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206

7. Research Articles Published

1. K.V. Sunooj, K. Radhakrishna, Johnsy George, & A.S. Bawa (2009).Factors

influencing the calorimetric determination of glass transition temperature in

foods: A case study using chicken and mutton Original Research Article, Journal

of Food Engineering, 91, 2, 347-352. (International Publication)

2. Kappat Valiyapeediyekkal Sunooj and Kolpe Radhakrishna (2013). Physico-

chemical changes in ready to eat pineapple chicken curry during frozen storage,

food and nutrition sciences journal, Accepted for publication in February issue.

(International Publication)-in Press

Papers presented

1. K.V. Sunooj, K. Radhakrishna “Effect of moisture content and annealing on glass

transition temperature of chicken” Nineteenth ICFOST on Health foods 31st Dec

2007 to 2nd Jan 2008 at IIT, Kharagpur (poster paper).

2. “Physico-chemical Changes in RTE pineapple chicken curry during frozen

storage” International symposium on emerging and novel food processing technologies,

19-20 Dec2007, DFRL (DRDO), India(poster paper).

3. K.V. Sunooj, K. Radhakrishna “Physico-chemical Changes in RTE mutton curry

during frozen storage International Livestock and Dairy Expo-2008, 22-24 Aug 2008,

New Delhi(poster paper).

Factors influencing the calorimetric determination of glass transition temperaturein foods: A case study using chicken and mutton

K.V. Sunooj *, K. Radhakrishna, Johnsy George, A.S. BawaDefence Food Research Laboratory, Freeze-Drying and Animal Products Technology Division, Siddarthanagar, Karnataka, Mysore 570 011, India

a r t i c l e i n f o

Article history:Received 16 April 2008Received in revised form 16 September2008Accepted 20 September 2008Available online 27 September 2008

Keywords:ChickenGlass transitionDifferential scanning calorimetryThermo gravimetric analysisAnnealing

a b s t r a c t

Glass transition temperature (Tg) of complex food systems like chicken and mutton depends on severalfactors. Hence the reported values of Tg for these food systems varied from sample to sample. Presentstudy was an attempt to investigate various parameters such as annealing temperature and timeemployed, moisture content of the sample, rate of heating during DSC scan etc. affecting the calorimetricdetermination of Tg. Varying one parameter at a time and by keeping all others constant, the influence ofthese parameters on Tg, were investigated. As annealing temperature decreased from �11 �C to �23 �C,Tg values for chicken and mutton shifted towards lower temperatures. Similarly annealing time up to 1 halso significantly influenced Tg values. Initial moisture content of the meat samples was also found toinfluence Tg values. Rate of heating employed during the DSC analysis, played a vital role in the Tg deter-mination. This study delineated the effect of these parameters on glass transition temperature and helpedin optimizing DSC protocol for calorimetric determination of Tg for chicken and mutton samples.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Chicken and mutton are consumed for their taste and nutritivevalue, and their demand is ever increasing through out the world.However, the safety of meat is of major concern to its manufac-tures as well as end users and hence strict quality control protocolsare required during in the processing and preservation. Freezing isone of the most reliable and easy methods for preserving meat(Goff, 1995). However the stability of frozen meat depends onthe state of water in the product and the stability of ice crystalsduring frozen storage (Goff and Sahagian, 1996). Hence mecha-nisms that govern the formation of glassy state and glass transitionconcepts are very much relevant during frozen storage (Rahman,1999). The concept of water activity alone is insufficient to predictshelf stability of frozen foods. The alternate complimentaryideas like storing meat below Tg, may help to improve the shelfstability.

Glass transition (Tg) is a second order thermodynamic transi-tion, helpful in elucidating the time–temperature related proper-ties as well as the state of water during freezing and storage.Molecular mobility of glassy materials is significantly reduced be-low Tg. This in turn delays various deteriorative changes such astexture loss, enzymatic spoilage, flavour loss etc. in foods duringstorage (Mitchell, 1998). Levine and Slade (1986) and Slade and

Levine (1988) postulated that Tg influences the stability of foods.Below Tg, water in the concentrated phases becomes kineticallyimmobilized and therefore does not support or participate in dete-riorative reactions. Studies indicated that the concept of Tg shouldbe added along with the existing concept of water activity, to get abetter understanding about the factors governing the stability offoods (Rahman et al., 2005; Sablani et al., 2007a,b). Rahman(2006) combined the glass transition and water activity conceptsin the state diagram in order to determine the stability of foods.

The most commonly used method to determine Tg of foods is byusing differential scanning calorimeter (DSC), which detects thechange in heat capacity, occurring over a range of temperatures.A step change occurring in the thermogram line of heat capacitycurve during heating of food samples can be taken as glass transi-tion temperature. In food samples, the glassy state conditions de-pend on the ice formation during freezing. Therefore the largenumber of variables influencing the freezing of foods can also af-fect the determination of glass transition temperature (Championet al., 2000). In order to determine Tg values effectively, conditionsthat promote maximum ice formation has to be employed. Henceannealing protocols during freezing are extremely important(Brake and Fennema, 1999).

Delgado and Sun (2002) reported the Tg of chicken meat (breastportion) as �16.83 �C using DSC by employing an annealing tem-perature of �20 �C for 1 h. However the Tg value can vary depend-ing on annealing temperature and the duration employed (Brakeand Fennema, 1999; Rahman et al., 2007). Parameters like anneal-ing temperature and time, rate of heating, moisture content,

0260-8774/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2008.09.014

* Corresponding author. Tel.: +91 821 2473390.E-mail address: sunooj4u@gmail.com (K.V. Sunooj).

Journal of Food Engineering 91 (2009) 347–352

Contents lists available at ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

processing conditions etc. have been found to affect Tg values ofvarious food systems like fish muscles, tomato, date flesh, carbohy-drate solutions etc. (Hashimoto et al., 2004; Telis and Sobral, 2002;Rahman, 2004; Ribeiro et al., 2003; Carrington et al., 1994; Goff,1994; Huang et al., 1994; Sahagian and Goff 1994). However re-ports on systematic study on the effect of individual parameterson Tg of food samples are scanty (Sablani et al., 2007a,b). The lackof common DSC protocols used in food systems are a hindrance inthe comparison of Tg values between the samples. Hence the mainobjective of this study was to focus on how various parametersinfluence the Tg determination of chicken and mutton and thusframe a protocol for DSC analysis.

