MEAT

SCIENCE
Meat Science 68 (2004) 363369
www.elsevier.com/locate/meatsci

Tenderization of bualo meat using plant proteases from

Cucumis trigonus Roxb (Kachri) and Zingiber ocinale roscoe
(Ginger rhizome)
B.M. Naveena
b

a,*

, S.K. Mendiratta b, A.S.R. Anjaneyulu

a
National Research Centre on Meat, CRIDA Campus, Santosh Nagar, Hyderabad, 500 059, Andra Pradesh, India
Department of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, 243 122, Bareilly, UP, India

Received 22 July 2003; received in revised form 15 March 2004; accepted 9 April 2004

Abstract
This study was conducted to develop a method for improving tenderness and overall qualities of tough bualo meat using plant
proteolytic enzymes from Cucumis trigonus Roxb (Kachri) and Zingiber ocinale roscoe (Ginger rhizome). Their tenderizing ecacy
was compared with the most popular enzyme papain. 3
3
3 cm chunks from Biceps femoris muscles of spent Murrah bualoes
(45 years age) were marinated with distilled water (control), 2% (w/w) powdered cucumis extract, 5% (w/v) ginger extract or 0.2%
(w/w) papain for 48 h at 4 C and subjected to various physico-chemical, histological and sensory evaluations. An increase
p < 0:01 in collagen solubility, sarcoplasmic and myobrillar protein solubility, and reduction p < 0:01 in shear force values
were observed in all enzyme-treated samples compared to control. Electrophoretic pattern of muscle proteins also revealed extensive
proteolysis and reduction in number of protein bands in all treated samples. Improvement p < 0:01 in avor, juiciness, tenderness
and overall acceptability scores were observed in all enzyme-treated samples compared to controls. Ginger extract-treated meat
samples received better scores for appearance, avor, tenderness and overall acceptability. From these results, it is shown that ginger
and cucumis can be used as an eective alternative to papain.
2004 Elsevier Ltd. All rights reserved.
Keywords: Bualo meat; Tenderization; Proteolytic enzymes

1. Introduction
The world bualo population is estimated to be
approximately 166.4 million spread in 129 countries
around the world (Food & Agricultural Organization,
2002), of which 161.4 million of them are found in Asia
(97.2%). More than 50% of these bualoes (94 million)
are found in India and have great economic importance, especially due to high export potential. However,
the majority of bualo meat in India is produced from
aged, spent or unproductive animals, which is coarse
and tough in texture and imparts poor organoleptic
characteristics. Although bualo meat has the advantage of lower cholesterol content than beef, the meat
*

Corresponding author. Tel.: +91-040-24536501; fax: +91-04024533381.

E-mail address: naveenlpt@redimail.com (B.M. Naveena).
0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2004.04.004

from aged bualoes is not preferred because of its

toughness (Paleari et al., 1997). Tenderness has been
identied as the most important factor aecting consumer satisfaction and perception of taste. There are
several means of tenderizing meat either chemically or
physically. Treatment by proteolytic enzymes is one of
the popular methods for meat tenderization. Proteolytic enzymes derived from plants, such as papain,
bromelain and cin have been widely used as meat
tenderizers in most parts of the world. One such
promising enzyme, cucumin from melon variety fruits
of Cucumis trigonus Roxb plant, has been reported to
have proteolytic activity (Hujjatullah & Baloch, 1970).
The cucumis plant is found growing wild throughout
the drier upland tracts of India, Afghanistan and Persia. Although dried, coarsely ground fruits of C. trigonus Roxb (locally known as kachri) are traditionally
used as a meat tenderizer in some parts of India, no

