moderate amounts of vitamin C,manganese and potassium (USDA NND 2012). Along withother fruits and vegetables, consumption of bananas maybe associated with a reduced risk of colorectal cancer(Deneo-Pellegrini et al. 1996) and in women, breast cancer(Zhang et al. 2009) and renal cell carcinoma (Rashidkhaniet al. 2005). The market quality and consumer acceptabilityof fresh banana and processed banana are significantlyinfluenced by the fruit color. For vacuum-fried, bananaslice products are prepared by peeling and slicing beforevacuum-frying. In this preparation step, as a result inslicing and waiting for processing, there is accumulation ofcell fluids, especially the phenolic compounds, on the cutsurface and their exposion to oxygen, leading to browning(Garcia and Barette 2002).

Phenolic compounds undergo oxidation to browncompounds that discolor fruits, reducing their quality(Rocha and Morais 2001). Discoloration is known asenzymatic browning which results from the action of agroup of enzymes called polyphenol oxidase (PPO). PPOhas been reported to occur in all plants and exists inparticularly high amounts in mushroom, banana, apple,

pear, potato, avocado and peach (Garcia and Barette 2002).PPO catalyzes, in the presence of oxygen, the oxidation ofmono-and di-phenols to o-quinones; these products arehighly reactive and can either polymerize spontaneously toform high-molecular-weight compounds or brownpigments, or react with amino acids and proteins toenhance the brownish color produced (Vamos-Vigyazo1981; McEvily et al. 1992).

Inhibition of enzymatic browning can be achieved by anumber of strategies that can be divided into three classes,depending on whether they affect the enzymes, substratesor reaction products, although in some cases, two or threetargets can be affected at the same time. In addition,enzymatic inhibition can be reversible or irreversible; thelatter case often achieved by physical treatment (heat),while chemicals may act in one or another way. Thecontrol of enzymatic browning has always been a challengeto the food industry. For using chemical treatments, severaltypes of chemicals are used in the control of browning;some act directly as inhibitors of PPO, others by renderingthe medium inadequate for the development of thebrowning reaction, still others act by reacting with theproducts of the PPO reaction before these can lead to theformation of dark pigments (Nicolas et al. 1994). Bananachips coated with an edible coating and produced using thehigher speed during the oil centrifuge step in the vacuum-frying process maintained a good quality with low oilcontent, representing a healthier snack for consumers(Sothornvit 2011).

Banana peel is a byproduct of the use of bananas thatcan be used as snack foods like banana peel chips. Thebanana peel contains a lot of water (68.90%) andcarbohydrate (18.50%). To produce chips with goodquality in terms of color, aroma, and taste, the temperaturesetting should not exceed 85◦C and vacuum pressurebetween 65-76 cm Hg (Dewantara 2012).

Kiwi fruit (Actinidia deliciosa)Kiwi fruit (Actinidia deliciosa [A. Chev.] C.F. Liang et

A.R. Ferguson) is native to southern China (Scott et al.1986). Kiwi fruit is a highly nutritional fruit due to its highlevel of vitamin C and it has a strong antioxidant activitydue to carotenoids, lutein, flavonoids and chlorophyllcontents (Cassano et al. 2006). Furthermore, kiwi fruitshave a very short shelf-life due to their highly perishablenature, and they are not only consumed as fresh fruits butalso as processed foods in the form of jams, juices, cannedfruits, frozen and dehydrated products (Abedini 2004;Emamjome and Alaedini 2005).

The color and the shrinkage of kiwi fruit chips weresignificantly (p<0.05) correlated with the fryingtemperature and time, while the crispiness was affectedonly by the frying temperature. There was no significantrelation between the vacuum pressure and the responsesexcept the shrinkage. Sensory evaluation indicated thatthere were no significant differences (p<0.05) between thevacuum fried kiwi slices and the dried kiwi chips exceptflavor. The optimum conditions for the vacuum frying ofkiwi slices were found to be: 105ºC, 62 mbar, and 8


minutes, for the frying temperature, the vacuum pressureand the frying time, respectively (Maadyrad et al. 2011).

Shallot (Allium cepa var. aggregatum)Shallot (Allium cepa L. var. aggregatum G. Don) is an

elementary spice of Southeast Asia as well as the world.Shallots appear to contain more flavonoids and phenolsthan other members of the onion genus (Yang et al. 2004).It was proven to increase high density lipoprotein (HDL)cholesterol, reduce low density lipoprotein (LDL), reducecholesterol in the blood and control blood sugar. Thus itwould be beneficial to develop a snack from shallot. Deep-fat fried snack is one of the most tasteful products.However, high fat content could reduce consumption dueto a health concern issue; and uses high temperature in anopened system speeds up oxidation and thereby ranciditydevelopment. Vacuum frying could be used to reduce fatcontent, frying temperature and slow down rancidity of oil.To minimize fat content of fried snack, shallot should befried under vacuum 551 mm Hg and 108°C for 13 minutes.The optimal vacuum frying condition was conducted tocompare with the deep-fat frying. Vacuum fried shallotshowed the improvement of product color as well as adecrease in fat content in the finished product. After 7continuous vacuum frying processes, a slight change inacid value for the oil was found. Therefore, the optimalvacuum frying condition could be applied to produce friedsnack from shallot (Therdthai et al. 2007).

Salaks (Salacca edulis)Salaks or snake fruit (Salacca edulis Reinw.) is one of

the horticultural commodities that have high potential to beexplored and developed in Indonesia. Salaks fruit containnutrients such as protein, carbohydrates, dietary fiber,calcium, phosphorus, iron, carotene, and thiamine that aregood for body health. The mass production of salaks makesan excess amount of salaks distributed in the market; salaksbecome wasted and priceless. To prevent the decreasingvalue of salaks, it can be proceed in to fruit chips. Theprocess of making salaks chips start from the frying stage.In order to pretend the composition and the taste of salakschips, vacuum frying is used. After fried, salaks must bepackaged with the suitable packaging to provide a longershelf life of salaks chips. Aluminum foil is the bestpackaging for salaks chips comparing to polypropylene andlaminated plastic because alumunium foil has the lowesttransmission rate of water and oxygen (Maulana 2012).

Melon (Cucumis melo)Melon (Cucumis melo L.) is an annual plant that is

pervasive or a year or vines. Melon fruit is an excellentsource of vitamin A and vitamin C, and a good source ofpotassium. Optimum temperature for the manufacture ofmelon chips is 75°C with a time of 55 minutes. Chips withthis variable has a sweet, brownish orange color, crisp, andhas aromas of melon and entrained water content is equalto 92.406% (Arum 2012).