2. Materials and methods

2.1. Sample preparation

Boneless chicken breast meat (class A) was procured from thelocal market; trimmed of any visible fat, chopped with a knife intosmall strips (5 cm length, 3 cm width and 2 cm thickness). Thesestrips were divided into two lots and the first lot was kept frozenuntil analyzed. While the second lot was freeze-dried, packed inAluminium foil based packaging material and kept frozen at�18 �C until analyzed. Before any experiment, the meat sampleswere thawed in a refrigerator at 4 ± 1 �C. Small sample cubesweighing 6–10 mg were carefully cut from the strips and usedfor thermal analysis. The moisture, crude fat, and protein contentswere determined using AOAC, 1984 procedures, (24.002, 24.005and 24.027, respectively).

2.2. Freeze drying

Freeze drying was carried out in a pilot scale freeze dryer, Epsi-lon 1/60 (Martin Christ GmbH & Co KG, Osterode, Germany)equipped with rapid freezing and drying facilities. Samples (Chick-en and mutton) were pre-frozen to �40 ± 2 �C for 4 h and dried un-der variable chamber pressure (100–300 Pa) and temperatures(30–60 �C). After freeze drying, the final moisture content of chick-en and mutton samples was reduced to 2–3%. Vacuum was re-leased and the product taken to low humidity room (23 ± 2%) forpacking. Meat samples were powdered using a mortar and pestlein a humidity controlled room. About 6–10 mg of these sampleswas used for thermal analysis.

2.3. Measurement of Tg by DSC

DSC 2010 differential scanning calorimeter (TA Instruments,New Castle, DE, USA) was used to determine the Tg values. Sampleswere enclosed in hermetically sealed aluminum pans just beforeanalysis and then loaded into the equipment at room temperature.An empty pan was used as reference and nitrogen gas at a flow rateof 60 ml/min was employed in the purge line to control the localenvironment around the sample. The cell was calibrated as perthe DSC manufactures recommendation. It consists of three proce-dures namely thermogram line, cell constant and temperature cal-ibration. The thermogram line calibration involved heating the cellthrough the entire temperature range i.e., from �50 to 20 �C. Thecell constant was determined by heating the standard material (in-dium) through its melting temperature. Temperature calibrationwas performed by using a multiple-point calibration with indium(mp 156.60 �C, DHm 28.71 J/g), distilled water (mp 0 �C, DHm

335 J/g) and heptane (mp �91.0 �C, DHm 140 J/g). In our experi-ments, samples were held at various annealing temperatures like�11 �C, �14 �C, �15 �C, �17 �C, �20 �C, and �23 �C by employingvariable annealing time (0–2 h) followed by cooling to �30 �C.

After holding isothermally for 1 min, the samples were rampedat various heating rates like 0.5,1, 2, 3, 4 and 5 �C/min to 20 �C.DSC measurements were replicated thrice. Experiments were alsoconducted by employing different annealing times like 0, 15, 30,45, 60, and 120 min, while keeping the annealing temperatureand rate of heating constant. Results of the experimental run wereanalyzed with the universal analysis software, provided with theDSC instrument. Mid point of the step change in the heat flowcurve was taken as glass transition temperature. DSC was also em-ployed to determine the freezing behavior of meat samples.

2.4. Thermo gravimetric analysis

Thermo gravimetric analyzer (TGA Q50, TA Instruments, DE,USA), was used in conjunction with a thermal analysis controller.TGA was employed to measure the amount and rate of change inweight of the material either as a function of increasing tempera-ture or time, under a controlled atmosphere. Initial weight of eachsample was approximately 20 mg. Samples were placed in plati-num pans and heated in a furnace flushed with N2 gas at the rateof 40 ml/min and heated from 30 �C to 105 �C at a rate of 10 �C/min and held isothermally for 60 min. In this study, TGA was usedto determine the accurate moisture content in all the samples byplotting percentage weight loss against time.

2.5. Statistical analysis

Statistical analysis was performed with SPSS software (SPSSInc., 1996) and used to test the significant effect of various param-eters on DH and Tg values at 1% level of significance (P < 0.01).

3. Results and discussion

The average chemical composition of chicken and mutton sam-ples has been given in Table 1.

Determination of Tg in food systems depends on the variousparameters employed during DSC scan. Meat samples such aschicken and mutton exhibit Tg at subzero temperatures and it isinfluenced by ice crystal formation in the freezable water present.The present study was aimed at determination of the temperatureat which ice crystal formation takes place in meat samples andhow it varies depending on the annealing conditions employed.Chicken and mutton samples were cooled using DSC from 20 �Cto �30 �C at the rate of 2 �C/min. Fig. 1 shows the freezing curveof the fresh chicken and mutton samples. It indicates that the onsetof enthalpy change corresponding to the ice crystallization startsaround�11 �C and ends at around�23 �C. The enthalpy of ice crys-tallization (DH) was determined by integrating the heat flowcurves, while peak maximum value was taken as the freezingpoint. In order to determine the effect of annealing on DH values,meat samples were annealed at different temperatures, in therange of �11 �C to �23 �C by keeping the annealing time of60 min as constant. The results have been given in Table 2. Statis-tical analysis (at 99% confidence level) showed that annealing tem-perature had a significant effect on the DH values. The DH values(134 ± 1.66 J/g) were found to be the minimum when the sampleswere annealed at �17 �C for chicken. This implies that maximum

Table 1Average chemical composition of chicken and mutton

Composition (%, w/w) Chicken Mutton

Moisture 71.4 ± 1.06 64.26 ± 1.23Protein 26.04 ± 0.49 29.2 ± 0.39Fat 2.01 ± 0.12 6.12 ± 0.22

Values are mean ± standard deviation.