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B.M. Naveena et al. / Meat Science 68 (2004) 363369

systematic studies have been undertaken to determine

its ecacy of tenderization in bualo meat and meat
products.
Another such plant proteolytic enzyme called zingibain was isolated from Zingiber ocinale roscoe
(ginger rhizome) by Thompson, Wolf, and Allen (1973).
The ginger protease is a thiol proteinase with an optimum activity at 60 C. Its proteolytic activity on both
collagen and actomyosin was reported to produce more
tender meat (Naveena & Mendiratta, 2001; Thompson
et al., 1973). However, ginger rhizome is used primarily
as a avoring agent for bakery products and sausage
seasoning. Its utilization as a meat-tenderizing agent is
not fully appreciated and the literature available is
scanty.
In spite of easy availability and many useful properties, cucumis and ginger have not been exploited by the
meat industry due to lack of scientic literature. Hence,
the current investigation was undertaken to identify the
tenderizing ecacy of C. trigonus Roxb and Z. ocinale
roscoe in comparison with the popular proteolytic enzyme papain and to elucidate the changes caused by
their proteolytic activity.

2. Materials and methods

Biceps femoris muscles from spent adult female
Murrah bualoes (45 years age) were collected within
23 h post-slaughter from a selected retail meat shop.
They were packed in low-density polyethylene (LDPE)
bags and stored in refrigerator at 4 C for 24 h. After
24-h chilling, muscles were taken out of refrigerator and
cut into small chunks of approximately 3 cm3 size and
were randomly allotted for dierent treatments.
2.1. Cucumis trigonus Roxb (Kachri)
Oval-shaped fruits longitudinally marked with dark
green strips of dots were collected from the Institute
farm. Fruits were washed, cut into halves and squeezed
to remove the seeds. Fresh peels obtained were dried in
an oven at 40 C and ground in a mixer to a ne powder.
Two grams of kachri powder was mixed with 15 ml of
chilled distilled water and kept overnight at 4  1 C to
facilitate swelling of cells and easy release of enzymes.
Then, it was homogenized and ltered through muslin
cloth.

2.3. Papain
Readily available papain enzyme powder from standard rm (SRL, Bombay, India) was used.
2.4. Enzyme treatment and marination
About 3
3
3 cm uniform-sized bualo meat
chunks were sprayed with either 2% w/w, 5% w/v or 0.2 %
w/w of powdered cucumis extract, fresh ginger extract
and papain, respectively. After thorough mixing by
hand, chunks were placed in polyethylene bags and kept
at 4  1 C for 48 h. Thus, there were four treatments:
(a) Control
: 15 ml distilled water
(b) 2% w/w cucumis : 2 g cucumis powder + 15 ml
distilled water
(c) 5% w/v ginger
: 5 ml fresh ginger extract + 10
ml distilled water
(d) 0.2% w/w papain : 0.2 g papain + 15 ml distilled
water
After 48 h of marination, the 27-cm3 meat chunks
were washed, drained and cooked in oven at 75  1 C
for 20 min. The cooked samples were evaluated for
cooking yield, pH, moisture, shear force values and
sensory attributes. Raw meat chunks (before cooking)
were also subjected to various types of physico-chemical
and histological studies.
2.5. Moisture, crude protein, pH and cooking yield
Moisture and crude protein content of meat samples
were determined by the AOAC (1995) method. For pH
determination, 10 g of muscle sample was homogenized
with 50 ml chilled distilled water and the pH values were
measured with a digital pH meter (Model CP-901,
Century Instruments Ltd., India). The weights of samples were recorded before and after cooking and the
cooking yield was expressed as a percentage.
2.6. WarnerBratzler shear force
The cooked samples were chilled at refrigerator
temperature overnight and used for objective determination of tenderness (after equilibration at room temperature). The WarnerBratzler shear force (WBSF)
was measured in 21 cores of 1-cm3 sizes with bres
perpendicular to the direction of the blade (Model
no.81031307, GR Elect. Mfg. Co., USA). The force required to shear the samples was recorded (N/cm2 ).