Papaya (Carica papaya)Papaya (Carica papaya L.) is pretty much cultivated in

Indonesia. Papaya fruit is a source of nutrients such as pro-vitamin A carotenoids, vitamin C, folate and dietary fiber.Papaya skin, pulp and seeds also contain a variety ofphytochemicals, including lycopene and polyphenols(Echeverri et al. 1997). Generally, processed papayaproducts on an industrial scale are still a household nataand candy. In fact, papaya has a huge production to beprocessed into other products, such as fruit chips. Invacuum frying process, administration of CaCl2 canimprove the texture of papaya chips. Calcium chloride iswidely used to improve texture of the processed fruit andvegetable products; it can also be used for texture chips,because it can reduce the decomposition of the cells thatcause tissue softening (Indera-Sari 2012).

Sweet potato (Ipomoea batatas)Sweet potatoes (Ipomoea batatas (L.) Lam.) have been

an important part of the diet in the world and are a staple ofhuman consumption, led by New Guinea at about 500 kgper person per year. Considering fiber content, complexcarbohydrates, protein, vitamins A and C, iron, andcalcium, the sweet potato ranked highest in nutritionalvalue to other vegetables (CSPI 1992). For vacuum frying,sweet potato was sliced into 2 mm thickness and pretreatedwith 1% (w/w) NaCl solution and 1% (w/w) CaCl2 solutionfor 1 hour prior to frying process. Pre-treated slices werefried at atmospheric condition (180°C) and vacuumcondition (120°C, 130°C, 140°C) for 5 minutes. In general,pre-treatments gave a significant effect to the texture, colorand oil contents of atmospheric fried crisps. NaClpretreated crisps showed the best crispness and colorquality compared to the control. For vacuum fried crisps,breaking force (N) increased with increasing oiltemperature. Oil absorption of control slices showed nosignificant difference (p<0.05) at all frying temperature,while NaCl pre-treated slices showed an increase withtemperature increased. In contrast, CaCl2 pre-treatmentincreased oil absorption with increasing frying temperature.As frying temperature increased, the lightness of crisps wasdecreased, while a* and b* value were increased in allpretreatment. The best texture of crisps was obtained at130°C vacuum frying temperature. Comparing betweenatmospheric and vacuum fried crisps, there is no significantdifference (p<0.05) in terms of fracturability of crisps.However, oil absorption of vacuum fried crisps is 7.12%less than atmospheric fried. The color of vacuum friedcrisps was also lighter than atmospheric fried. Sensoryevaluation revealed that, consumer can accept the qualityattributes of vacuum fried crisps. There is no significantdifference between vacuum fried and atmospheric friedcrisps in terms of color, crispness and overall acceptability(binti Ismail 2011). In Cilembu sweet potato chips, theoptimum quality of the vacuum frying is 35 minutestreatment; and is obtained flavor, color, and crispness to theoptimum water content of 17.4% (Abdullatif 2012).

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Okra (Abelmoschus esculentus)Okra (Abelmoschus esculentus L. Moench) is an

important economic vegetable of the world. Okra is apopular health food due to its high fiber, vitamin C, andfolate content. Okra is also known for being high inantioxidants. Okra is also a good source of calcium andpotassium (Duvauchelle 2011). Processed okra is animportant agricultural product. During processing, many ofimportant quality compounds in okra maybe lost. Thevacuum frying treatment reduced the physical and chemicalquality of okra, but increased beta-carotene content. Themoisture heating and vacuum fry processing affected thequality of okra chips. The processing appeared to affect thechemical quality of organically grown okra less thanconventionally grown okra, especially the vitamin C andbeta-carotene contents. The rate of chemical decline waslower with the blanching process, especially the vitamin Ccontent, whilst vacuum frying resulted in the highest levelsof beta-carotene. The growing area, environmentalconditions and climate where the different okras grew maypartially affect to those of physical and chemical qualities(Arlai 2009).

Gembili (Dioscorea aculeata)Gembili (Dioscorea aculeata L.) is one of the types of

tubers that have not been cultivated and not many peopleknow. The nutritional value is not known yet. This plant iswidely grown in the rural areas which are usually used as asubstitute food for rice, snack, even just left alone to grow.In this time, the processing gembili as food only to theprocess of boiling or steaming, so the need for theutilization gembili processed into new products that havehigh sales value through the manufacture of chips such asfood diversity efforts. From the experiments conductedwith gembili weight 300 g, frying temperature 75◦C andchanging variables such as frying time of 20, 25, 30, 35 and40 minutes in the manufacture of vacuum frying chips ofgembili showed that the longer the frying pan, the watercontent is more and more vaporized. Water contentcontained in the chips greatly affect the quality of the chipswhich the less water content of chips have a longer shelflife and more crisp (Wibowo 2012).

Beans (Phaseolus vulgaris)Bean (of the Dutch, boontjes, Phaseolus vulgaris L.) is

a kind of beans that can be eaten. Bean is high in starch,protein and dietary fiber and is an excellent source of iron,potassium, selenium, molybdenum, thiamine, vitamin B6,and folate. The fruits, seeds, and beans are rich in proteinand vitamin that helps lower blood pressure and escortblood sugar metabolism and very suitable food by thosewho suffer from diabetes or hypertension. The optimumtemperature for the manufacture of chips beans by usingvacuum fryer is 90oC for 30 minutes. Chips with thisvariable has a low bitter taste, greenish brown, very crisp,and has a very strong smell of beans and water content are8.62% (Septiyani 2012).

Manggo (Mangifera indica)Mango (Mangifera indica L.) is a horticultural

commodity in Indonesia. Mango fruit is rich in vitamin Cand carbohydrate. Much-loved mango consumers becauseit can be consumed fresh or in processed form. Mango is aseasonal fruit which the product will be abundant in theharvest season and rare outside of the harvest season.Mango is a perishable commodity (have a relatively shortshelf life), hence the need for an alternative treatment thatmango production in large quantities can be consumed tothe all year round. Vacuum frying mango chips with fryingtemperature 80◦C, for 45 minutes is the best yield thatproduces low water content and good organoleptic chips(Sulistyaningrum 2012).

Chickpea (Cicer arietinum)Chickpeas (Cicer arietinum L.) are a source of zinc,

folate and protein. Chickpeas are low in fat and most of thisis polyunsaturated. Chickpeas also provide dietaryphosphorus (168 mg/100 g), which is higher than theamount found in a 100 grams serving of whole milk (NDL-USDA 2008). They can assist in lowering cholesterol in thebloodstream (Pittaway et al. 2008).