348 K.V. Sunooj et al. / Journal of Food Engineering 91 (2009) 347–352

freeze concentration can be achieved by employing the sameannealing temperature. This observation is in agreement with thereports of Brake and Fennema (1999), who suggested that the mosteffective annealing temperature employed should be near to the Tgof the food samples. The degree to which maximum freeze concen-tration (Levine and Slade, 1986) can be achieved in meat samples isinfluenced by exposure time at a particular temperature. Hencesufficiently long holding time (60 min) was employed as reportedby Delgado and Sun (2002) at various annealing temperatures, soas to attain maximum freeze concentration. Storage of samples atannealing temperatures for long durations leads to spontaneousice crystallization, which in turn reduces the enthalpy content. Inthe case of mutton, annealing at �15 �C for 60 min gave a mini-mum DH value of 164 ± 2.23 J/g was observed.

3.1. Effect of annealing on glass transition

The effect of annealing on the Tg of chicken and mutton sampleshas been studied by employing various annealing temperaturesand time. Figs. 2 and 3 depict the effect of annealing temperatureon Tg values. As the annealing temperature was decreased from�11 �C to �23 �C, Tg values shifted towards lower temperatures.When annealing temperatures for chicken and mutton sampleswere around �17 �C and �15 �C, respectively, step changes occur-ring to heat flow curves were more prominent. This resulted in aneasy determination of Tg. Similarly, various annealing times like 0,15, 30, 45, 60, and 120 min were also employed by keeping theannealing temperature constant. The effect of annealing time on

Tg values of fresh chicken samples has been shown in Fig. 4. It isevident that as the annealing time increases Tg becomes more vis-ible and the values shift towards lower temperatures. Table 2 givesthe effect of annealing time on Tg values of fresh and freeze-driedchicken and mutton samples. DSC studies revealed that the Tg wasnot traceable without sufficient annealing time and as the anneal-ing time increased, the likelihood of detecting Tg also increased.Statistical analysis (P < 0.01) showed that the annealing time also

(a)

(b)

-10

0

10

20

30

40

Hea

t Flo

w (m

W)

-30 -10 10

Temperature (°C)Exo Up Universal V3.0G TA Instruments

Fig. 1. Freezing curves of fresh (a) chicken and (b) mutton.

Table 2Summary of enthalpy change and freezing point of chicken and mutton at variousannealing temperatures

Annealingtemperature (�C)

Chicken Mutton

DH (J/g) Freezingpointa (�C)

DH (J/g) Freezingpointa (�C)

�11 145 ± 1.61* �17.19 ± 0.21* 170 ± 1.38* �12.12 ± 0.13*

�14 143 ± 1.31* �16.86 ± 0.14* 167 ± 1.49* �11.83 ± 0.72*

�15 140 ± 1.98* �16.88 ± 0.86* 164 ± 2.23* �9.12 ± 0.36*

�16 137 ± 1.88* �16.89 ± 0.53* 168 ± 1.33* �9.77 ± 0.28*

�17 134 ± 1.66* �16.93 ± 0.15* 171 ± 1.28* �9.32 ± 0.53*

�18 136 ± 1.34* �15.63 ± 0.41* 175 ± 1.39* �9.48 ± 0.45*

�19 139 ± 1.21* �12.43 ± 0.32* 174 ± 1.44* �9.78 ± 0.91*

�20 148 ± 1.45* �11.70 ± 0.61* 175 ± 1.67* �10.92 ± 0.71*

�23 157 ± 1.48* �11.83 ± 0.71* 176 ± 1.53* �10.01 ± 0.27*

Values are mean ± standard deviation.* Values are statistically significant at P < 0.01.a Peak maximum values.

-16.63 C

(a)

(b)(c)

(d)(e)

-9.98 C-13.02 C

-17.12 C-18.25 C

-32

-24

-16

-8

0

Hea

t Flo

w (m

W)

-35 -25 -15 -5 5 15

Temperature (°C)Exo Up Universal V3.0G TA Instruments

Fig. 2. Effect of annealing temperature on ((a) �11 �C, (b) �14 �C, (c) �17 �C, (d)�20 �C, and (e) �23 �C) Tg of fresh chicken.

-12.91 C

-19.34 C-17.66 C

(d)

-10.84 C

-8.07 C(a)

(e)

(b)(c)

-24

-16

-8

0

Hea

t Flo

w (m

W)

-35 -25 -15 -5 5 15

Temperature (°C)Exo Up Universal V3.0G TA Instruments

Fig. 3. Effect of annealing temperature on ((a) �11 �C, (b) �14 �C, (c) �15 �C,(d) �17 �C, and (e) �20 �C) Tg of fresh mutton.

(e)(f)

(a)(b)(c)

Tg

(d)

-20

-10

0

Hea

t Flo

w (m

W)

-30 -10 10Temperature (°C)Exo Up

Fig. 4. Effect of annealing time ((a) 0 min, (b) 15 min, (c) 30 min, (d) 45 min,(e) 60 min, and (f) 120 min) on glass transition temperature.

K.V. Sunooj et al. / Journal of Food Engineering 91 (2009) 347–352 349

had a significant effect on Tg values up to 1 h. Hence the optimumannealing conditions were employed for fresh chicken and muttonsamples before the measurement of Tg. Conditions employed were�17 �C for 1 h and �15 �C for 1 h for the fresh chicken and mutton,respectively. Tg values for the samples were �16.63 �C and�12.97 �C, respectively. Freeze-dried chicken and mutton sampleswere also subjected to DSC analysis at different annealing temper-atures (Figs. 5 and 6) and annealing time (Table 3). Interestingly Tgvalues shifted to higher temperatures like �13.21 �C and �9.24 �Cin case of freeze-dried chicken and mutton, suggesting that themoisture may have played a vital role in determination of Tg.

