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Effect of Application of Papain on Spent Hen by Marination in Combination with Vacuum Tumbling

Bhaskar Kanchi Robinson J. J. Abraham V. Appa Rao R. Narendra Babu A. Serma Saravana Pandian S. Wilfred Ruban
Vol 7(8), 153-159

A study on improving the tenderness of spent hen meat was carried out to determine the optimum level of ultra refined papain powder and time period of marination and vacuum tumbling. The physico-chemical characteristics viz. pH, Shear force value, myofibrillar fragmentation index, protein solubility, collagen solubility and organoleptic characteristics were analyzed at 30 and 120 minutes. The results revealed that marination for half an hour with vacuum tumbling for 15 minutes with 100 TU of Ultra Refined Papain Powder (URPP) for chicken breast and 125 TU (URPP) for chicken thigh would be optimum for improving the tenderness of spent hen meat.

Keywords : Spent Hen Meat Tenderness Vacuum Tumbling Marination


The utilization of spent hens in further processing is one of the most neglected areas in the poultry industry. Spent hens are the by-product of egg industry. The meat from these birds is considered to be of low in quality because of its age and relative toughness and therefore sold at a lower price than broiler meat in the market. Meat from spent hen is a good protein source, highly enriched with omega-3 fatty acids and low in cholesterol content in particular the breast muscle which has been shown to have health promoting benefits. Therefore, strategies for utilization of spent hen need to be developed to increase the value of its meat.

Experiment I Determination of Optimum Level of Ultra Refined Papain Powder (URPP)

Based on preliminary studies conducted in the Department of Livestock Products Technology (Meat Science) with trained sensory panel with (50, 75, 100, 125, 150, 175 and 200 TU) it was concluded that 100 TU, 125 TU, and 150 TU (Tyrosine Unit) was selected for tenderization of Carcasses of spent hen.

Experiment II Effect of Application of Papain on Spent Hen by Marination in Combination with Vacuum Tumbling

In this phase the effect of marination of spent hen carcasses in different concentrations of papain solution followed by vacuum tumbling was evaluated. Three spent hen carcasses were marinated (immersed) with 1000 ml of enzyme solutions containing three different concentrations of viz.100 TU, 125 TU and 150TU in a sterile plastic container. One carcass was marinated in 1000 ml of distilled water and was kept as control. All the marinated samples were stored at refrigerated temperature (4±1˚C) for a period of 2 hours. In all the treatment, after marination for 30 and 120 minutes, the carcasses were vacuum tumbled (20 psi) at a speed of 8rpm for 15 minutes and samples were drawn for analysis of pH, shear force value, myofibrillar fragmentation index, total protein, collagen content and collagen solubility.

Materials and Methods


The pH of the chicken meat samples marinated and injected with papain was measured using a digital pH meter (Digisun Electronic System, Model: 2001). The shear force values were assessed following the standardized protocol of Wheeler et al. (1996). Fragmentation index values were determined by the procedure of Davis et al. (1980). Protein solubility was determined according to procedure of Joo et al. (1999). Sarcoplasmic proteins were extracted from 2 g sample using 20 ml of ice-cold 0.025 M potassium phosphate buffer (pH 7.2). The samples were homogenized using Homogenizer (SILVER SON Machine LTD., England) and kept overnight at 4 °C with frequent shaking. Samples were centrifuged at 4000 rpm for 20 minutes and concentration of protein in the supernatant was determined by the Biuret method. Collagen content was determined using hydroxyproline assay for determination of the amino acid hydroxyproline as described by Neuman and Logan (1950) with slight modifications as suggested by Naveena et al. (2004). Collagen solubility was determined by method described by Mahendrakar et al. (1989).The sensory evaluation of cooked chicken meat samples were assessed by a trained and semi-trained taste panel drawn from the from the Department of Livestock Products Technology (Meat Science), Madras Veterinary College based on a 9 point Hedonic scale. The data was subjected to statistical analysis in SPSS (version 20.0) software as per the standard procedure outlined by Snedecor and Cochran (1994).

Results and Discussion


Higher (P<0.01) pH values were observed in papain treated spent hen meat and in marination time followed by vacuum tumbling compared to control. Spent hen thigh showed significantly higher pH values than breast. The higher pH value in this experiment reveals that tumbling causes more uniform distribution of papain there by resulting in increase in pH. Babji et al. (1982) reported that the process of tumbling seemed to produce meat with a slightly higher pH and tumbled meat had significantly higher pH values as compared to non tumbled meat. Similar findings were reported by Lengkey et al. (2014) who observed an increase in pH in spent hen meat as the marination time was increased.