In processing technology of young chickpea, it issoaked in CaCl2 solution (1000 ppm, t=30’). For wetflavoring method, young chickpea was boiled with flavor,meanwhile for dry flavoring method, young chickpea wassteam blanched. Then, young chickpea vacuum fried at 65,75, and 85°C with vacuum pressure 72 cm Hg, andpackaged in alumunium foil. Yield of young chickpeachips were 13.58-14.17% with vacuum frying time rangefrom 1.08-1.41 hours. For both flavoring methods onyoung chickpea chips, moisture was 6.33-7.39%; ash 4.45-6.10%; fat 33.95-42.93%; protein 10.86-12.24%; crudefiber 11.94-14.10%; free fatty acid (FFA) 0.62-0.70%;vitamin C 0.27-0.46 mg/100g; and vitamin A 135.54-265.39 ppm. Sensory evaluation showed that differenttreatment of flavor and temperature did not have significanteffect (P>0.05) to all parameter (color, odor, texture, taste,crispiness and acceptability of chickpea chips). But, flavorhad significant effect (P<0.05) to chickpea chips taste, andtemperature had significant effect (P<0.05) to crispiness ofchickpea chips (Widaningrum et al. 2008)

Apple (Malus domestica)Apple (Malus domestica Borkh.) is one of the most

widely cultivated tree fruits, and the most widely known ofthe many members of genus Malus that are consumed byhumans. Apple peels are a source of variousphytochemicals with unknown nutritional value (Boyer andLiu 2004) and possible antioxidant activity in vitro (Lee etal. 2004). The predominant phenolic phytochemicals inapples are quercetin, epicatechin, and procyanidin B2 (Leeet al. 2003.

Shidqiana (2012) had been conducted a study to lookfor water content and organoleptic on apple chipsprocessed by vacuum fryer. The study was conducted in 5variables change, the longer of frying time were 35, 40, 45,50 and 55 minutes, while the fixed variable was 750 gheavy material and frying temperature was 80°C. The


results indicates that water content of the chips haddeclined 9.45%, 7.46%, 6.44%, 5.47%, and 4.97respectively, and of the organoleptic test the most preferredapple chips were processed with a temperature of 80°C andtime of 50 minutes; at this variable the color was well, theflavor was delicious and the crispness was crisp.

Shyu and Hwang (2001) studied the effect of processingconditions on the quality of vacuum fried apple chips. Theyused a single vacuum pressure condition, 3.115 kPa, andthree levels of temperature, 90, 100, and 110 ºC to fry thechips. After frying, the chips were centrifuged for 30minutes at 350 rpm to remove the surface frying oil andthen packed in polyethylene bags and stored at 30ºC. Usingtexture (hardness) as an indicator of product quality, theoptimum conditions were vacuum frying temperature of100-110ºC, vacuum time of 20-25 minutes, and aconcentration of immersing fructose solution of 30-40%.

Sapodilla or sawo (Manilkara zapota)Sapodilla (Manilkara zapota (L.) P.Royen) is a meso

American tropical fruit crop which is widespread inIndonesia, and can harvest throughout the year. Sapodillafruit is generally consumed as a table fruit, rarely furtherprocessing. The fruit has an exceptionally sweet, maltyflavor. The unripe fruit is hard to the touch and containshigh amounts of saponin, which has astringent propertiessimilar to tannin, drying out the mouth. After ripening,sapodilla cannot survive long, easily damaged and rot. Forvacuum fried chips, acquired conditions which make goodchips, starting with slicing fruit with a stainless steel bladein a uniform thickness, immersing the slices in a brownsolution of sodium bisulfite (1000 ppm) to preventenzymatic browning reactions.

Frying temperatures cause decreased significantly (p<0.05) on water content, hardness and brightness (L value).Water content ranged from 3.45 to 5.15% (dry basis).Hardness ranged between 2.73-4.50 kg/7mm. While Lvalues ranged between 42.58 and 50.92. Factors of fryingtemperature, frying time and the interaction between thesetwo factors did not affect significantly (p> 0.05). On theother observations that yield, fat content, and colorparameters (a and b values), yield provided the rangebetween 24.05-26.01%. 27.35; fat content ranged from31.05% (dry basis); value of a (redness) was 3.79-8.46while the value of b (yellowness) was 14.78-20.00(Paramita 1999).

Eggplant (Solanum melongena)Eggplant (Solanum melongena L.) is one of the favorite

fruit among the people that it tastes good. Nutritionally,eggplant is low in fat, protein, and carbohydrates. It alsocontains relatively low amounts of most important vitaminsand minerals. Eggplant juice can significantly reduceweight, cholesterol levels, and aortic cholesterol (Jorge etal. 1998). In general, eggplant is consumed in the freshform or cooked vegetables. One great way to reduce thewater content is to process them into fruit crisps. Toimprove the crispness of the product, the freezing processis conducted. Freezing process increase the level ofcrispness and reduce the shrinkage. Besides the freezing

process, soaking the product in CaCl2 is also needed inorder to maintain the texture of the product during heatprocess (Virgiawan 2011; Nur-Aeny 2012). Meyer (1987)states that CaCl2 including material hardening or firmingagent for fruits and vegetables. CaCl2 significantly affectthe fracture but does not affect the color, flavor and yield.Immersion on CaCl2 significantly affects the water contentand crispy chips and all organoleptic parameters (taste,color, appearance, crispness) (Nur-Aeny 2012). Thesoaking of the prepared eggplant in CaCl2 has significanteffects on the breaking strength but has no significanteffects on water content and yield. The CaCl2 soakingfactor combined with freezing time have significant effectson all organoleptic parameters. Best treatment was chosenusing effective index method and marks the soaking ofproduct in 1.5% CaCl2 and the freezing time of 12 hours asthe best treatment (Virgiawan 2011).

Jackfruit (Artocarpus heterophyllus)Jackfruit (Artocarpus heterophyllus Lam.) is commonly

used in Southeast Asian cuisines. It can be eaten raw whenripe, but as the raw unripe fruit is considered inedible, it isbest cooked. The ripe jackfruit is naturally sweet withsubtle flavoring, and contains a lot of energy (95calories/100 g) and the antioxidant vitamin C (13.7mg/100g) (NDL 1998). For vacuum frying of jackfruit, thefrying condition is vacuum pressure of-70 cm Hg andtemperature level of 75◦C and 80◦C. Such condition wasdone to minimize the heat used and therefore reducechanges in composition, color, taste and flavor of thejackfruit. The fried product was 22% and the product haslow water content of 3.58% (wet basis) with the taste,flavor, color and volume similar to the fresh jackfruit.Financial analysis of the jackfruit production capacity of 30kg per day showed that NPV (Net Present Value) was IDR52.391.000 which was bigger than investment cost, IRR(Internal Rate Return) was 51% and PBP (Pay BackPeriod) was 1.95 years, thus jackfruit fried chips was viableto be established (Alamsyah et al. 2002).