2.2. Zingiber ocinale roscoe (ginger rhizome)

2.7. Water-holding capacity
Fresh ginger rhizome from a local market was peeled,
sliced and blended with equal quantity of chilled, distilled water for 12 min. The homogenate was squeezed
with ngers through four layers of muslin cloth.

Water-holding capacity (WHC) was determined according to Wardlaw, Maccaskill, and Acton (1973).
Minced meat (20 g) was placed in a centrifuge tube

B.M. Naveena et al. / Meat Science 68 (2004) 363369

containing 30 ml of 0.6 M Nacl and was stirred with

glass rod for 1 min. The tube was then kept at 4  1 C
for 15 min, stirred again and centrifuged at 3000g (R-24,
Remi Instruments, India) for 25 min. The supernatant
was measured and WHC was expressed in percentage.
2.8. Muscle bre diameter
Muscle bre diameter was determined as described
by Tuma, Venable, Wuthier, and Henrickson (1962).
A core (2.54 cm) of muscle tissue was xed in formal
saline for 24 h and was blended in a micro blender at low
speed for 30 s. A drop of the homogenate was placed
over a glass slide, covered with cover slip and observed
under a microscope with 10
eyepiece containing a
calibrated micrometer. The diameter of 21 bres was
measured and the average muscle bre diameter was
expressed in microns.
2.9. Protein solubility
Protein solubility was determined according to procedures of Joo, Kauman, Kim, and Park (1999). Sarcoplasmic proteins were extracted from 2-g minced
muscle using 20 ml of ice-cold 0.025 M potassium
phosphate buer (pH 7.2). The samples were homogenized and kept overnight at 4 C with frequent shaking.
Samples were centrifuged at 1500g for 20 min and
protein concentration in the supernatant was determined by the Biuret method. Total protein (sarcoplasmic + myobrillar) was extracted from 2-g muscle using
40 ml ice-cold 1.1 M potassium iodide in 0.1 M phosphate buer (pH 7.2). Homogenization, centrifugation
and protein determination were carried out as described
above. Myobrillar protein concentrations were obtained by dierence between total and sarcoplasmic
protein solubility.
2.10. Hydroxyproline estimation
Hydroxyproline content of the meat sample was
determined based on the procedure of Nueman and
Logan (1950). Two-gram meat samples were hydrolyzed with 40 ml of 6 N HCl for 18 h. The hydrolysate
was ltered and the volume adjusted to 50 ml with
distilled water. An aliquot was used for hydroxyproline
estimation. Absorbance was measured at 540 nm and
the hydroxyproline content was determined by referring to a standard graph. Collagen content was calculated by multiplying by 7.14 and was expressed in mg/g
tissue.
2.11. Collagen solubility
Collagen solubility was determined by the method
described by Mahendrakar, Dani, Ramesh, and Amla

365

(1989). Five grams of muscle tissue was taken in a

250-ml beaker and immersed in a water bath after covering the beaker with petri dish. The water bath was
then heated to boiling temperature and held for 30 min.
The cooked meat was then taken out of the beaker and
cut into small pieces and homogenized with 50 ml distilled water at 4  1 C in a blender for 2 min. The extract was then centrifuged at 1500g for 30 min. Aliquots
of cooked out juice and centrifugate were hydrolyzed for
18 h and soluble hydroxyproline was calculated. Collagen solubility was calculated as:
% Collagen solubility
7:14
% hydroxyproline solubilized:
2.12. Sensory evaluation
Cooked meat chunks were evaluated by seven semitrained panel members consisting of scientists and students of the division with previous experience. Three
training sessions were held during preliminary trials
before initiation of this experiment. The appearance,
avor, juiciness, tenderness and overall acceptability of
the cooked chunks were evaluated using 8-point descriptive scale (where 8 extremely desirable, 1 extremely undesirable) (Keeton, 1983).
2.13. Electrophoresis
Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) was carried out using the
methods of Laemmli (1970) and See and Jackowski
(1993) with electrophoresis apparatus (Model: Protean
Xi Cell, BioRad, USA). Five grams of minced meat
was mixed with 50 ml of 0.01 N sodium phosphate
buer (pH 7.0) containing 1% SDS and 1%
2-mercaptoethanol and incubated at 37 C for 2 h.
The mixture was centrifuged at 1500g for 30 min. An
aliquot of supernatant was dialyzed overnight at
room temperature (26 C) against 0.1 N sodium
phosphate buer containing 0.1% 2-mercaptoethanol.
About 50 lm of dialyzed solution was used for
loading the gel. Electrophoresis was performed at a
constant voltage mode of 100 V/slab at 30 mA for
56 h or until the tracking dye reached the lower end
of the gel. The gel was removed and stained with
Coomassie blue for 45 h. The gels were then destained and photographed.
2.14. Statistical analysis
The data generated were analyzed by statistical software package using standard procedures (Snedecor &
Cochran, 1989) for analysis of variance and multiple
range test to compare the means and determine the eect
of treatments.