Table 1: Mean ± SE values of pH, Shear force value and MFI, Protein solubility, collagen content and solubility of spent hen breast and thigh marinated with Ultra refined papain powder (URPP) at different concentration and vacuum tumbled

Breast Thigh
Parameters Treatments 30 minutes 120 minutes F value (treatments) 30 minutes 120 minutes F value (treatments)
pH Control 5.86Aa± 0.02 5.92Ab ± 0.04 1354.13** 6.06Aa ± 0.13 6.11Aa± 0.16 56.07**
100TU 6.07Ba± 0.02 6.19Bb ± 0.06 6.39Ba± 0.14 6.46Bb± 0.17
125TU 6.16Ca± 0.05 6.21Ba ± 0.08 6.50Ca± 0.20 6.55Bb± 0.22
150TU 6.20Da± 0.13 6.25Ca ± 0.07 6.58Ca± 0.22 6.63Cb± 0.18
Shear Force (kg/cm2) Control 4.85Cb± 0.23 4.30Ca± 0.25 32156.60* 5.20Ba± 0.30 5.00Ca± 0.28 29.27*
100TU 4.35Bb± 0.26 3.20Ba± 0.12 4.55Ab± 0.34 3.95Ba± 0.37
125TU 4.10Bb± 0.24 2.94Aa± 0.27 4.35Ab± 0.31 3.60Ba± 0.29
150TU 3.75Ab±0.27 2.70Aa ± 0.28 4.10Ab ± 0.32 3.24Aa ± 0.37
MFI Control 716.00Db±0.89 701.00Ca± 0.24 64463.82** 923.00Da± 0.89 911.16Ca± 0.36 1479.23**
100TU 674.00Cb± 0.45 622.83Ba± 0.28 873.33Cb± 0.20 821.00Ba± 0.43
125TU 657.50Bb± 0.28 607.00Ba± 0.20 857.00Bb± 0.35 804.00Ba± 0.26
150TU 635.00Ab± 0.44 588.00Aa± 0.32 804.00Ab± 0.11 768.00Aa± 0.38
Sarcoplasmic protein solubility (mg/g) Control 85.11Aa ± 0.29 86.87Aa ± 0.19 412386.41* 86.15Aa ± 0.28 88.14Aa ± 0.22 3141.50*
100TU 86.16Aa± 0.25 91.15Bb ± 0.23 89.76Ba± 0.21 91.87Ba± 0.24
125TU 87.88Aa± 0.30 92.88Bb ± 0.22 90.04Ca± 0.23 94.00Bb± 0.02
150TU 88.15Ba± 0.34 94.87Cb ± 0.32 90.89Ca± 0.28 96.13Cb± 0.22
Myofibrillar protein solubility (mg/g) Control 124.53Aa ± 0.17 124.98Aa± 0.10 183669.49** 124. 04Aa±0.17 125.26Aa± 0.26 10606.46*
100TU 124.10Aa± 0.00 127.23Bb± 0.17 125.89Aa± 0.31 128.11Bb± 0.39
125TU 125.05Ba± 0.17 128.10Bb± 0.10 126.85Ba± 0.35 128.92Bb± 0.22
150TU 125.89Ba± 0.18 129.00Cb± 0.10 127.55Ca± 0.38 129.15Cb± 0.29
Collagen content (mg/g) Control 4.14Aa ± 0.16 4.25Aa ± 0.19 964.32* 4.73Aa± 0.19 4.77Aa ± 0.22 172.76*
100TU 4.18Aa± 0.17 4.57Bb± 0.22 4.77Aa± 0.36 4.80Ba± 0.19
125TU 4.26Ba± 0.26 4.72Bb± 0.23 4.83Ba± 0.17 4.88Bb± 0.40
150TU 4.31Ba ± 0.20 4.83Cb± 0.18 4.89Ba± 0.16 4.93Cb± 0.24
Collagen solubility (%) Control 31.82Aa ± 0.27 32.53Aa ± 0.31 8709973.43** 33.74Aa ± 0.13 34.15Aa ± 0.02 13355.78*
100TU 44.86Ba± 0.19 49.67Bb± 0.22 46.16Ba± 0.18 50.21Bb± 0.35
125TU 48.13Ba± 0.16 57.27Bb± 0.26 50.38Ba± 0.22 54.45Bb± 0.47
150TU 52.47Ca± 0.15 63.24Cba± 0.23 53.02Ca± 0.18 64.14Cb± 0.24