Cassava (Manihot esculenta)Cassava (Manihot esculenta Crantz) root is essentially a

carbohydrate source. Its composition shows 60-65%moisture, 20-31% carbohydrate, 1-2% crude protein and acomparatively low content of vitamins and minerals.However, the roots are rich in calcium and vitamin C andcontain a nutritionally significant quantity of thiamine,riboflavin and nicotinic acid. Cassava starch contains 70%amylopectin and 20% amylose. Cooked cassava starch hasa digestibility of over 75% (Tewe 2004).

After harvest, cassava conditions quickly change, thatneeds processing to longer shelf life, such as for makingvacuum fried chips. Cassava varieties very significanteffect on the water content, HCN levels, fat content, yield,broken power and color of chips. Blanching and freezingtreatments significantly affect on water contents, levels ofHCN, starch content, fat content, the fracture, taste, color,appearance and crispness of cassava sticks. The besttreatment was obtained from treatment of butter, withfreezing and blanching with HCN levels of 3.54 ppm, and

5 (2): 86-103, November 2013100

the chips have starch content of 32.43%, the fracture2284.19 N/m, flavor score 4.2 (good), appearance score3.22 (rather dense) and crispness score 4.04 (crispy)(Aprillia 2007).

Another product of cassava is tapioca chips fromcassava starch. In atmospheric frying, NaCl pre-treatmenthad greatly reduced the oil absorption of tapioca crisps butdid not provide improvement on color and texture. Undervacuum frying, the oil absorption for control and pre-treated sample shows a significant different (p<0.05) at allfrying time range. For color values, L*, a* and b* was notaffected by the NaCl pre-treatment, but was affected by thefrying time. While for texture, NaCl pre-treatmentincreases hardness and breaking force as the frying timeincreases. The most suitable time of vacuum frying fortapioca crisps is at 2 minutes as it gives the best quality ofcrisps in terms of oil absorption, color and texture. Therewas a significant different in all physical and sensoryproperties between atmospheric and vacuum fried tapiocacrisps. The vacuum fried tapioca crisps had absorbed53.36% less oil compared to atmospheric fried crisps.Vacuum fried tapioca crisps also had lighter color andbetter texture compared to atmospheric frying. However forsensory evaluation, consumer prefers the atmospheric friedcrisps rather than vacuum fried crisps (Binti Zahroni 2012).

CONCLUSION

Vacuum fried chips are potential to increase the addedvalue of fruits and vegetables, both nutritionally andeconomically. Fruits and vegetables are processed withvacuum frying have better nutritional value than traditionaldeep-fat frying, as well as the texture, color, and othersensory character is also better. This process prevent orreduce the formation of harmful substances in thetraditional deep-fat frying, such as acrylamide andexcessive saturated oil, thus meet the demands of modernpublic health. This process also adds value to fruits andvegetables are not eligible to be sold because of defects andprevent the waste of fruits and vegetables during theharvest because of the large supply.

ACKNOWLEDGEMENTS

This paper was generously supported by the SebelasMaret University through IbM BOPTN for financial year2012 and IbM Dikti for financial year 2013. Without thekindness, this paper would not have been possible. Theauthors thanks to the administrative village head (lurah)and residents of Kampung Kejiwan in Wonosobo, CentralJava, Indonesia for supporting the application of vacuumfrying program.

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Short Communication:Global warming – Problem with environmental and economical impacts

SHIVANI M. RAIKesharbai Lahoti College of Commerce, Amravati-444 602, Maharashtra, India. Tel. +91-9737057775, ♥email: [email protected],

[email protected]

Manuscript received: 16 April 2013. Revision accepted: 10 May 2013.

Abstract. Rai SM. 2013. Short Communication: Global warming – Problem with environmental and economical impacts. NusantaraBioscience 5: 102-105. The present article is focused on global warming, which is an important global problem being faced by thehumankind. The article discusses about the causes of the global warming, such as green house gases. The earth receives energy from theSun in the form of solar radiations with small amount of infra red and ultraviolet rays. A part of these radiations is absorbed by greenhouse gases which results into warming of the earth. These radiations increase temperature on the universe and are one of the mostimportant global problems. The efforts from all the countries of the world are required for reduction of emissions of green house gases.

Key words: economy, environment, global warming, green house gases

Abstrak. Rai SM. 2013. Komunikasi singkat: Pemanasan global – Permasalahan dengan dampak lingkungan dan ekonomi. NusantaraBioscience 5: 102-105. Artikel ini difokuskan pada pemanasan global, yang merupakan masalah global penting yang dihadapi oleh umatmanusia. Artikel ini membahas tentang penyebab pemanasan global, seperti gas rumah kaca. Bumi menerima energi dari mataharidalam bentuk radiasi surya dengan sejumlah kecil sinar infra merah dan ultraviolet. Sebagian dari radiasi ini diserap oleh gas-gas rumahkaca yang mengakibatkan pemanasan bumi. Radiasi-radiasi ini meningkatkan suhu alam semesta dan merupakan salah satu masalahglobal yang paling penting. Upaya dari semua negara di dunia diperlukan untuk mengurangi emisi gas rumah kaca.

Kata kunci: ekonomi, lingkungan hidup, pemanasan global, gas rumah kaca

The temperature of the earth is rising with fast pace.Since 1975, the global surface temperature has beenincreased by 0.5oC (Hansen et al. 1999; Jones et al. 1999;Mann et al. 1999; Jansen et al. 2000), The main reasons forrise in temperature include green house gases,deforestation, etc. According to the U.S. EnvironmentalProtection Agency (2009): “Global warming is an averageincrease in the temperature of the atmosphere near theEarth’s surface and in the troposphere, which cancontribute to changes in global climate patterns”. Globalwarming can occur from a variety of causes, both naturaland human induced. In common usage, “global warming”often refers to the warming that can occur as a result ofincreased emissions of greenhouse gases from humanactivities.” In fact, due to global warming sea-level is risingand has become a great puzzle (Woodsworth 1990;Douglas and Peltier 2002; Woodworth and Player 2003;Holgate and Woodworth 2004).

The basic cause of global warming is increase intemperature due to the greenhouse gases. A certain amountof these greenhouse gases maintain the earth's climatecongenial to live but it has been observed that over theyears these gases have increased and do not allow the solarheat to escape into space keeping the earth's temperaturewarmer than needed and allowing the polar caps to melt alittle more each year causing a rise in the oceans.