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B.M. Naveena et al. / Meat Science 68 (2004) 363369

3. Results and discussion

3.1. Analysis of raw meat chunks (after 48 h of
marination)
The results of various physico-chemical and histological changes of raw meat chunks treated with dierent enzymes are shown in Table 1.
3.1.1. Moisture, crude protein, pH and WHC
The moisture and crude protein content did not
dier signicantly between control and treated samples
(Table 1). There was a marked reduction p < 0:05 in
pH of cucumis-treated samples compared to other
treated samples. This could be due to low pH of powdered cucumis extract (4.85.0) compared to ginger and
papain extracts (6.50 and 6.25, respectively). There was
a signicant p < 0:05 reduction of WHC in cucumistreated samples compared to others. However, control,
ginger- and papain-treated samples did not dier
p < 0:05, although slightly higher values were observed in samples treated with ginger and papain. The
reduction in WHC of cucumis-treated samples might be
due to lower pH and this drop in pH may be responsible
for an overall reduction in reactive groups of proteins
available for water-holding (Forrest, Aberle, Hedrick,
Judge, & Merkel, 1994). The linear increase in WHC of
ginger- and papain-treated samples with increase in pH
were in agreement with Bouton, Carrol, Harris, and
Shorthose (1973). Comparatively lower WHC values in
our experiment might be due to meat from very old
animals that have lower WHC (Syed Ziauddin, 1994)
and also may be due to slight denaturation of sarcoplasmic proteins, which play an important role in determining WHC (Joo et al., 1999).
3.1.2. Collagen content and solubility
Between each treatment, no signicant dierences
were observed in collagen content. However, slightly
higher collagen content recorded in treated samples

diered a little from other reports. Signicantly negative

correlation between collagen content and tenderness of
meat was reported by Adam, Harrison, and Hall (1960).
Higher (p < 0:01) collagen solubility values were observed in all enzyme treated samples compared to control. The increased collagen solubility of ginger-treated
samples in our experiment was consistent with the
ndings of Thompson et al. (1973), who reported
a signicant increase in collagen solubility of ovine
B. femoris muscle with ginger extract treatment. They
found that proteolytic activity of ginger protease on
collagen was many fold greater than on actomyosin and
the combined proteolysis of these two muscle proteins
resulted in signicantly more tender meat. Takagi,
Arafuka, Inouye, and Yamasaki (1992) also reported
signicantly higher collagen solubility in beef meat
treated with papain compared to water-treated control
and alkaline elastase-treated sample.
3.1.3. Protein solubility
Signicantly p < 0:01 higher myobrillar and total
protein solubility values were observed in all enzymetreated samples compared to control. On the other
hand, sarcoplasmic protein solubility values of enzymetreated samples increased only marginally in comparision to control. Increase in protein solubility with
enzyme treatment was also reported by Buckley, Gann,
Price, and Spink (1974) and Kim, Lee, Lee, Cheng, and
Kim (1981). Increase in solubility of enzyme-treated
samples might be due to increase in permeability of
myobrils, which will disintegrate easily. In control
samples, regularly aligned laments of myobrils prevent buer penetration, thus making action seemingly
resistant to extraction (Davey & Gilbert, 1968).
Lower solubility of sarcoplasmic proteins in our experiment was in agreement with Kang and Rice (1970)
who reported that water soluble proteins are more resistant to enzyme degradation than other fractions. Joo
et al. (1999) reported that water soluble protein solubility increases with increasing pH, but salt soluble