3.2. Effect of moisture content on glass transition

Effect of moisture content on Tg was determined by conductingDSC scans for freeze-dried samples with different moisture levels.In order to vary the moisture content of chicken and mutton, thesamples were drawn at regular intervals from the freeze-drier.The moisture content of the samples was approximately 10%,20%, 30%, 40%, 50%, and 60%. Actual moisture content of these sam-ples was determined by thermo gravimetrically. Figs. 7 and 8 de-pict thermograms of the chicken and mutton samples drawn atvarious time intervals. It is clear from the figures that fresh chickenand mutton samples had moisture content of 71.74% and 64.26%,respectively, while those in case of freeze-dried samples were2.376% and 2.78%. While conducting DSC scans for all the samples,other parameters like annealing temperature and time, rate ofheating etc. were kept constant in order to keep the moisture con-tent as the only variable. Fig. 9 depicts the variation of glass tran-sition temperatures for the samples with different moisturecontents. In both chicken and mutton samples, Tg values werefound to decrease with increased moisture content. A similar resulthas been earlier reported during thermal analysis of tomatoes (Tel-is and Sobral, 2002). Generally water acts as a plasticizer and canthus affect the Tg of food samples. Water is a mobility enhancer,which leads to large increase in free volume and decreased viscos-ity. Slade and Levine (1991) reported that the direct plasticizing ef-fect of increased moisture content leads to increased segmentalmobility of chains in amorphous region of glassy and partially crys-talline systems and tends to reduce the Tg value. In the present

(a)-7.18 C

(b)-10.25 C

(c)-13.21 C

(d)

-13.37 C(e)

-17.75 C

-5.0

-2.5

0.0

2.5

5.0

Hea

t Flo

w (m

W)

-30 -15 0 15Temperature (°C)Exo Up

Fig. 5. Effect of annealing temperature on ((a) �11 �C, (b) �14 �C, (c) �17 �C,(d) �20 �C, and (e) �23 �C) Tg values of freeze-dried chicken.

(a)

-6.62 C-7.51 C

(b)

-13.36 C

(e) -12.18 C

(d) -9.24 C

(c)

-0.03

0.01

0.05

0.09

Hea

t Flo

w (m

W)

-25 -20 -15 -10 -5

Temperature (°C)Exo Up

Fig. 6. Effect of annealing temperature on ((a) �11 �C, (b) �14 �C, (c) �15 �C,(d) �17 �C, and (e) �20 �C) Tg of freeze-dried mutton.

Table 3Effect of annealing time on the glass transition temperature of fresh and freeze-dried(FD) chicken and mutton sample

Annealingtime (min)

Tg values (�C)

Fresh chicken FD chicken Fresh mutton FD mutton

0 Not traceable Not traceable Not traceable Not traceable15 �13.33 ± 0.03* �10.52 ± 0.06* �10.49 ± 0.05* �7.83 ± 0.07*

30 �14.31 ± 0.07* �11.47 ± 0.07* �11.03 ± 0.03* �8.02 ± 0.06*

45 �15.79 ± 0.04* �12.66 ± 0.04* �11.42 ± 0.06* �8.73 ± 0.02*

60 �16.63 ± 0.01* �13.21 ± 0.03* �12.91 ± 0.04* �9.24 ± 0.01*

120 �16.72 ± 0.2 �13.23 ± 0.02 �12.93 ± 0.03 �9.25 ± 0.02

Values are mean ± standard deviation.* Values are statistically significant at P < 0.01.

(h) 71.74%

(a)

(g)59.97%

(e)

(c)

(b)

(d) 30.79%

40.11%(f) 50.46%

0

20

40

60

80

100

Wei

ght (

%)

10 20 30 40 50 60 70

Time (min) Universal V4.1D TA Instruments

2.376%9.623%

20.28%

Fig. 7. TGA analysis of fresh and freeze-dried chicken samples drawn at differenttime intervals.

50.65 deg C59.38 deg C

39.72 deg C

2.77 deg C(b)10.34 deg C(c) 20.43 deg C

(d)(e)

(f)(g)

30.22 deg C

(h).64.26 deg C

20

40

60

80

100

120

Wei

ght (

%)

2 12 22 32 42 52 62 72

Time (min)Universal V4.1D TA Instruments

(a)

Fig. 8. TGA analysis of fresh and freeze-dried mutton samples drawn at differenttime intervals.

350 K.V. Sunooj et al. / Journal of Food Engineering 91 (2009) 347–352

study, the Tg values were also found to be influenced by the initialmoisture content of chicken and mutton samples.

3.3. Effect of rate of heating on glass transition

Since the rate of heating is also a significant factor affecting thecalorimetric determination of Tg (Rahman, 2006), six differentheating rates viz; 0.5, 1, 2, 3, 4, and 5 �C/min were employed duringDSC scan. Fig. 10 shows the effect of rate of heating on Tg values forfresh as well as freeze-dried chicken and mutton samples. As therate of heating increased, Tg values were also showed a linear in-crease. Rahman et al. (2007) have also reported a similar trend,while determining the Tg of spaghetti. In case of freeze-dried sam-ples, Tg values changed marginally due to reduced moisture con-tent. As rate of heating increases, the duration of exposure to aparticular temperature decreases. So at lower heating rates(Fig. 10) the characteristic step change in the heat flow curvesoccured at lower temperatures leading to lower Tg values. As therate of heating decreases, this step change becomes more visibleand thus any rate of heating at 2 �C/min or below can be recom-mended for an easy and accurate detection of Tg.

4. Conclusions

Various factors influencing the calorimetric determination ofTg in chicken and mutton samples were investigated. Freezing

behaviors of these samples were studied by employing differentannealing temperatures and time. Proper selection of annealingconditions is very critical in the determination of Tg. The presentstudy revealed that an annealing temperature of �17 �C and anannealing time of 1 h were the best suited for chicken samples,as it helped to attain the maximum freeze concentration at re-duced time interval. While in case of mutton, annealing condi-tions like �15 �C for 1 h gave similar results. The effect ofmoisture content as well as the rate of heating on Tg was alsodetermined. The rate of heating like 2 �C/min or below was foundto be acceptable, while moisture content had to be estimated cor-rectly before reporting the Tg values. The various parametersinfluencing Tg determination were identified and it helped inframing a DSC protocol for determining Tg in the case of meatsamples.

Acknowledgements

The authors are grateful to Dr. V.A. Sajeevkumar and K.R. Vijay,Scientists, Defence Food Research Laboratory, Mysore, for theirvaluable guidance and help rendered during the study.