Agriculture is the main occupation across the globe with1.2-1.5 billion hectare as a crop land and 3.5 billion as agrass land (Howden et al. 2007; Thangarajan et al. 2013).Agriculture contributes up to 10-12% of the total emissionsof green house gas emissions (IPCC 2007).The higherinput of the modern chemical fertilizers have createdproblems like degradation of quality of soil, loss ofbiodiversity and contamination of ground water. Excessiveirrigation is also the cause of climate change and globalwarming (Puma and Cook 2010).

According to a survey, in the past three decades globalwarming is 0.6°C and 0.8°C in the past century. It is notappropriate to claim that ‘‘most global warming took placebefore 1940”. Up to 1975, there was slow global warming,with large fluctuations, over the century up to 1975,followed by rapid warming at a rate 0.2°C per decade.Global warming was 0.7°C between the late 19th century(the earliest time at which global mean temperature can beaccurately defined) and 2000, and continued warming inthe first half decade of the 21st century is consistent withthe recent rate of 0.2°C per decade (Hansen et al. 2006).Around the areas of ocean, quite away from theanthropogenic activities warming occurs.

The gradual rise in earth temperature is a matter ofgreat concern. General public, politicians andenvironmentalists are interested to solve this issue at global

RAI – Environmental and economical impacts of global warming 105

level. There are various reports of efforts made to solve thisproblem all over the world. There have been publicawareness and a great concern towards the problem ofglobal warming and it is realized that it should be tackledmeticulously in order to save the mankind.

This article is aimed to discuss the global warmingproblem, its causes and pragmatic approach to solve thisproblem.

Causes of global warmingThere are many greenhouse gases (GHGs) responsible

for warming. Due to the anthropogenic activities the gasesare emitted in different ways. Most of these gases areproduced by modern agricultural practices, from thecombustion of fossil fuels in industries, cars, and bygeneration of electricity. The most important among thesegases is carbon dioxide. The carbon dioxide (about 20 %)produced due to the anthropogenic activities remains in theenvironment for thousands of years. Other gas whichcontributes a major part includes methane released fromland and agriculture, nitrous oxide from fertilizers, gasesused in refrigerators and freezers. There has been muchdeforestation which is really a cause of worry becausethese forests are responsible for binding carbon-dioxide.

There are ten primary green house gases includingwater vapor, carbon dioxide, methane, and nitrous oxidewhich are occurs naturally. Perfluorocarbon,hydrofluorocarbons, and sulfur hexafluoride are found inthe atmosphere due to emissions from different kind ofindustries. Among these, water vapor is the most abundantkind of green house gases present in the atmosphere.Carbon-dioxide is the primary anthropogenic greenhousegas, accounting for 77% of the human contribution to thegreenhouse effect.

It is estimated that from 10,000 years ago until 150years ago, atmospheric concentrations of carbon-dioxide,methane and nitrogen-dioxide were relatively stable.Unfortunately, during the last 150 years, concentrations ofmethane and nitrogen dioxide increased 148% and 18%,respectively. There are various sources of Greenhouse GasEmissions. Due to human activities (anthropogenic source)carbon-dioxide is emitted from burning fossil fuels, cementindustries and due to rapid deforestation. Methane andnitrogen dioxides emissions are both man-made andnatural. Agriculture accounts for major contribution ofmethane and nitrous dioxide gases. Manyhydrofluorocarbons used in refrigeration, cooling, and assolvents in place of ozone depleting chlorofluorocarbons.

There are different heat-trapping abilities of the greenhouse gases. It is worthy of note that a molecule ofmethane gas produces more than 20 times the warming of amolecule of carbon dioxide. Another example is nitrousoxide, which is 300 times more powerful than carbondioxide. There are other gases also which includechlorofluorocarbons. These have been banned in most ofthe countries of the world because they are responsible fordegradation of the ozone layer. This ozone layer has heat-trapping capacity -thousand of times greater than carbondioxide. There are various reports, which provide evidencethat carbon dioxide is a major contributor of global warming.

There was no concrete decision in Copenhagen inDecember 2009 to reach to final conclusion to extend andbroaden the Kyoto Protocol raises the prospect thatattempts to limit atmospheric concentrations of carbondioxide (CO2) and other greenhouse gases (GHGs), as aconsequence of which global temperature increases. It isreally difficult politically. Nordhaus (2010) reportedimproved estimates of the likely trajectories of globaloutput, GHG emissions, climate change, and damages inthe coming decades.

Deforestation is a major problemForests play a major role in balancing the carbon-

dioxide in the atmosphere in several ways. The plants ofthe forests remove carbondioxide from the atmosphere andabsorb carbon into different parts of the plants, such aswood, leaves, where it can be stored for a large period.However, due to deforestation, stored carbon may bereleased into the atmosphere, depending in part on howmuch of the wood is destroyed. For example, forest firesdestroy many plants. In addition human also fell trees fortimber and other uses. Deforestation is sometimes man-made because for construction purpose cleaning of forestsis required. A huge amount of carbon stored in forestsworldwide indicates the significant role of forests inclimate change and global warming. According to anestimate, the forest trees are estimated to store theequivalent of roughly 760 billion metric tons of carbon-dioxide worldwide over one hundred times the UnitedStates’ emissions of Carbon-dioxide and other greenhousegases in 2009.

Ecological effects of global warmingDroughts and floods

Between the ninth and fourteenth centuries (MedievalWarm Period) the global temperatures rose up to 2oC(Acemoglu et al. 2012). Fagan (2008) stated that thisbrought bounty to some areas, but others suffered fromdroughts. There will be drastic changes of tropical rainfallon a regional basis (Allen and Ingram 2002) Due to the risein temperature water evaporates rapidly. This evaporatedwater will quickly condense to form clouds and fall on theearth as rains.. Unfortunately, this rainfall is not evenlydistributed. The rapid evaporation of water may generateseveral problems particularly in developing countrieswhere availability of water is a great problem. Plant lifedepends on water from rivers and lakes if water evaporateswith faster rate; the life is threatened due to drought. Thedrought will affect indirectly the crops and if there will notbe proper crop yield, there will be food problem. On theother hand in wet area the evaporation would be muchhigher and this would cause untimely rainfall and flood.Drought is a main natural cause of agricultural, economic,and environmental damage (Burton et al. 1978; Wilhite andGlantz 1985; Wilhite 1993).

Rising sea levelConstant increase in temperature would be responsible

for melting ice in North and South poles (Steffensen et al.2008). There are various reports concerning sea level rise(Douglas 1991, 1992; Maul and Martin, 1993; Church et al.

