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Romanian Journal of Food Science

Official Journal of the Romanian Association of Food Professionals

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Romanian Journal of Food Science 2011, 1(1): 7480 74

Content evaluation of some vegetal pigments of wheat shoots obtained by germination consecutive to zinc treatment Camelia MOLDOVAN 1,*, Eugeniu CRINICEANU 2, Nicoleta-Gabriela HDRUG 1, Delia-Gabriela DUMBRAV 1, Mrioara DRUG 1, Mirela POPA 1, Mihai DRUG 1 and Bogdan RDOI 1 University of Agricultural Sciences and Veterinary Medicine of Banat, 119A Calea Aradului, 300645 Timioara, Romania 1 Faculty of Food Processing Technology; 2 Faculty of Veterinary Medicine Received 14 October 2011; received in revised form 17 November 2011; accepted 15 December 2011

Abstract

There are many experiments of zinc effect on wheat final leaf or wheat germination percent. Soil constraints affect potential grain yield of wheat. Among these constraints, acidic soils are especially important due to their combined effect on zinc. The originality of the present study consists of the evaluation of the response of wheat shoots (not final leaf) obtained by grain germination consecutive to zinc feeding, as acetate or sulphate on textile support (not soil). The content quantification of chlorophyll, carotenes and xanthophylls of wheat shoots obtained in the presence of zinc has demonstrated the requirements and benefits of that at 50 ppm doses. Zinc effects at 100 ppm have not so efficiently raised the content of these pigments. Therefore, zinc sulphate proved to be more stimulant for pigments synthesis, in comparison with zinc acetate.

Keywords: wheat shoots, zinc, chlorophyll, carotene, xanthophylls.

1. Introduction

It is well-known that strong processed and refined food intake has negative effects on health. The awareness of the public over this fact is growing and slowly leads to a general change in the attitude of the average consumer. The recent tendency is to reduce the consumption of this type of food and upsurge the vegetable diets, especially those based on biological agriculture.

Since ancient times, the beneficial effects of germinated plants and their shoots have been known.

By germination, the plant activates some enzyme systems, particularly important for the proper * Corresponding author: Tel.: +40 256 277.455 E-mail address: kmimol@gmail.com

unfolding of the biochemical reaction in the human body: the proteins transforming in aminoacids, the fats leading to essential acids. Proteins, vitamins, enzymes and minerals can multiply up to 1200%.

In winter time, when the offer of green vegeta-bles is poor or obtained by greenhouse production, our body is deprived of numerous vitamins, enzymes and minerals. An alternative to this shortcoming would be to harvest homemade shoots by seeding grain and consuming them such as salad or green juice rich in chlorophyll, carotenes and xantho-phylls. Plants like wheatgrass or wheat fresh shoots have high content of chlorophyll. Vegetal pigments synthesis starts with seeds sprout, which uses water, minerals from earth and light to create life. Chlorophyll-rich plants have a high percentage of active enzymes and B vitamins (Walters, 2009).

Content evaluation of some vegetal pigments of wheat shoots obtained by germination consecutive to zinc treatment

Romanian Journal of Food Science 2011, 1(1): 7480 75

Chlorophyll is considered to be the life fluid, having also a chemical structure similar to heme (from haemoglobin). Its absorption by animal body is instant; it provides brain, organs and blood with an important amount of oxygen, helping them to function at optimum capacity.

The role of the chlorophyll is undoubtedly positive for the human consumer, being very efficient in chronic dysfunctions, possessing characteristics of formation and rejuvenation of erythrocytes. It can prevent the multiplication of pathogenic bacteria and helps to remove toxins from the body, to fortify the immune system (Lai et al., 1978; Walters, 2009) and protects against harmful action of electromagnetic radiation, microwave and nuclear waste.

Because chlorophyll is destroyed above 40C, raw food with high chlorophyll content is important for human nutrition (as various salads, green smoothies or juice).

Carotenes and xanthophylls (hydroxicarotenoids, aldehydic and cetonic carotenoids) in green plants, not only the chlorophyll, are particularly important for animal nutrition (they are involved in the process of seeing (Dumbrava, 2007), growing, reproduction (Dumbrava, 2007); they have anticancer effects (Lai et al., 1978; Walters, 2009) and antioxidant effects (Dumbrava, 2005, 2007, Moldovan et al., 2010); they are regulators of the immune response system (Ben-Arye et al., 2002, Dumbrava, 2005); they have special efficiency in repair of damage tissues (Dumbrava, 2005, Dumbrava et al., 2007; and wound healing (Dumbrava, 2005, Dumbrava et al., 2007, Walters, 2009), they are skin photo-protectors (Dumbrava et al., 2007), etc.

Due to their properties, these natural pigments are frequently used in various domains (medicine, livestock, agriculture, pharmacy, food industry, cosmetics) (Dumbrava, 2005, Moldovan et al., 2010).