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-20

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Tg (D

eg C

)

Chicken

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-7.5

-5

0 20 40 60 800 20 40 60 80

Moisture content (Wt %)Moisture content (Wt %)

Tg (D

eg C

)

Muttona b

Fig. 9. Effect of moisture content on Tg of (a) chicken and (b) mutton.

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eg C

)

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fresh mutton

a b

Fig. 10. Effect of rate of heating on Tg of (a) chicken and (b) mutton.

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Food and Nutrition Sciences, 2013, 4, ***-*** Published Online February 2013 (http://www.scirp.org/journal/fns)

Physico-Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage

Kappat Valiyapeediyekkal Sunooj1, Kolpe Radhakrishna2

1Department of Food Science & Technology, Pondicherry University, Puducherry, India; 2Defence Food Research Laboratory, Freeze-Drying and Animal Products Technology Division, Mysore, India. Email: sunooj4u@gmail.com Received *************** 2013

ABSTRACT

RTE Pineapple chicken curry, a traditional Kerala recipe, was prepared and standardized by using de-boned broiler meat chunks, pineapple and spices. The product having both meat and gravy (1:1.9) was packed in polyethylene pouches and stored at −18˚C ± 2˚C for 6 months. During frozen storage, the free fatty acid (FFA) values were 0.28 - 0.46 (as percentage oleic acid) and thiobarbituric acid (TBA) values were 1.68 - 2.45 (mg of malonaldehyde/Kg of sample) increased in both meat and gravy. Meat and gravy pH were in the range of 5.5 to 6. Marginal decrease in shear force values (43.4 - 39.6 N) were also observed. During storage the SPC was found to be decreasing over period of stor- age (100, 40, 20, <10 respectively). Psychrophiles were within acceptable limit and pathogens were absent. Microbi- ological data showed that the product was microbiologically safe. The sensory score indicated that the RTE pineapple chicken curry is acceptable after storage at −18˚C ± 2˚C for 6 months. Keywords: Chicken Curry; Pineapple; Frozen Storage; GC

1. Introduction

Globally several chicken recipes are very popular. Indian traditional meat based recipes are well known for its delicious and appetizing taste worldwide, but these foods require number of unit operations and long preparation time [1]. In order to minimize the tedious operations involved in preparation of food and to save time in- cluding all the required nutrition and to fulfill satiety of consumer especially for working population there is a need to develop several products that involve minimum processing. Ready to eat product has a budding industry in India and overseas, since needs of population is changing with time. Quality and safety of food is a major concern of both consumer and manufacturer that is in- fluenced by several factors.

Indians have a variety of meat preparations that involves profuse use of spices that adds to the flavor as well as imparts antioxidant, antibacterial and various other functional roles. There are a number of products that vary in terms of preparation, kinds of meat, spices etc. Selection of suitable packaging material has been a great area of interest in order to maintain the shelf life and produce better products. Foods that have a short shelf life are processed for longer shelf life in variety of packaging materials such flexible films, multilayer packages, glass, cans etc. Canned products are available

in market but has several limitations such as metal imparts an undesirable taste to the product during storage, is an expensive method and prone to a high incidence of leakage through seams [2]. Similarly retort pouches also need an additional processing before storage that adds to the cost but has an advantage that can be stored at room temperature for several months. Hence in the present study polythene pouches were used for packaging the product that were of low cost and also maintained the quality of food at frozen storage without any damage to the packaging material that usually is seen with other polymer materials such as polypropylene. Anon [3] suggested that curry products can be frozen and marketed in polythene pouches. Changes in quality during chilled or frozen storage have been studied in cod and haddock fillets [3], muscle of volador [4], buffalo meat burgers [5], chicken nuggets [6,7] etc.

Kerala is known for its heavily spiced non vegetarian foods and pineapple chicken curry is one of the tradi-tional recipes. A traditional recipe, pineapple chicken curry was prepared and packed in polyethylene bags and changes in its physico-chemical quality during frozen (−18˚C ± 2˚C) storage (for 6 months) were studied. An attempt was made in the present study to bring forth a standardized recipe for the pineapple chicken curry and also to cater the needs of working population and single

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Physico- Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage 2

person staying alone. These days a number of RTE are already available in market but still there is a need of proper marketing and awareness among the consumer for judicial utilization of upcoming technology and im- proved products so that both the consumer and the manufacturer can be benefited. Changes in the physico- chemical quality of product were also determined in the course of technological processes of freezing and during storage of frozen products.

2. Materials and Methods

2.1. Preparation of RTE Pineapple Chicken Curry

Broiler chicken of 6 - 8 weeks age were procured from the local market, dressed conventionally and were brought to the FD-APT division lab, Mysore, India. On the day of preparation, the carcasses (1.10 to 1.20 kg) were washed under running tap water and deboned. Breast and leg muscles were cut into cubes of 2 - 3 cm and were marinated in curd for 1 - 2 hours. This was cooked for 10 - 15 min at 95˚C - 100˚C and cooled to 30˚C - 40˚C. The ingredient composition for the prepara- tion of RTE pineapple chicken curry is given in Table 1. Cooking oil was heated in a stainless steel vessel to 110˚C - 120˚C, added clove, cinnamon and cardamom and roasted for 1 - 2 minutes. Sliced onion, ginger, garlic, coriander leaves and green chillies were added and sau- téd till light golden colour. Then added tomato puree, red chilli powder, coriander powder, turmeric powder, salt and pineapple cubes and cooked for 4 min on low flame. To this gravy mix, added cooked chicken cubes along with cooked out juice and mixed well. This was further heated for 5 min at 85˚C - 90˚C. The product cooled to 30˚C - 40˚C for 40 - 50 min and separated the meat chunks from gravy.

The product was packaged in polythene pouches (300 gauge) with 100 g meat and 250 g gravy in each pouch. The sealed packets placed in wax coated carton and fro- zen in a plate freezer until the product temperature reached −45˚C to −60˚C (115 - 125 min). A digital tem- perature recorder with metallic probes (Aptec, Chennai, India) was used to ensure adequate temperature decline of the product. The frozen product was then stored at a freezer maintained at −18˚C ± 2˚C.