5 (2): 104-107, November 2013106

2001; Chambers et al. 2002; Douglas and Peltier 2002;Brohan et al 2006). The report of melting ice in Antarcticais a burning example. The surface melting was recorded inice sheet (Velicogna 2010; Buis and Cole 2012; Vinas2012) The glaciers melts and causes land-slide as aconsequence of which the sea level is rising. It is estimatedthat in future 1-4 m water level will rise. If the ice sheets ofGreenland and Antarctica fully melts, the sea level will riseby 64 m. During the period of 1993-2003 there has beenloss of 50-100 tons of ice. The low-lying areas in generaland coastal areas in particular will be flooded and may besubmerged. It is really a matter of great concern that 29%of the world’s population which lives in coastal areas willbe affected. According to Church and White (2006)between 1870 and 2004, global average sea levels rose195 mm.

The results of the sea-level rise would affect badlyparticularly coastal flooding and storm damage, erodingshorelines, salt water contamination of fresh watersupplies, flooding of coastal wetlands and barrier islands,and an increase in the salinity of estuaries are all realities ofeven a small amount of sea level rise (Lambeck andChappell 2001). There are about 30 countries which wouldbe affected by rising sea level. There is an alarming repotby Bennartz et al. (2013) in July 2012, an historically raremelting was recorded across the entire Greenland ice sheet,raising questions about the frequency and spatial extent ofsuch events.

Extreme weatherClimate is the average of many weather events over of a

span of years (Huber et al. 2011). In fact, climate changecan be described in terms of average changes intemperature or precipitation (Karl et al. 2008). After 1880,globally 2005 and 2010 were the warmest year (NCDC-NOAA 2010), both years are known for exceptionallydamaging weather events, for example, Hurricane Katrinain 2005 and the deadly Russian heat wave in 2010. Theyear 2005 have been considered as the warmest yearglobally, 19 countries set new national high-temperaturerecords. In 2010, global precipitation was also far abovenormal and it was the wettest year since 1900 (Huber et al.2011). It was Rio de Janeiro which received the heaviestrainfall in 30 years causing nearly 300 mudslides andkilling at least 900 people (Cabral 2010).

There would be a remarkable change in weather owingto temperature rise. The high temperature can increasewinds, rains and storms and finally there would change inoverall climate of Earth. The climate of the future will beentirely different from the one we are having now. We arealready experiencing the change of weather all over theworld and it is a matter of discussion among scientists,politicians and common people.

Economic impactAs far as the global warming is concerned, it’s essential

economic elements can be explained in a simple economicmodel which include four elements: (i) the consumption ofthe present generation, (ii) the consumption of futuregenerations, (iii) the conventional capital stock resulting

from the investment of the current generation, and (iv) theclimatological capital stock representing the reduction inthe stock of greenhouse gases in the atmosphere due toinvestments of the current generation in the mitigation ofglobal warming (Foley 2007).

There are severe economic impacts of global warmingand climate change. The loss of crops, forests, and animalsare most important. Due to the sea-level rise there will behuge migration of the inhabitants of as low-lying countrieswhich would be affected with flood. Moreover, there willbe disruptions to global trade, transport, energy suppliesand labor markets, banking and finance, investment andinsurance, would all create havoc on the stability of bothdeveloped and developing nations. Consequently, there willbe adverse effect on markets by increased volatility.

Due to rise in temperature and change in climaticconditions, several diseases, such as, Malaria, dengue andviral diseases will spread, which will be responsible forhuge economic loss in treatment of the diseases. There willbe excessive economic loss due to hurricane, floods anddiseases. The problem will not only be faced by developingcountries, it will be the problem of the developed worldalso. The main problem in coastal areas particularly wouldbe of potable water, energy and transportation. These allproblems would indirectly affect the economy of thepeople.

Public perceptionIt is really very important for public to understand that

global warming is manmade According to a recent surveyin the U.S. by Rabe and Borick (2012) provides evidencethat public opinion for the global warming depends mainlyon their perceptions of local climate variations.

In 1988, Hansen and his coworkers suggested that bythe early 21st century the informed public should be able torecognize that the frequency of unusually warm seasonshad increased. In 2011, heat waves in Texas and Oklahomain the summer raise the question of whether these extremeevents are related to the on-going global warming trend,which has been attributed with a high degree of confidenceto human-made greenhouse gases.

The change of global temperature may have the greatestpractical impact via effects on the water cycle. Indeedclimate changes occurring with global warming involveintimate interactions of the energy and water cycles.

The extreme rise in temperature causing heat waves andfrequent floods has received public attention. However, thecommon public has no perception of why the globalwarming is taking place. It is the need of the hour togenerate awareness about the environmental problems ingeneral and rise in temperature.

SolutionsInvesting in clean energy industries, such as wind and

solar, as well as energy efficiency programs, can lead usout of crisis and into a new clean energy economy. Weshould focus on vehicles which can be run on solar light. Itputs about 10,000 miles a year on the car, running it purelyon sunlight. The solar panels that provide all the electricityhomes also charge the car battery. By using solar energy-

RAI – Environmental and economical impacts of global warming 107

based cars, the use of oil can be reduced. Governmentshould focus on use of solar energy and it should beimperative that new buildings should meet energy-efficiency standards that maximize energy savings. Itshould be mandatory for new buildings existing andcommercial spaces to save energy by installing energyefficient heating, cooling and lighting systems. Thegarbage should be recycled in order to avoid methane gasproduction. Garbage should not be burnt because it releasescarbon dioxide and hydrocarbons into the atmosphere.There should be plantation program at large scale. There isa greater need to develop non-fossil fuel energy sources.Solar, wind and hydroelectric power, which are the free giftof almighty can reduce greenhouse gases.

It can be concluded that the greenhouse effect is one ofthe most important global problems. The efforts from allthe countries of the world are required for reduction ofemissions of green house gases. However, it has beenexperienced from the past that there are meetings,conventions, and discussions by the scientists andpoliticians regarding the global warming and climatechange but the efforts are not focused. The most importantis that if reductions are not controlled, we should try to gofor mass plantation programs by public participations.

