Food Chemistry: Chen Qihe, He Guoqing, Jiao Yingchun, Ni Hui
Food Chemistry: Chen Qihe, He Guoqing, Jiao Yingchun, Ni Hui
Food Chemistry: Chen Qihe, He Guoqing, Jiao Yingchun, Ni Hui
Chemistry
Food Chemistry 98 (2006) 624629
www.elsevier.com/locate/foodchem
a
Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China
b
Department of Agricultural Science, Qinghai University, Xining 810003, China
Received 12 January 2004; received in revised form 15 June 2005; accepted 15 June 2005
Abstract
The tenderization eect of a new elastase from Bacillus sp. EL31410 was investigated on beef meat. Meat tenderization was done
by dipping the meat cut in a solution containing proteolytic enzymes after freeze-dehydration. It was found that a marination time
of 4 h was enough for enzyme adsorption. The samples were treated for 4 h in dierent enzyme solutions and then was stored at 4 C
for 24, 48, 72 h, and subjected to texture measurement, sensory evaluations, biochemical analysis and histological observations. A
marked decrease in hardness, by texture measurements, was observed in the meats with papain and elastase and higher sensory
scores for tenderness were observed in the meats treated with enzymes than in the control. The papain-treated beef meat received
the highest score for tenderness, but the scores given for juiciness and taste were lower than that of the control. Rapid increases of
fragmentation of myobrils from the enzyme-treated meat were observed in the rst 24 h of storage, especially for papain-treated
meat. Meantime, elastin of myobrilar structure was selectively degraded by elastase compared with the control when stored at 4 C
for 48 h as shown by electron microscopy. These ndings suggest that Bacillus elastase (EL31410) is a promising substitute for
papain as a favourable meat tenderizer.
2005 Elsevier Ltd. All rights reserved.
Keywords: Meat tenderization; Papain; Elastase; Freeze-dehydration; Myobrils; Intramuscular connective tissue
0308-8146/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.06.043
C. Qihe et al. / Food Chemistry 98 (2006) 624629 625
achieved during cooking (Gerelt, Ikeuchi, & Suzuki, ing glucose and casein and corn steep our, at 37 C for
2000). 30 h. Ammonium sulfate precipitation of the culture
Although most studies have focussed on the identi- uid was performed to obtain the partially puried en-
cation and purication of elastase-producing strains, zyme, and this fraction was then further puried, using
these enzymes are not successful in meat tenderization, SephadexG-100 column chromatography.
mainly because of safety problems, such as pathogenic-
ity, or other disadvantageous eects. We isolated a new 2.2.3. Enzyme assay
elastase from Bacillus sp. EL31410 (Chen & He, 2002) Elastolytic activity was assayed by the colorimetric
this enzyme was a protease with very high elastolytic method of Sachar (1955). Enzyme preparation was incu-
activity. Therefore, it is interesting to further study its bated with 20 mg of Congo-red elastin in 2 ml of 0.2 M
structural and functional relationships, including the boric acid buer (pH 7.4) with shaking for 20 min at
dierence in substrate specicity. In the present study, 37 C. The reaction was stopped by adding 2 ml of
we investigated elastase, applied to beef meat tenderiza- 0.7 M sodium phosphate buer (pH 6.0), and immedi-
tion, in comparison with other non-specic proteases, ately ltered. Absorbency of the ltrate was read at
such as papain, and evaluated the feasibility of using it 495 nm against a control (no enzyme). One unit of elas-
for this purpose. tase activity was dened as the amount of enzyme re-
quired to solubilize 20 mg elastin-congo-red under the
tested conditions.
2. Materials and methods
2.2.4. Preparation of myobrils and fragmentation index
2.1. Materials Myobrils were made from each muscle according to
the procedure described by Busch, Stromer, Goll, and
The elastase was puried from Bacillus sp. EL31410 Suzuki (1972). The ground muscle was suspended in 5
culture. Papain (salt precipitation, 200,000 U/ml) was volumes (w/v) of 50 mM TrisHCl buer (pH 7.6) con-
obtained from the Biochemical Co., Ltd. Of Guangxi, taining 100 mM KCl and 5 mM EDTA by using a blen-
China. The Congred elastin from bovine neck ligament der for 1 min. The myobrils were sedimented in a
was purchased from the Sigma Company. Milk casein centrifuge at 1000g for 10 min and suspended again in
was purchased from Shanghai Biochemical Co., Ltd. 5 volumes of the same buer by use of a blender for
(Shanghai, China). Beef meat was excised from the 1 min. The re-suspended myobrils were sedimented at
shoulder part of a culled-cow carcass after slaughter 1000g for 10 min, and the resuspension-sedimentation
and stored at 25 C (Hangzhou meat process factory, process was repeated three more times. After the fth
China). Before use, it was tempered overnight in a cold wash, the myobrils suspended in the same buer were
room (4 C) and cut into small pieces (50 50 30 mm). passed through a 20-mesh nylon net to remove connec-
Three small pieces of beef meat were prepared for each tive tissue. The strained myobrils were sedimented at
experiment. 1000 g for 10 min washed three times in 1000 mM KCl
and nally suspended in 100 mM KCl. After adjusting
2.2. Analytical methods the protein concentration to 0.5 mg/ml of 100 mM
KCl, turbidity at 540 nm of the solution was measured
2.2.1. Dehydration and enzyme treatment as fragmentation index by HP spectrophotometer
Each piece of beef meat was freeze-dried at 50 C 751GW.