The specialty literature reveals that healthy plants develop only on soils with optimum minerals content. One of these minerals is zinc essential for vegetal and animal organisms (Hacisalihoglu et al., 2003).

The effects of zinc have been studied mainly for its toxicity, deficiency or essentiality in mature wheat (Salama et al., 2002; Cakmak, 2008, Genc et al., 2009, Peleg et al., 2009). There were also studied the effects of zinc on germination percentage of wheat (Stankovi et al., 2010). The scarcity of studies on how to enrich the biologically active substances in wheat seedlings led to the necessity of

this study. Thus, the aim of this study is the quantification of chlorophyll, xanthophyll and carotene in wheat shoots obtained through grain germination consecutive to zinc feeding as acetate or sulphate. 2. Materials and methods

To germinate the wheat seeds a textile support was used. The seeds were placed and the zinc solutions were added to this support. The study of the germination action used distilled water and some zinc solutions: ZnSO4 (50 respectively 100 ppm Zn), Zn (CH3COO)2 (50 respectively 100 ppm Zn), thus forming 5 groups: group 1 (WhDW) distilled water; group 2 (WhSU50) ZnSO4 solution with

50 ppm Zn; group 3 (WhSU100) ZnSO4 solution with

100 ppm Zn; group 4 (WhAC50) Zn(CH3COO)2 solution with

50 ppm Zn; group 5 (WhAC100) Zn(CH3COO)2 solution with

100 ppm Zn; The germination phases were monitored during

all the germination periods. The addition of these solutions was made at every 24 h, in equal quantities for all groups.

When the shoots have approximately 1015 cm, they are harvested in order to evaluate the chlorophyll, carotenes and xanthophylls content. a. Chlorophyll content evaluation

Despite the fact that the zinc role in plant development is well-known, its influence on the germination process has been less studied.

To determine the chlorophyll content in wheat green shoots, they are triturated with quartz sand in the presence of acetone 80%. The homogenate obtained was centrifuged at 3000 rot/min, and the supernatant was collected in a glass bottle. The process was repeated by adding acetone until the extract was transparent. The supernatants collected were finally joined and their absorbance measured at 646 nm, respectively 663 nm using the UV-VIS Perkin Elmer spectrophotometer. The obtained extract with acetone was spectrophotometrically compared with a control sample (reference acetone 80%) at both wavelengths.

Albeit the literature contains many calculus formulas for chlorophyll quantification (Sims & Gamon, 2002; Porra, 2002; Brezeanu, 2005; Saupe,

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Romanian Journal of Food Science 2011, 1(1): 7480 76

2009), the equations presented by Ianculov et al. (2005) have been used here due to their simplicity and reliability. Therefore, the chlorophyll concentration was calculated using formulas:

Chl a = 12.21A663 - 2.81A646 (1)

Chl b = 20.13A646 - 5.03A663 (2)

Chltotal = 17.32A646 + 7.18A663 (3)

where: Chl a is chlorophyll a, in mg/L Chl b chlorophyll b, in mg/L Chltotal total chlorophyll content, in mg/L A663 sample absorbance at 663 nm A646 sample absorbance at 646 nm (Ianculov et al., 2005). b. Carotenes and xanthophylls content evaluation

To determine the carotenes and xanthophylls content, the absorbance of the same samples obtained as result of the above-mentioned method (chlorophyll dosing) was measured at 470 nm wavelength. Then the carotenes and xanthophylls content was calculated using the following formula (Brezeanu, 2005):

22904.127.31000 470 bChlaChlAXC

=+ (4) where: C is the carotenes content, in mg/L; X xanthophylls content, in mg/L; A470 ample absorbance at 470 nm.

To calculate only the carotenes content, Lichtenthaler & Welburn relation was used, quoted by Porra (2002):

227

04.127.31000 470 bChlaChlAC= (5)

3. Results and discussion

Visible spectra of the acetone extracts obtained from wheat shoots consecutive to zinc treatment are presented in Figure 1.

An increasing content of chlorophyll a and b in groups treated with 50 ppm zinc (WhSU50 and WhAC50) was observed for both acetate and sulphate compared with the control group (WhDW), which was treated only with distilled water (see Figures 2 and 3). Similar results were obtained by Salama et al. (2002), who found that zinc treatment of chickpea and maize plants deficient in zinc increased the content of chlorophylls.

Figure 1. VIS-curves of wheat extracts germinated with zinc addition (WhDW control sample with distilled water; WhSU50 ZnSO4 with 50 ppm Zn; WhSU100 ZnSO4 with 100 ppm Zn; WhAC50 Zn(CH3COO)2 with 50 ppm Zn; WhAC100 Zn(CH3COO)2 with 100 ppm Zn; Whpw wheat treated with

distilled water whose extract was colorimetered without being diluted).