Table 1
Physico-chemical and histological qualities of bualo meat chunks treated with cucumis, ginger and papain for 48 h
Parameters

Control

Cucumis (2% w/w)

Ginger (5% w/v)

Papain (0.2% w/w)

pH
Moisture (%)
Crude protein (%)
Water-holding capacity (%)
Collagen content (mg/g tissue)
Collagen solubility (% total collagen)
Sarcoplasmic protein solubility (mg/g)
Myobrillar protein solubility (mg/g)
Total protein solubility (mg/g)
Muscle bre diameter* (lm)

5.69  0.05ab
76.51  0.44
20.08  0.33
13.26  0.74b
6.58  0.27
6.58  0.19a
19.13  0.69a
62.11  2.07a
81.25  1.94a
60.76  1.05

5.57  0.03a
76.75  0.48
20.06  0.33
11.16  0.38a
7.06  0.41
10.91  0.44b
21.45  0.65bc
84.80  0.91c
106.42  0.80c
58.14  2.44

5.71  0.04b
77.18  0.44
19.24  0.40
14.22  0.61b
7.04  0.38
12.86  0.45c
20.54  0.87ab
75.70  0.54b
95.45  0.80b
57.66  2.26

5.72  0.04b
77.39  0.14
19.42  0.19
13.55  0.55b
7.06  0.47
12.71  0.55c
23.30  0.77c
74.80  0.74b
98.11  1.51b
58.57  1.81

Means bearing same superscripts row-wise do not dier signicantly p < 0:01.
Number of observations 6, *21.

B.M. Naveena et al. / Meat Science 68 (2004) 363369

367

protein solubility showed the weakest correlation. Increase in protein solubility with ginger and papain
treatment was also reported by Naveena and Mendiratta (2001) in spent hen meat and Kang and Rice (1970)
in beef, respectively.
3.1.4. Muscle bre diameter and electrophoretic pattern
of muscle proteins
Although muscle bre diameter values were slightly
lower in all enzyme treated samples, they were not signicantly dierent than control.
A representative SDSPAGE gel for the dierent
treatments can be seen in Fig. 1. There was increased
proteolysis of muscle proteins in all enzyme-treated
samples as evidenced by reduction in the number of
protein bands. From the gure it is also evident that
break down or cleavage of high molecular weight
proteins into low molecular weight proteins of 30 kDa
and below (Hu-Lonergan et al., 1996) had occurred
in all enzyme-treated samples, which resulted in increased concentration of low molecular weight protein
bands. The breakdown of proteins in the higher range
was more clear in cucumis-treated sample than ginger
and papain-treated sample, indicating more pronounced proteolysis. Increased proteolysis in cucumistreated samples can be correlated with signicantly
higher protein solubility. Jorgova, Danchev, and
Kostov (1989) reported that bacterial proteolytic enzyme treatment of muscle proteins showed reduction
in the level of higher molecular weight fractions due
to degradation of myosin, thus increasing meat
tenderness.
3.2. Analysis of cooked meat chunks
The results of dierent physico-chemical characteristics and sensory attributes of cooked meat chunks
treated with dierent enzymes are shown in Table 2.

Fig. 1. Electrophoretic pattern of muscle proteins treated with

cucumis, ginger and papain. Lane M, molecular weight marker (lowrange); lane 1, control; lane 2, cucumis-treated sample; lane 3, gingertreated sample and lane 4, papain-treated sample.