overnight until it appeared porous. After the dehydra-
tion, each sample was dipped for dierent times (in a 2.2.5. Mechanical texture measurement
cold room) in 5 volumes of a solution containing prote- A Rheometer, NRM 2002, measured meat tenderness
olytic enzymes, such as papain (from plant) and elastase with a conical plunger according to the procedure de-
(Bacillus sp. EL31410). The concentrations of enzyme scribed by Okabe (1979).
solution were 0.1% and 1% for papain and 1% for elas-
tase. Elastase activity of the preparation was 300 U/ml 2.2.6. Electron microscopic studies
by column chromatography. Papain activity of prepara- Specimens for scanning electronmicrograph (JSM-
tion was 200,000 U/ml. An untreated sample (control) T300JEOL) of intramuscular connective tissue were pre-
was dipped in deionized water instead of an enzyme pared by the cell-maceration method of Ohtani, Ushiki,
solution after freeze-dehydration. Taguchi, and Kikuta (1988). Briey, small pieces from
the control and enzyme-treated muscles were xed in
2.2.2. Enzyme preparation 2.5% glutaraldehyde in 0.1 M phosphate buer, pH
Crude elastase and puried elastase were prepared as 7.0, for 24 h, the NaOH solution being replaced every-
described by Takagi et al. (1992). Briey, a Bacillus sp. day by a fresh one and then rinsed in distilled water
EL31410 was aerobically cultured in a medium contain- for 5 days at room temperature. Then the pieces were
626 C. Qihe et al. / Food Chemistry 98 (2006) 624629
Table 2
3. Results and discussion
Adsorption ratio of enzyme solution for beef meat at dierent dipping
times
3.1. Specic activity of elastin and collagen
Treats Absorption ratio (%)
3h 4h 8h
Elastin is an important connecting component of stri-
ated muscle such as beef meat, which is veried to be the Control 75.0 1.21 76.5 1.78 77.1 2.01
Papain (1%) 80.2 1.45 82.5 1.96 83.1 2.11
main factor aecting animal muscle tissue tenderness
Papain (0.1%) 76.7 1.32 78.2 1.43 78.9 1.65
(Thomas & Patridge, 1966). Consequently, it is neces- Elastase 82.6 1.58 85.9 1.69 86.2 1.87
sary to investigate the actions of dierent tenderizers
on elastin. Bacterial elastase, belonging to the family
of metalloproteinases, has been studied for many years.
90
These proteinases require Zn2+ atoms for activity, and
in contrast to the serine proteinases, they cleavage pep- 3h
85
tide bonds on the amino-terminal side of the amino acid 4h
absorbance ratio(%)
elastase izer. On the other hand, the elastase from Bacillus sp.
60 EL31410 seems better as a meat tenderizer to this point.
In view of these data, it was apparent that elastase pre-
40
fers elastin and/or collagen to myobrillar proteins as a
20 substrate, whereas papain tends to degrade, not only
0
collagen, but also myobrillar proteins, which could re-
0 20 40 60 80 100 120 sult in the over-tenderization of meat.
storage time (h)
Table 5
Fig. 2. Changes in hardness of enzyme-treated beef meat. Results of Duncans multiple range test between means of the xed
eects for the two factors (A and B) under elastase treatment
Sources Mean Signicant Highly signicant
Table 3 value level (5%) level (1%)
Sensory evaluations of dierent enzyme-treated meatsa
A (treatments)
Traits Treatments A2 0.50 a A
A1 (elastase) A2 (papain) A3 (the control) A1 0.2708 a A
A3 0.6667 b B
B1 (apperance) 0.250 0.250 0.125
B2 (tenderness) 0.125 0.250 0.500 B (traits)
B3 (juciness) 0 0.125 0.500 B2 0.3337 a A
B4 (bitterness) 0.375 0.125 0 B5 0.1667 a A
B5 (avor) 0.125 0.250 0.375 B6 0.1250 a A
B6 (taste) 0.500 0 0.250 B4 0.0833 a A
a B1 0.0837 a A
A means dierent treatments; B shows dierent meat traits; The
B3 0.25 a A
data from each meat sensory trait were means from eight panellists.