Means bearing different superscripts between columns (a, b, c) between rows (A, B, C) differ significantly (p<0.05) or (p<0.01). ** – highly significant (P<0.01) difference, * – significant (P<0.05) difference, NS – Non significant (P>0.05) difference

Shear Force Value

The shear force values were significantly (P<0.01) reduced in all enzyme-treated samples compared to control. Spent hen breast showed significantly lower shear force values than thigh followed by longer marination time and tumbling. Morgan et al. (1991) also reported a decrease in shear force value when spent cow meat was marinated with 0.3 M CaCl2 followed by tumbling. Similar findings were reported by Weiss (1973) who opined that short-term tumbling could be beneficial to the industry in reducing marination time.

Myofibrillar Fragmentation Index

MFI is linked to meat tenderness and is used as a method to estimate the degree of tenderness (Taylor et al., 1995).The myofibrillar fragmentation index was significantly (P<0.01) reduced in all enzyme-treated samples compared to control. Spent hen breast meat showed significantly lower myofibrillar fragmentation index than thigh. Pearson and Tauber (1984) stated that tumbling was known to improve brine penetration and distribution and also induces mechanical damage to the myofibrillar integrity thereby improving tenderness. In addition myofibrillar fragment index depends on ageing, species and process of blending according to (Dosler et al.,2007).

Protein Solubility

Myofibrillar proteins hydrolysis is considered to be a key factor in determining the meat tenderness (Ha et al., 2012). Spent hen meats showed a significant and gradual increases P<0.05) in the solubility of sarcoplasmic and myofibrillar proteins following marination compared to control samples. The increase in myofibrillar protein solubility may be due the increased amount of extractable salt-soluble protein caused by destruction during tumbling. Increased tissue disruption during tumbling allows increased protein extractability, which results in greater solubilisation of myofibrillar proteins. Similar findings were observed by (Xargayo and Lagares, 1992).

Collagen Content

A highly significant (P>0.05) difference in collagen content was observed between the different treatments but a significant (P<0.05) difference between the marination time in breast and thigh s of spent hen. The collagen content of the thigh muscles were higher compared to breast muscle and the results are in accordance with the findings of Naveena et al. (2004) who observed similar results.

Collagen Solubility

The highest collagen solubility was observed in the thigh treated with 150 TU of papain for a period of 120 minutes marination in combination with vacuum tumbling. The lowest collagen solubility was observed in the breast treated with 100 TU of papain for a period of 30 minutes marination followed by 15 minutes of vacuum tumbling. The solubility of proteins especially myofibrillar proteins and collagen is greatly associated with marination of meat with tenderizers (Aktas et al., 2003). The results of the present study are in agreement with Liu et al. (1994) who was observed an increase in collagen solubility in spent chicken and attributed it to the weakening of intramuscular connective tissue, including collagen and elastin. The observed increase in collagen solubility of papain-treated samples was in accordance with the findings of Badr 2008.

Sensory Evaluation

The spent hen meat treated with different concentrations of papain presented in Table 2.

Table 2: Mean ± SE values of sensory characteristics of spent hen breast and thigh marinated with ultra refined papain powder (URPP) at different concentration and vacuum tumbled