3.2.1. Cooking yield, moisture, pH and shear force

Signicant p < 0:01 reduction in pH and cooking
yield was observed in cucumis-treated samples compared to others. However, in samples treated with ginger
and papain, pH and cooking yield increased. Shear force
values were signicantly p < 0:01 reduced in all enzyme-treated samples compared to control. Signicant
reduction in shear force values of spent hen meat treated
with ammonium sulphate extracted cucumis powder was
reported by Kumar and Berwal (1998). Reduction in
shear force values with ginger extract treatment was also

Table 2
Physico-chemical characteristics and sensory attributes of cooked bualo meat chunks treated with cucumis, ginger and papain for 48 h
Parameters

Control

Cucumis (2% w/w)

Ginger (5% w/v)

Papain (0.2% w/w)

Physico-chemical characters*
Cooking yield (%)
pH
Moisture (%)
Shearforce value** (N/cm2 )

52.8  0.70b
5.84  0.02ab
54.29  0.50
40.52  1.78b

50.36  0.58a
5.77  0.03a
54.10  0.32
22.25  1.53a

53.86  0.54b
5.90  0.03b
54.94  0.26
21.70  1.71a

53.78  0.74b
5.89  0.04b
54.98  0.71
21.73  1.50a

Sensory attributes**
Appearance
Flavor
Juiciness
Tenderness
Overall acceptability

6.69  0.09
6.21  0.12a
6.21  0.10a
5.73  0.14a
5.90  0.11a

6.73  0.10
6.78  0.11b
6.76  0.11b
7.00  0.06b
6.90  0.80b

6.85  0.09
7.26  0.08c
6.85  0.11b
7.14  0.07b
7.11  0.09b

6.76  0.10
6.78  0.11b
7.00  0.08b
7.02  0.08b
6.90  0.07b

Means bearing same superscripts row-wise do not dier signicantly (p < 0:01).
Number of observations *6 and **21.

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B.M. Naveena et al. / Meat Science 68 (2004) 363369

reported in sheep (Mendiratta, Anjaneyulu, Lakshmanan, Naveena, & Bisht, 2000; Thompson et al., 1973),
bualo (Syed Ziauddin, Rao, & Amla, 1995) and spent
hen meat (Naveena & Mendiratta, 2001).
3.2.2. Sensory evaluation
The meat chunks treated with dierent enzyme extracts received signicantly p < 0:01 higher scores for
avor, juiciness, tenderness and overall acceptability as
compared with that of control (Table 2). The ginger
extract-treated samples received better scores for appearance, avor, tenderness and overall acceptability
compared to others. However, no signicant dierence
in the scores of meat treated with cucumis, ginger and
papain was observed. The sensory evaluation scores for
tenderness in all enzyme-treated samples are in good
agreement with the results of WBSF.
Signicant increase in tenderness and overall acceptability scores of spent layer hens treated with cucumis
powder was reported by Kumar and Berwal (1998).
Improvement in avor, juiciness, tenderness and overall
acceptability scores with ginger extract treatment in our
experiment is consistent with some earlier reports
(Mendiratta et al., 2000; Syed Ziauddin et al., 1995).

4. Conclusions
The results obtained in this experiment clearly indicate the tenderizing eect of cucumis, ginger and papain.
In general, there was a signicant increase in collagen
solubility, sarcoplasmic and myobrillar protein solubility, and a signicant reduction in shear force values in
all enzyme-treated samples compared to controls. Electrophoretic pattern of muscle proteins also depicts
proteolysis and degradation of muscle proteins. Samples
treated with ginger were rated superior and most preferred by the panelists, which can be attributed to the
desirable ginger avor. Cucumis- and papain-treated
samples scored almost equally. Therefore, cheaper and
easily available cucumis and ginger can be eectively
utilized at household or industrial level and they can be
used as better alternatives to papain for tenderization of
tough meat.

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