2.2. Quality Evaluation

Physical, chemical and microbiological studies were conducted for 6 months of frozen storage. The product was thawed at 26˚C ± 2˚C for 30 min and was subjected to the following analyses.

2.2.1. Physical and Chemical Parameters Separated the meat chunks and gravy and homogenized

Table 1. Ingredients composition of pineapple chicken curry.

Ingredients Amount

Chicken Cubes 2.22 Kg

Pine apple 300 g

Onion 200 g

Green Chilli 32.0 g

Salt 22.0 g

Garlic 23.0 g

Corn Flour 10.0 g

Ginger 23.0 g

Pepper 2.00 g

Vegetable Oil 75.0 ml

Vinegar 6 t sp

Soy sauce 3 t sp

Tomato sauce 100 g

Chili sauce 20.0 g

Jaggery 20.0 g

in a mixer for sampling. Proximate composition and NaCl content in chicken chunks and gravy were deter- mined [8]. FFA, pH and TBA of both chicken chunks and gravy were determined periodically during the storage period. Free fatty acid (FFA) [7] and thiobarbituric acid (TBA) were determined by aqueous extraction procedure [9] and pH by immersing a glass calomel electrode di- rectly into the sample using a pH meter (Cyberscan 1000, Eutech Instruments, Singapore). Shear values were meas- ured in a Lloyds Texturometer (LR5K, Lloyd Instru- ments Ltd., Hampshire, UK) in 100 kg load cell at a speed of 50 mm/min with a 1 mm thick blade using chicken chunks of 1 × 1 × 1.5 cm strips.

2.2.2. Free Fatty Acid Content (FFA) The FFA content of the samples was estimated as per AOAC (1990) [8]. The FFA content of the samples was estimated as per AOAC (1990). A known quantity of fat extracted was taken in to a 100 ml flask and 50 ml of hot neutralised alcohol was added followed by 1 - 2 ml of phenolphthalein reagent. The flask was shaken vigor- ously to dissolve all the fat content and titrated against 0.25 N NaOH solutions to get a faint pink colour. From the titre value FFA content was calculated as follows:

Free Fatty Acid % as Oleic acidml of alkali of alkali 28.2

Weight of fat g

N

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Physico- Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage 3

2.2.3. Thiobarbituric Acid Reactive Substance (TBARS) Determination

TBARS values in meat/poultry samples were determined as per Taraldgis method [10]. Taraldgis method (1960) is one of the most widely used tests to evaluate the extent of lipid oxidation in meats. This is based on the reaction between important oxidation product malnaldehyde with TBA reagent to produce a colour complex. The chro- mogen results from the condensation of two molecules of TBA with one molecule of malonaldehyde.

20 g of blended sample was accurately weighed and transferred into a RB flask. To that 2.5 ml of conc. HCl was added along with 97.5 ml of distilled water. pH was adjusted to 1.5. Mixture was steam distilled and 50 ml distillate was collected in 10 min. From this 25 ml of distillate was transferred into stoppered glass tubes and 5 ml of TBA reagent was added. The test tubes were kept in boiling water bath for 35 min and it was cooled and OD was measured at 538 nm. The TBARS values were calculated using the standard curve.

2.2.4. Total Fatty Acid Analysis by Gas Chromatography

2.2.4.1. Esterification of Fatty Acids The samples were esterified as per the procedure of Metcalfe et al. [11] with slight modifications.

About 150 mg of lipid was accurately weighed into a clean and dry stoppered test tube. 4 ml of 0.5 N alcoholic sodium hydroxide solutions was added and heated for 5 min over a water bath at 90˚C. On cooling 5 ml of Boron trifluoride-methanol reagent (14%) was added and heated for 5 min at 90˚C over a water bath, followed by addition of 10 ml of saturated sodium chloride solution. The sam- ples were thoroughly cooled to room temperature and 5 ml of hexane was added to each tube. It was shaken well and kept undisturbed. The upper hexane layer was drawn out into clean dry conical flask and dried over anhydrous sodium sulphate to remove the traces of moisture if pre- sent. The samples were filtered and transferred to stop- pered clean dry tubes for gas chromatographic analysis.

2.2.4.2. Quantification of Fatty Acid Analysis by Gas Chromatography

Analysis of total fatty acids was carried out by ceres 800, Chemito model Gas chromatograph fitted with BPX 70 column (25 mt, 0.32 mm ID) and flame ionisation detec- tor. Temperature gradient programming was employed from 150˚C to 220˚C. Split ratio was adjusted to 1:25 and capillary flow of carrier 2 ml/min. Injector and detector port temperatures were adjusted as 230 and 240 respec- tively. For FID, Hydrogen and Oxygen was used and the flow was adjusted as 45 ml/min and 45 ml/min respect- tively. Along with samples standard esters of fatty acids were also injected and the fatty acids were detected by

comparing the retention time of the standard esters of fatty acids. The quantification of the fatty acids was car- ried out by evaluating with the standard fatty acid esters area corresponding to each peak in the chromatogram. Iris 32 software is used to integrate and evaluate the chromatogram in the analysis.

2.2.5. Microbiological Quality Chicken chunks in the curry were cut using a sterile knife and mixed with the gravy. Placed a 50 g sample of the mixture in a sterile stomacher bag containing 450 ml sterile saline (0.85% NaCl) solution and blended in a stomacher (Seward Stomacher 400, Seward Medical, London, UK). The blended samples were tested for stan-dard plate counts (SPC), coliforms, yeast and mould (Y&M), srtaphylococci, salmonella and E. coli by pour plate method [12].

2.2.6. Sensory Quality Packets of RTE chicken curry was thawed by holding them at 26˚C ± 2˚C and warmed the product in a hot pan maintained at 80˚C - 90˚C for 3 - 4 min. The coded sam- ples were subjected to sensory evaluation by 10 in-house trained panelists using a 9-point hedonic scale [6,13] and recorded the mean score of each attribute (colour, flavor, mouthfeel, consistency of the gravy, meat texture and overall acceptability).