Breast Thigh
Sensory characteristc Treatments 30 minutes 120 minutes F value (treatment) 30 minutes 120 minutes F value (treatments)
Appearance Control 6.00Aa ± 0.28 6.05Aa ± 0.23 0.925NS 6.00Aa ± 0.09 6.00Ca± 0.03 1.04NS
100TU 6.28Ca± 0.25 6.50Bb± 0.10 6.17Bb± 0.02 5.96Ba± 0.05
125TU 6.15Ba± 0.42 6.48Bb ± 0.45 6.10Bb± 0.07 5.90Ba± 0.02
150TU 6.04Aa± 0.44 6.50Bb± 0.38 6.04Ab± 0.04 5.84Aa± 0.05
Flavor Control 5.00Aa ± 0.18 5.10Ba± 0.22 3539.80** 5.00Aa ± 0.17 4.90Aa ± 0.20 453.86*
100TU 5.70Cb± 0.28 5.00Ba± 0.26 5.55Ca± 0.19 5.80Db± 0.22
125TU 5.62Cb± 0.37 4.80Aa± 0.21 5.40Ca± 0.03 5.40Ca± 0.08
150TU 5.29Bb± 0.29 4.75Aa ± 0.38 5.19Ba± 0.21 5.21Bb± 0.33
Juiciness Control 5.50Aa± 0.22 6.00Aa ± 0.28 14437.28* 5.00Aa ± 0.04 5.35Ab ± 0.03 99.84*
100TU 5.84Aa± 0.31 6.80Ab± 0.36 5.10Aa± 0.16 6.66Bb± 0.05
125TU 6.00Ba± 0.18 7.02Bb± 0.29 5.27Ba± 0.08 6.85Bb± 0.20
150TU 6.25Ba± 0.24 7.32Bb± 0.35 5.42Ba± 0.09 7.00Cb± 0.17
Tenderness Control 5.40Aa ± 0.18 5.85Aa± 0.017 15199.54* 5.25Aa ± 0.05 5.45Aa ± 0.03 425.27**
100TU 5.90Aa ± 0.16 6.65Bb ± 0.23 5.45Ba± 0.16 6.45Bb± 0.19
125TU 6.21Ba± 0.21 6.98Bb± 0.34 5.64Ca± 0.21 6.57Bb± 0.26
150TU 6.46Ba± 0.27 7.23Cb± 0.23 5.80Da± 0.19 6.67Cb± 0.54
Overall acceptability Control 5.25Ab ± 0.19 4.85Aa ± 0.22 8848.15* 5.00A ± 0.14 5.30A ± 0.15 508.45**
100TU 6.22Bb± 0.27 5.85Ba± 0.19 5.80B± 0.25 5.65B± 0.33
125TU 6.00Bb± 0.38 5.65Ba± 0.18 6.10C± 0.18 5.40B± 0.20
150TU 5.80Ab ± 0.26 5.30Ba ± 0.37 5.75B± 0.14 5.20A± 0.16

Means bearing different superscripts between columns (a, b, c) between rows (A, B, C) differ significantly (p<0.05) or (p<0.01). ** – highly significant (P<0.01) difference, * – significant (P<0.05) difference, NS – Non significant (P>0.05) difference

All the papain treated samples received significantly (P<0.01) higher scores for flavour, juiciness, tenderness and overall acceptability at 30 minutes of marination time as compared with that of 120 minutes of marination followed by 15 minutes vacuum tumbling. Higher sensory scores were observed in breast as compared with thigh and the difference in sensory scores could be due to difference in light and dark muscles (McCready and Cunningham, 1971). Flavour scores was higher in samples marinated at 30 minutes in combination with 15 minutes vacuum tumbling whereas with an increase in concentration of papain at the time of marination in combination with vacuum tumbling for 15 minutes a significant decrease in flavour scores was observed at 120 minutes due to development of off flavours. This could be attributed to the fact that papain causes degradation of tissue resulting in release of free amino acids which upon cooking liberated off flavour Prusa et al. (1981).

Lowest scores of tenderness were observed at 100 TU concentrations at 30 minutes marination in combination with vacuum tumbling for 15 minutes. The results indicated that marination time for 30 minutes in combination with vacuum tumbling for 15 minutes with different concentration of papain was insufficient to tenderise the spent hen meat, whereas 120 minutes of marination time in combination with vacuum tumbling caused over tenderization of the meat surface, leading to undesirable ‘‘mushy” textured meat. The results were in agreement with (Ashie et al., 2002) who reported that higher concentration of papain resulted in over tenderized meat resulting in mushy texture. A highly significant (P<0.01) increase in juiciness was observed with increase in concentration as well as the time of marination in combination with vacuum tumbling for 15 minutes. Among the treatments, 100 tyrosine units of papain treated breast marinated for 30 minutes and vacuum tumbled had better overall acceptability scores compared to 125 and 150 tyrosine units of papain treated breast meat. However, thigh meat treated with 125 tyrosine units of papain for 30 minutes marination in combination with vacuum tumbling for 15 minutes scored significantly (P<0.05) higher scores for overall acceptability compared to 100 and 150 tyrosine units of papain.


Based on the results of physico-chemical and sensory characteristics, it was observed that the optimum concentration of papain and marination in combination with vacuum tumbling for tenderization of spent hen breast was 100 TU for 30 minutes marination time plus 15 minutes vacuum tumbling and that for spent hen thigh was 125 TU for the same period of time mentioned above. This method of marinating poultry meat will provide an opportunity to the food processing plant to market ready-to-cook product.


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