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Content evaluation of some vegetal pigments of wheat shoots obtained by germination consecutive to zinc treatment

Romanian Journal of Food Science 2011, 1(1): 7480 77

The groups which are treated with 100 ppm zinc (WhSU100 and WhAC100) have less content of chlorophyll a and total chlorophyll than the control group (Whdw). Sagaroy et al. (2009) found similar results after zinc treatment of sugar beet (Beta vulgaris L.) with 100-300 ppm Zn. The treatment of plants under 100 ppm zinc has a negative effect on the chlorophyll content.

The highest amount of chlorophyll a 2.44 mg/L was determined in group WhSU50 treated with 50 ppm zinc as sulphate. Similar, the highest amount of chlorophyll b was registered in group WhAC50 treated with zinc acetate with 50 ppm Zn.

Total chlorophyll amount (Figures 2 and 3) was correlated with the chlorophyll a content (it has the highest percentage in the calculating relation).

Zinc treatment of wheat shoots (during their germination process) had manifested beneficial effects on them in terms of carotenes and xanthophylls content. Thus, zinc solutions (as sulphate or acetate) led to increases of carotenes and xanthophylls content in experimental groups WhSU50 6.288 mg/L, WhSU100 6.051 mg/L, WhAC50 5.286 mg/L respectively WhAC100 3.443 mg/L comparing to control group WhDW 1.568 mg/L (Figures 4 and 5).

0

0,5

1

1,5

2

2,5

3

chlorophyll a (mg/L) total chlorophyll (mg/L)

Wh DW

Wh SU50

Wh SU100

Wh AC50

Wh AC100

Figure 2. Chlorophyll a and total chlorophyll content of wheat shoots obtained

by germination consecutive to zinc treatment.

0

0,005

0,01

0,015

0,02

0,025

0,03

chlorophyll b (mg/L)

Wh DWWh SU50Wh SU100Wh AC50Wh AC100

Figure 3. Chlorophyll b content of wheat shoots obtained

by germination consecutive to zinc treatment.

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Romanian Journal of Food Science 2011, 1(1): 7480 78

However, high Zn doses led to decrease in carotene and xanthophyll content. These results are directly correlated with Sagardoys (2009) data, who reported that leaves of sugar beet plants treated with 50 and 100 ppm Zn developed symptoms of Fe deficiency, including decreases in Fe, chlorophyll and carotenoid concentrations. The same results were obtained by Shi and Cai (2009) on peanut leaves after zinc stress induced by foliar treatment with 200, 500 and 1000 ppm Zn as ZnSO4.

The treatment of wheat with zinc acetate led to lower levels of carotenes and xanthophylls than treatment with zinc sulphate for the same zinc doses.

Compared with the control group, zinc treatment of wheat has a much higher content of carotenes and xanthophylls.

According to Lichtenthaler i Welburn (1983), the carotenes content is dominant, the xanthophylls content being a little percent of the sum of these (Figure 5).

0

0,01

0,02

0,03

0,04

0,05

0,06

Xanthophylls, mg/L

Wh DW

Wh SU50

Wh SU100

Wh AC50

Wh AC100

Figure 4. Xanthophylls content of the wheat shoots obtained

by germination consecutive to zinc treatment.

0

1

2

3

4

5

6

7

carotenes, mg/L carotenes and xanthophylls, mg/L

Wh DW

Wh SU50

Wh SU100

Wh AC50

Wh AC100

Figure 5. Xanthophylls and carotene content of the wheat shoots obtained

by germination consecutive to zinc treatment.

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Content evaluation of some vegetal pigments of wheat shoots obtained by germination consecutive to zinc treatment

Romanian Journal of Food Science 2011, 1(1): 7480 79

4. Conclusions

Zinc addition in doses of 50 ppm in germination solutions leads to a major increase in these three vegetal pigments (chlorophyll, carotenes and xanthophylls).

The 100 ppm Zn doses negatively affected the chlorophyll pigments synthesis, the amount of these being less than that of the control group. One explanation of this fact would be the onset of oxidative stress caused by too high doses of zinc, which can be toxic for wheat shoots.

The registered amount of carotenes and xanthophylls had higher values in comparison with the control group, for both applied doses (50 respectively 100 ppm Zn) or zinc solution (acetate or sulphate).

These results showed that, by applying 50 ppm of zinc, the obtained amount of carotenes and xanthophylls was higher than the dose of 100 ppm of zinc: WhSU50 - 6,288 mg/L > WhSU100 - 6,051 mg/L respectively WhAC50 - 5,286 mg/L > WhSU100 - 3,443 mg/L.

The solution of zinc sulphate in comparison with zinc acetate was more efficient in increasing the carotenes and xanthophylls synthesis.

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