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A-1

Authors Index

Abdelazim H 35Abou-Leila BH 65Ahmed AM 57Al-Hammady MAM 75Ameh JB 51Ammar MSA 35, 75Ashour F 35Atawodi SE 51Azadfar D 30Borah RK 1El-Mergawi RA 22Hendawy SF 22Hussein MS 22Ibrahim MM 70Khalid KA 15, 65, 70Khattab HI 57Khazaeian A 30Kumar R 1Malik N 8Metwally SA 65

Negedu A 51Obuid-Allah AH 75Pandey S 1Pyasi A 44Rai MK 51Rai SM 104Setyawan AD 86Singh RK 8Singh S 8Solichatun 86Soni KK 44Sugiyarto 86Susilowati A 86Talaat IM 57Tapwal A 1Umoh VJ 51Verma RK 44Youssef AA 22Zoghi Z 30

A-2

Subject Index

Acropora humilis 35, 37, 39, 41, 75, 76, 77, 78,80, 81, 82, 83, 84

amino acids 57, 58, 59, 60, 62, 63, 64Ammi visnaga 57, 58, 59, 60, 62, 63, 64Anise 15, 16, 17, 18, 19, 20autoclaving 51, 52, 53, 54, 55, 56B. cepacia 8, 9, 10, 11, 12, 13bio fertilization 22, 23biometry properties 30, 31bleaching 36, 38, 39, 41, 75, 76, 82, 83,

84Calendula officinalis 25, 65, 66, 68, 69,carbohydrate 1, 3, 6, 7, 15, 17, 19, 20, 25,

45, 53, 54, 55, 92, 96, 97, 99carpophores 1castor seeds 51, 52, 53, 54, 55chips 86, 87, 88, 89, 90, 91, 93, 95,

96, 97, 98, 99citronella 70, 71, 72, 73coral disease 35, 36, 37, 38, 40, 41, 42coriander 15, 16, 17, 18, 19, 20distribution 6, 35, 42, 88economy 104, 106ectomycorrhiza 44, 46, 47, 48environment 7, 15, 30, 31, 35, 36, 40, 41,

42, 65, 73, 75, 76, 83, 84, 94,97, 104, 105, 106

essential oil 15, 16, 17, 18, 19, 20, 27, 57,58, 59, 60, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73

ethnomycology 1, 2Fagus orientalis 22, 30, 31, 32,fiber 2, 3, 30, 31, 32, 33, 34, 54,

97, 98fixed oil 15, 16, 18, 19, 20flower yield 65, 66, 67, 68, 69food 1, 2, 6, 7, 16, 65, 83, 86, 87,

88, 89, 90, 91, 92, 93, 95, 96,98, 99, 104

free fatty acids 51, 54, 55, 56, 89, 95, 98,frying 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99, 100global warming 7, 9, 104, 105, 106, 107green house gases 104, 105, 107growth 1, 6, 7, 8, 9, 10, 11, 12, 13,

15, 16, 17, 18, 19, 20, 22, 23,24, 25, 26, 27, 28, 30, 31, 33,36, 38, 39, 40, 41, 42, 44, 45,46, 48, 65, 66, 67, 68, 69, 70,71, 72, 73, 76, 83, 84, 86, 87

growth criteria 57

Gulf of Aqaba 35, 36, 38, 40heritability 70, 71hormones 57, 59, 62, 64inoculum 44, 46irrigation intervals 22, 23, 24, 25, 26, 27, 28,mycorrhiza 1, 23, 24, 25, 26, 27, 28, 44,

45, 46, 48nitrate 8, 9, 11, 12, 13, 24nutrient uptake 12, 44peroxide value 51, 54, 55phenolic compounds 57, 58, 59, 60, 62, 63, 64plant growth 8, 12, 18, 20, 23, 25, 27, 28,

68plus trees 30, 32, 33, 34preservation 52, 53, 65, 86, 87, 90, 92, 93,

96proline 2, 65, 66, 67, 68, 69protein 1, 2, 3, 6, 7, 11, 15, 16, 18,

19, 20, 51, 52, 53, 54, 56, 76,82, 88, 95, 97, 98, 99

proximate analysis 1, 2proximate composition 6, 7, 51, 52R. undicola 8, 9, 10, 11, 12, 13Ras Mohammed 35, 36, 37, 38, 40Red Sea 35, 36, 38, 40, 41, 76Red Sea corals 75, 84Rhizobiumleguminosarum bv.phaseoli

8, 9, 11, 13

sal forest 44, 46, 48,sedimentation 22, 40, 41, 42, 75, 76, 80, 81,

82, 83, 84selection 30, 31, 32, 33, 34, 66, 70, 71,

72, 73, 93, 94silymarin 22, 24, 26, 27, 28Stylophora pistillata 38, 39, 42, 75, 76, 78, 79, 81,

82, 83, 84sweet fennel 15, 16, 17, 18, 19, 20Sylibium marianum 22, 23, 25, 26, 27,synthesis 8, 15, 18, 20, 23, 25, 40, 43,

48, 65, 68, 76, 77, 83,temperature 2, 6, 9, 17, 18, 41, 51, 52, 67,

75, 76, 77, 78, 79, 81, 82, 83,84, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99,103, 104, 105

vacuum frying 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 100

vase life 65, 66, 67, 68, 69water regime 65, 66, 67, 68, 69Zea mays 8

A-3

List of Peer Reviewer

Abdala G. Diédhiou Laboratoire Commun de Microbiologie, IRD/ISRA/UCAD, Centre de Recherche de Bel-Air, BP 1386 CP 18524 Dakar-Sénégal

Ahmad Dwi Setyawan Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas MaretUniversity, Surakarta 57126, Central Java, Indonesia

Andrea Patriarca Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales,Universidad de Buenos Aires, Ciudad Universitaria, Pab II, Piso 3, 1429-Buenos Aires,Argentina

Ashok Kumar Department of Botany, Dr. Bhim Rao Ambedkar Government Degree College,Maharajganj, Maharajganj-273303, Uttar Pradesh, India

Claudia L. Prins Plant Production Laboratory, Centro de Ciências e Tecnologias Agropecuárias,Universidade Estadual do Norte Fluminense Darcy Ribeiro (CCTA/UENF), 28013-602,Campos dos Goytacazes, RJ, Brazil

Daniel Isidoro Unidad de Suelos y Riegos (unidad asociada EEAD-CSIC), CITA, Avda. Montañana930, 50059-Zaragoza, Spain

Federica Aureli Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità,Viale Regina Elena, 299, 00161 Rome, Italy

Goodarz Hajizadeh Department of Forestry, Faculty of Natural Resources, Sari University of AgriculturalSciences and Natural Resources, Sari, Mazandaran, Iran

Jay P. Verma Institute of Environment and Sustainable Development, Banaras Hindu University,Varanasi-221005, Uttar Pradesh, India

Kateryna Kon Department of Microbiology, Virology, and Immunology, Kharkiv National MedicalUniversity, 61022 Pr. Lenina, 4, Kharkiv, Ukraine

Mahdi Reyahi Khoram Department of Environment, Hamadan Branch, Islamic Azad University, P.O. Box65155-184, Hamadan, Iran

Mahendra K. Rai Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati444602, Maharashtra, India

Sugiyarto Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas MaretUniversity, Surakarta 57126, Central Java, Indonesia

Wiryono Department of Forestry, Faculty of Agriculture, University of Bengkulu. Bengkulu38371A, Bengkulu, Indonesia.