2.3. Statistical Analysis

Statistical analysis was performed with SPSS software (SPSS Inc., 1996) and used to test the significant effect of various parameters at 5% level of significance (P > 0.05). Each test was performed in replication of three.

3. Results and Discussion

3.1. Physical and Chemical Quality

In the present RTE recipe pineapple used were different from other regular chicken curry recipe. Pineapple had certain advantages and made the recipe much more ac- ceptable. Pineapple has exceptional juiciness that added to the curry and vibrant flavor with several health bene- fits. The enzyme bromelain present enhanced the texture of chicken by tenderizing and maintained it for longer also the highly acidic nature of fruit lowered the pH thus extending the shelf life. Chicken chunks were found to have less moisture, fat, ash and salt compared to gravy i.e. 69.40%, 11.08%, 1.80% and 1.58% respectively in chunks whereas in gravy a higher values were observed i.e. 74.5%, 13.43%, 1.9% and 1.8% respectively. The higher values in gravy can be contributed due to the added spices, salt and water during cooking of the chicken curry but since chicken is a good source of pro- tein whereas in gravy no ingredient that has as high pro-

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Physico- Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage

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4

tein content in chunks (19.43%) thus curry had a signifi- cant lower value for protein i.e. 2.7%. The physio-che- mial changes during frozen storage of pineapple chicken curry were depicted in Table 2. There was marginal fall in the pH (Table 2) of meat and gravy from 5.7 to 5.51 and 6.02 to 5.68 respectively during 6 months of frozen storage. FFA values (as % of oleic acid) were increased marginally from 0.28 to 0.453 in meat and 0.29 to 0.46 in gravy during frozen storage for 6 months (Table 2).

Lipase activity has been reported as the cause of increased FFA values in meat products during storage by many authors. But the increase in FFA did not increase the rancidity in pork sausages [14,15], fried chicken [16], buffalo meat burgers [6] and chicken nuggets [7]. In- creased level of FFA does not cause any toxicological effects [17].

TBA is a measure of oxidative rancidity of the product. TBA values (mg of malonaldehyde/kg sample) fluctuated non-significantly between 1.68 and 2.64 for meat and 2.21 and 2.45 for gravy during the storage period (Table 2). This effect is attributed to the very low temperature storage and the antioxidant effect of the spices used [18-20].

A significant (P ≤ 0.05) increase in TBA value during frozen storage was reported in chicken nuggets [6,21], Iberian ham [22] (Martin et al., 2000), and fish fingers [23] and buffalo meat burgers [2003]. Wang et al. [24] has reported TBA values of around 2.0 in fresh chicken meat after vacuum packaging and storage for 7 weeks at 7˚C.

3.2. Texture

The shear values for meat pieces observed a marginal decrease (17.64%) during 6 month frozen storage (Table 2), indicating that the freezing and storage in frozen con- dition has little effect on the textural qualities of meat in the curry. Initially, the samples required higher shear

force i.e. 34 N but during storage the force decreased to 28 N. This decrease in shear force can be attributed to both cooking and freezing process. Combes et al. [25] suggested that with advancement in cooking process several changes occur due to heat application as well as mechanical properties are influenced. Similarly, freezing also leads to textural changes as during the process of freezing ice crystals are formed in between the fibres that causes stretching and ruptures of connective tissues thus inducing higher tenderization. Hence there is a decrease in shear force values during freezing. Also, Shanks et al. [26] have reported that tenderization in meat is highly dependent on freezing rate, storage temperature and fro- zen storage duration that affect the amount of intracellu- lar crystal formation and physical disruption occurring in muscle.

3.3. Microbiological Quality

Various microbiological tests were carried out have shown that freshly prepared chicken curry (before freezing) had microbial counts of 1 × 102 and 2 × 101 (in log cfu/g) for SPC, yeasts and molds respectively (Table 3). Banwart [27] suggested that microbial specifications for cooked poultry products should be in the range of 4 - 5 log cfu/g for aerobic plate counts. During freezing mi- crobes become dormant thus, there is limited or no microbial spoilage at low temperature [28]. Studies have revealed that beef frozen for up to 90 days did not spoiled due to microbial growth [29]. S. aureus, coliform, salmonella and E. coli could not be detected in these products.

After a period of 6 months a decrease in both SPC and yeasts & molds count were observed to <10 and 10 re- spectively that can be due to decrease in pH that was observed with an increase in storage period. Absence of S. aureus, coliform, salmonella and E. coli can be con- tributed by thermal processing initially followed by hy-

Table 2. Changes in physicochemical quality of chunks and gravy.

pH FFA (as percentage oleic acid)TBA (mg malonaldehyde per

Kg fat) Shear force (N) Storage period

(in months) In Meat Sample In Gravy In Meat Sample In Gravy In Meat Sample In Gravy In Meat Sample In Gravy

0 5.73 ± 0.03 6.02 ± 0.11 0.28 ± 0.02 0.29 ± 0.05 1.68 ± 0.11 2.21 ± 0.06 - -

1 5.65 ± 0.05 5.90 ± 0.05 0.31 ± 0.02 0.35 ± 0.04 2.00 ± 0.23 2.32 ± 0.04 34 ± 1.52 -

2 5.40 ± 0.03 5.60 ± 0.30 0.29 ± 0.07 0.42 ± 0.10 2.26 ± 0.12 2.36 ± 0.07 33 ± 1.00 -

3 5.68 ± 0.09 5.70 ± 0.10 0.28 ± 0.04 0.37 ± 0.04 2.03 ± 0.25 2.25 ± 0.02 32 ± 1.20 -

4 5.60 ± 0.17 5.70 ± 0.10 0.34 ± 0.01 0.35 ± 0.04 2.08 ± 0.09 2.25 ± 0.22 31 ± 1.52 -

5 5.55 ± 0.03 5.69 ± 0.02 0.41 ± 0.05 0.44 ± 0.06 2.19 ± 0.20 2.30 ± 0.20 29 ± 1.23 -

6 5.51 ± 0.01 5.68 ± 0.09 0.45 ± 0.07 0.46 ± 0.04 2.34 ± 0.13 2.45 ± 0.08 28 ±1.92 -

Mean± SD, n = 3.