628 C. Qihe et al. / Food Chemistry 98 (2006) 624629
Fig. 3. Scanning electronmicrographs of intramuscular connective tissue prepared from the elastase-treated beef meats and the control (a, control
200; b, control 5000; c, elastase 200; d, elastase 5000; the treatment time is 48 h, other tenderization conditions shown in methods).
C. Qihe et al. / Food Chemistry 98 (2006) 624629 629
In this study the structure from papain-treated meat was Chen, Q. H., & He, G. Q. (2002). Optimization of medium compo-
not investigated, consequently it is dicult to compare sition for the production of elastase by Bacillus sp. EL31410 with
response surface methodology. Enzyme and Microbial Technology,
with the eect of the papain-treated and the elastase-trea- 5, 667672.
ted. However, disruption of the structure of intramuscu- Cronlund, A. L., & Woychik, J. H. (1986). Eect of microbial rennet
lar connective tissue is another reason for meat on meat protein fractions. Agricultural Food Chemistry, 34,
tenderization by the proteolytic enzymes. 502505.
Cronlund, A. L., & Woychik, J. H. (1987). Solubilization of collagen in
restructured beef with collagenase and a-amylase. Journal of Food
4. Conclusion Science, 52, 857860.
Gerelt, B., Ikeuchi, Y., & Suzuki, A. (2000). Meat tenderization by
proteolytic enzymes after osmotic dehydration. Meat Science, 56,
In conclusion, some ndings in the present experi- 311318.
ment showed that the new elastolytic enzyme produced Kang, C. K., & Rice, E. E. (1970). Degradation of various meat fractions
by a Bacillus sp. is a promising meat tenderizer. It had by tenderizing enzymes. Journal of Food Science, 35, 563565.
a marked preference for elastin and collagen, which Liu, Lu, & Tang, Hongjun (2001). Study advancement of meat
tenderization technology. Meat Industry, 11, 4042.
can contribute to meat hardness, over other myobrillar
Moller, A. J., Vestergaard, T., & Wismer-Pedersen, J. (1973).
proteins at the pH of meat, usually ranging from 5.5 to Myobril fragmentation in bovine longissimus dorsi as an index
6.0. This elastase had the same tenderization eect on of tenderness. Journal of Food Science, 38, 824825.
beef meat as papain, based on the results of texture, sen- Ohtani, O., Ushiki, T., Taguchi, T., & Kikuta, A. (1988). Collagen
sory and structure analyses. However, much still re- brillar networks as skeletal frameworks: a demonstration by the
cell-maceration/scanning electron microscope method. Archives of
mains to be done before this elastase can be put to
Histology and Cytology, 51, 249261.
practical use; for example, studies of the changes in var- Okabe, T. (1979). Texture measurement by rheometer. Shokuhin
ious proteins in muscle and connective tissues and deter- Kogyo, 22, 19.
mination of the optimum conditions for the enzyme are Prusa, K. J., Chambers, E., IV, Bowers, J. A., Cunningham, F., &
necessary. Furthermore, there were many problems un- Dayton, A. D. (1981). Thiamin content, texture, and sensory
evaluation of postmortem papain-injected chicken. Journal of Food
der investigation if this enzyme, is to be applied to large-
Science, 46, 16841686.
scale meat industry, such as elastase safety and elastase Robert, P. M., Thomas, J. B., Catherine, J. F., Steven, D. S., Howard,
stabilization in the meat tendering process. G. W., & Robert, M. S. (1997). Elastin degradation by matrix
metalloproteinases. Journal of Biological Chemistry, 272(29),
1807118076.
Acknowledgement Sachar, L. A. (1955). Photometry method for estimation of elastase
activity. Proceedings of the Society for Experimental Biology and
Medicine, 90, 323325.
We thank the Experimental Center of Zhejiang Uni-
Takagi, H., Kondou, M., Tomoaki, H., Nakamori, S., Tsai, Y.-C.
versity (China) for technical assistance. H., & Yamasaki, M. (1992). Eects of an alkaline elastase
from an alkalophilic Bacillus strain on the tenderization of
beef meat. Journal of Agricultural and Food Chemistry, 40,
References 23642368.
Tang, Q. Y., & Feng, M. G. (1997). The practical statistical analysis
Busch, W. A., Stromer, M. H., Goll, D. E., & Suzuki, A. (1972). Ca2+- and its computer treat desktop. Beijing: Chinese agriculture press.
specic removal of Z line from rabbit skeletal muscle. Journal of Thomas, J., & Patridge, S. M. (1966). The chemistry of connective
Cell Biology, 52, 367381. tissues. Biochemical Journal, 74, 600607.