A-4

Table of Contents

Vol. 5, No. 1, Pp. 1-49, May 2013

Macro-fungal diversity and nutrient content of some edible mushrooms of Nagaland, IndiaRAJESH KUMAR, ASHWANI TAPWAL, SHAILESH PANDEY, RAJIB KUMAR BORAH

1-7

Impact of rhizobial inoculation and nitrogen utilization in plant growth promotion of maize (Zea mays L.)RAMESH K. SINGH, NAMRATA MALIK, SURENDRA SINGH

8-14

Effect of nitrogen fertilization on morphological and biochemical traits of some Apiaceae crops underarid regions in EgyptKHALID ALI KHALID

15-21

Response of Silybum marianum plant to irrigation intervals combined with fertilizationSABER F. HENDAWY, MOHAMED S. HUSSEIN, ABD-ELGHANI A.YOUSSEF,REYAD A. EL-MERGAWI

22-29

Study of altitude and selection on fiber biometry properties of Fagus orientalis LipskyZOHREH ZOGHI, DAVOUD AZADFAR, ALI KHAZAEIAN

30-34

Coral disease distribution at Ras Mohammed and the Gulf of Aqaba, Red Sea, EgyptMOHAMMED SHOKRY AHMED AMMAR, FEKRY ASHOUR, HODA ABDELAZIM

35-43

Effect of ectomycorrhizae on growth and establishment of sal (Shorea robusta) seedlings in central IndiaABHISHEK PYASI, KRISHNA KANT SONI, RAM KEERTI VERMA

44-49

Vol. 4, No. 2, Pp. 51-107, November 2013

Effects of autoclaving on the proximate composition of stored castor (Ricinus communis) seedsANTHONY NEGEDU, JOSEPH B. AMEH, VERONICA J. UMOH, SUNDY E. ATAWODI,MAHENDRA K. RAI

51-56

Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treated with somebioregulatorsIMAN M. TALAAT, HEMMAT I. KHATTAB, AISHA M. AHMED

57-64

Effect of water regime on the growth, flower yield, essential oil and proline contents of Calendula officinalisSAMI ALI METWALLY, KHALID ALI KHALID, BEDOUR H. ABOU-LEILA

65-69

Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus) grown inEgyptMOHAMED M. IBRAHIM, KHALID A. KHALID

70-74

Experimental effect of temperature and sedimentation on bleaching of the two Red Sea corals Stylophorapistillata and Acropora humilisMOHAMMED S.A. AMMAR, AHMED H. OBUID-ALLAH, MONTASER A. M. AL-HAMMADY

75-85

Review: Physical, physicochemical, chemical and sensorial characteristics of the several fruits andvegetables chips by low-temperature vacuum frying machineAHMAD DWI SETYAWAN, SUGIYARTO, SOLICHATUN, ARI SUSILOWATI

86-103

Short Communication: Global warming – Problem with environmental and economical impactsSHIVANI M. RAI

104-107

Guidance for Authors

Aims and Scope Nusantara Bioscience (Nus Biosci) is an officialpublication of the Society for Indonesian Biodiversity (SIB). The journalencourages submission of manuscripts dealing with all aspects ofbiological sciences that emphasize issues germane to biological and natureconservation, including agriculture, animal science, biochemistry andpharmacology, biomedical science, ecology and environmental science,ethnobiology, genetics and evolutionary biology, hydrobiology, micro-biology, molecular biology, physiology, and plant science. Manuscriptswith relevance to conservation that transcend the particular ecosystem,species, genetic, or situation described will be prioritized for publication.

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Journal:Saharjo BH, Nurhayati AD. 2006. Domination and composition structure

change at hemic peat natural regeneration following burning; a casestudy in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

The usage of “et al.” in long author lists will also be accepted:Smith J, Jones M Jr, Houghton L et al. 1999. Future of health insurance. N

Engl J Med 965: 325–329

Article by DOI:Slifka MK, Whitton JL. 2000. Clinical implications of dysregulated

cytokine production. J Mol Med. Doi:10.1007/s001090000086

Book:Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam.

Book Chapter:Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization,

biogeography, and the phylogenetic structure of rainforest treecommunities. In: Carson W, Schnitzer S (eds) Tropical forestcommunity ecology. Wiley-Blackwell, New York.

Abstract:Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th

annual symposium of the International Association for VegetationScience, Swansea, UK, 23-27 July 2007.

Proceeding:Alikodra HS. 2000. Biodiversity for development of local autonomous

government. In: Setyawan AD, Sutarno (eds) Toward mount Lawunational park; proceeding of national seminary and workshop onbiodiversity conservation to protect and save germplasm in Javaisland. Sebelas Maret University, Surakarta, 17-20 July 2000.[Indonesia]

Thesis, Dissertation:Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping

plants productivity in agroforestry system based on sengon.[Dissertation]. Brawijaya University, Malang. [Indonesia]

Online document:Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH,

Qu ak e SR , You L. 2 0 0 8 . A s yn th e t i c Esch er i ch ia co l ip red a to r -p rey ec os ys t em. Mol S ys t B i o l 4 : 1 8 7 .www.molecularsystemsbiology.com

Tables should be numbered consecutively and accompanied by a title atthe top. Illustrations Do not use figures that duplicate matter in tables.Figures can be supplied in digital format, or photographs and drawings,which can be ready for reproduction. Label each figure with figurenumber consecutively.

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NOTIFICATION: All communications are strongly recommended to be undertaken through email.


51-56

Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treatedwith some bioregulatorsIMAN M. TALAAT, HEMMAT I. KHATTAB, AISHA M. AHMED

57-64

Effect of water regime on the growth, flower yield, essential oil and proline contents ofCalendula officinalisSAMI ALI METWALLY, KHALID ALI KHALID, BEDOUR H. ABOU-LEILA

65-69

Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus)grown in EgyptMOHAMED M. IBRAHIM, KHALID A. KHALID

70-74

Experimental effect of temperature and sedimentation on bleaching of the two Red Sea coralsStylophora pistillata and Acropora humilisMOHAMMED S.A. AMMAR, AHMED H. OBUID-ALLAH, MONTASER A.M. AL-HAMMADY

75-85

Review: Physical, physical chemistries, chemical and sensorial characteristics of the severalfruits and vegetables chips produced by low-temperature of vacuum frying machineAHMAD DWI SETYAWAN, SUGIYARTO, SOLICHATUN, ARI SUSILOWATI

86-103


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51-56


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Nusantara Bioscience vol. 5, no. 2, November 2013

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