Physico- Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage 5

Table 3. Changes in microbiological quality (counts in log cfu/g) of pineapple chicken curry during frozen storage (−18˚C ± 2˚C).

Samples SPC Coliform Yeast &

Mold S. aureus Salmonella E. coli

Fresh 1 × 102 Nil 2 × 101 Nil Nil Nil

2 Months 4 × 101 Nil 1 × 101 Nil Nil Nil

4 Months 2 × 101 Nil 1 × 101 Nil Nil Nil

6 Months <10 Nil 1 × 101 Nil Nil Nil

gienic packaging in addition to the antibacterial effect of spices [30,31]. Finally before storage, samples were sub- jected to low temperature that also had an inhibitory ef- fect on the growth of undesirable microbes and prevent- ing spoilage. Narasimha et al. [32] suggested that meat products are spoiled when an off odor, slime formation along with the microbial population of 8 log cfu/g on the surface is evident.

3.4. Fatty Acid Estimation by Gas Chromatography

The individual fatty acid composition of pineapple chicken curry were estimated to find out the effect of pineapple on fat oxidation during storage .The stability of various saturated , monounsaturated and polyunsaturated fatty acids during 6 months storage at −18˚C were evaluated. The results of this study were depicted in Table 4. From the table it could be observed that the sample contains a mixture of fatty acids both saturated and unsaturated fatty acids. Unsaturated fatty acids constitute both monounsaturated (MUFA) and polyun- saturated (PUFA). Monounsaturated fatty acids are dominant and constitute approximately 52%. The oxi- dation of lipid is one of the primary causes of deterio- ration of meat during processing and storage leading to development of off-flavour, decrease in nutritive value, loss of colour, texture etc. So the degradation of un-

saturated fatty acids have been investigated by GC. From the data, it could be clear that, out of saturated

fatty acids lauric, myristicand palmitic found in low quantities. Stearic contribute the major mono unsaturated fatty acids. The PUFA i.e., linoleic, linolenic and arahi- donic acids were also present in small quantities. From the studies, it was observed that saturated fatty acids do not vary significantly during storage, where as MUFA and PUFA showed a significant change during storage.

3.5. Sensory Quality

Mean sensory scores given by panel members for 0-day samples for overall acceptability were in the range of 8.34 ± 0.32 on a 9-point hedonic scale (Table 5). During the storage period of 6 months a gradual decrease in scores were observed from 8.34 to 7.46 on 0 day and after 6 months of frozen storage respectively. The scores given by panel members have shown that the samples

Table 4. Fatty acid profile of sample.

Fatty acid Gram percentage Gram percentage

Initial 6 Months

Lauric (C12:0) 0.76 ± 0.03 0.75 ± 0.06

Mysitic (C14:0) 5.40 ± 0.21 5.32 ± 0.19

Palmitic (C16:0) 17.61 ± 0.12 17.49 ± 0.15

Palmitoleic (C16:1) 3.02 ± 0.09 2.85 ± 0.07

Stearic (C18:0) 20.23 ± 0.24 19.91 ± 0.21

Oleic (C18:1) 32.34 ± 0.14 31.51 ± 0.17

Linoleic (C18:2) 11.4 ± 0.17 11.06 ± 0.20

Linolenic (C18:3) 1.5 ± 0.02 1.24 ± 0.04

Arachidonic(C20:4) 0.82 ± 0.12 0.64 ± 0.13

Mean ± SD, n = 3.

Table 5. Changes in sensory quality of pineapple chicken curry product during frozen (−18˚C ± 2˚C) storage.

Storage period in months

0 1 2 3 4 5 6

Colour 8.6 ± 0.23 8.5 ± 0.23 8.3 ± 0.32 8.2 ± 0.21 8.0 ± 0.26 7.9 ± 0.35 7.8 ± 0.43

Flavour 8.3 ± 0.27 8.3 ± 0.27 8.1 ± 0.32 8.0 ± 0.29 7.8 ± 0.46 7.6 ± 0.36 7.4 ± 0.32

Mouth feel 8.4 ± 0.23 8.1 ± 0.32 8.0 ± 0.45 7.9 ± 0.42 7.7 ± 0.42 7.6 ± 0.24 7.5 ± 0.28

Consistency 8.2 ± 0.32 8.3 ± 0.42 8.0 ± 0.78 7.9 ± 0.64 7.7 ± 0.74 7.6 ± 0.74 7.5 ± 0.64

Texture 8.2 ± 0.54 8.4 ± 0.42 8.0 ± 0.68 7.7 ± 0.24 7.4 ± 0.32 7.3 ± 0.86 7.1 ± 0.32

OAA 8.34 ± 0.32 8.32 ± 0.33 8.08 ± 0.51 7.94 ± 0.36 7.72 ± 0.44 7.6 ± 0.51 7.46 ± 0.4

Mean ± SD.

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Physico- Chemical Changes in Ready to Eat Pineapple Chicken Curry during Frozen Storage 6

maintained the quality even after the storage period of 6 months under frozen conditions since all the scores were observed above 7 that are in an acceptable range. Simi- larly a decrease in trend was observed for beef patties [33], chicken nuggets [7] and egg loaf [34] and chicken curry [1].

4. Conclusion

Consumer’s rising interest in RTE has motivated re- searchers to standardize the process of traditional prod- ucts that are long being known as well as are highly consumed. In the present study recipe of pineapple chicken curry was standardized that was packed and analysed for six months which revealed that even after a storage period of 6 months the product was accepted by the sensory panelists as well as was found to be micro- biologically stable. A decreased pH further contributed to decrease in stability. FFA and TBA values have shown that there was no adverse affect on the nutritional quality due to the combined effect of low temperature and anti- oxidant effect of spices used. Fatty acid profile has been established by GC and several fatty acids were quanti- fied. Thus, the product quality was maintained and it can be concluded that it can be stored without marked loss in quality for a period of 6 months at −18˚C ± 2˚C.

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