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The effects of transglutaminase on the functional properties of the myofibrillar protein concentrate obtained from beef heart
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MEAT SCIENCE Meat Science 79 (2008) 278–284 www.elsevier.com/locate/meatsci
The effects of transglutaminase on the functional properties of the myofibrillar protein concentrate obtained from beef heart Aurelia Ionescu a, Iuliana Aprodu a
b,* ,
Aura Daraba˘ a, Lenutßa Porneala˘
a
Department of Biochemistry, ‘‘Dunarea de Jos’’ University, 111 Domneasca Street, 800201 Galati, Romania b Department of Biomechanics, Politecnico di Milano, P.za L. da Vinci, 32, 20133 Milano, Italy Received 26 July 2007; received in revised form 25 September 2007; accepted 26 September 2007
Abstract The aim of the present study was to evaluate the effect of bacterial transglutaminase on the functional properties of the myofibrillar protein concentrate from beef heart. The degrees of hydration and aggregation and emulsifying properties were studied. The degree of polymerization of the myofibrillar proteins depended on the enzyme concentration and setting time; the best results in terms of functional properties were obtained with 0.3 g transglutaminase/100 g protein with 60 min setting at 35 C. This investigation confirms that transglutaminase may be used for the production of myofibrillar protein aggregates with enhanced functional properties. 2007 Elsevier Ltd. All rights reserved. Keywords: Myofibrillar proteins; Bacterial transglutaminase; Functional properties; Rheological behavior
1. Introduction Myofibrillar proteins play an important role when processing meat, being responsible for the formation of cohesive structures and a firm texture following thermal treatment (Xiong, 1997). Functional behavior of myofibrillar proteins is influenced by their ability to form viscous gels via protein–protein interactions, to retain water and, in the case of emulsions, to form resistant films on the surface of the fat droplets. The properties of processed meat products such as, tenderness, juiciness and perception of flavors during mastication are based on these functional properties of the proteins. In order to improve the functional properties of meats, processors are currently using a wide range of vegetable additives, especially soy protein derivates. Often, consumers reject these additives as being chemically modified or as being obtained from genetically modified plants. *
Corresponding author. E-mail address: [email protected] (I. Aprodu).
0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.09.011
Nowadays, interest in developing new technologies to obtain functional extracts, ingredients and additives from slaughter-house by-products, is increasing (McKeith, Bechetel, Novakofski, Park, & Arnold, 1988; Srinivasan, Xiong, & Decker, 1996; Wan, Xiong, & Decker, 1993). Due to their functional properties, myofibrillar protein concentrates obtained from mechanically deboned poultry meat and beef heart can be used in various meat products (Desmond & Kenny, 1998; Ionescu, Aprodu, Zara, & Porneala, 2006; Ionescu, Aprodu, Zara, Vasile, & Gurau, 2003). Functional properties of proteins, such as gelling capacity, gel strength, viscosity, thermal stability and waterholding capacity are directly related with the proteins’ composition and structure. The use of enzymatically catalyzed reactions to modify protein structure is an important way of improving their functional properties (Sakamoto, Kumazawa, & Motoki, 1994; Seguro, Kumazawa, Ohtsuka, Toiguchi, & Motoki, 1995; Seki et al., 1990). One of the most studied enzymes used for structural modification of myofibrillar proteins is bacterial transglutaminase. Transglutaminase (TGase; EC 2.3.2.13) is an enzyme widely spread in nature; in animal organisms it is localized
A. Ionescu et al. / Meat Science 79 (2008) 278–284
in liver, muscular tissue and body fluids. Transglutaminase catalyses the intra- and intermolecular transverse crosslinking of proteins by an acyl transfer reaction between glutamine (Glu) and lysine (Lys) residues. The structure of the proteins is stabilized by the strong covalent (e-(c-Glu)Lys) cross-links between the peptide chains. Jiang, Hsieh, Ho, and Chung (2000) proved, through SDS–PAGE analysis, that bands corresponding to H-meromyosin disappeared and bands corresponding to cross-linked polymers appeared. The affinity of TGase for different types of protein depends on the distribution of Glu residues as well on the secondary and tertiary structures of the proteins (Matsumura, Chanyongvorakul, Kumazawa, Ohtsuka, & Mori, 1996). Casein, soy proteins, conalbumin, rabbit and carp myosin, beef actin and myosin, ovomucin are examples of proteins which are suitable substrates for TGase (Christensen, Sørensen, Højrup, Petersen, & Ramussen, 1996; Kato, Wada, Kobayashi, Seguro, & Motoki, 1991; Muguruma, Sakamoto, Numata, Yamada, & Nakamura, 1990; Nonaka et al., 1989; Nonaka, Toiguchi, Sakamoto, Kawajiri, Soeda & Motoki, 1994; Sakamoto et al., 1994; Tanaka, Nonaka, & Motoki, 1990). Moreover, Carrascal and Regenstein (2002) found the effect of microbial TGase on the functional properties of chicken meat proteins. They report an enhancement of emulsion stability and water uptake of chicken muscle proteins treated with TGase. Our study was focused on bacterial TGase obtained by industrial fermentation of Streptoverticillium mobarense, which is commercialized under the name Activa TG, WM, and MP. The Activa products offer various possibilities for the processed food industry (dairy products, meat products, fish and surimi based foods) by (i) standardizing the texture and water-holding capacity, (ii) improving gel consistency and increasing the firmness, elasticity and juiciness of hams, salamis and products based on concentrates of myofibrillar proteins, (iii) improving the nutritive value of the raw materials of low biological value, and (iv) substituting polyphosphates in hams and salamis while maintaining the taste and flavor. There is little information about the interaction of MTGase with myofibrillar proteins from beef heart. We may expect some variations regarding the interaction between MTGase and myofibrillar proteins from beef heart with respect to those ones reported for chicken muscle proteins, due to differences in the structural organization within the fibers. Myosin of the red fibers, which are the main fibers of cardiac muscle, generally form shorter filaments than the myosin of white muscular fibers. Xiong (1997) found that at protein concentrations of 4–10 mg/ ml, the consistency index of breast poultry protein suspensions (white fibers) was higher than one of poultry thighs (red fibers). The objectives of the present paper were to obtain a functional ingredient based on beef heart myofibrillar pro-
279
teins and to determine the effect of MTGase on its functional properties. 2. Materials and methods 2.1. Materials Activa TG-1 MTGase, provided by Ajinomoto (Inc. Teanec, NJ, USA) was used. The enzymatic product is made up of 99% maltodextrin and 1% transglutaminase with a declared enzymatic activity of about 100 UE/g. The enzyme is active over large ranges of temperature (2– 60 C) and pH (5–8), and is inactivated at high temperatures, depending on the composition of the food. Lygamme EFI80, which is a mixture of 30% xanthan gum and 70% guar gum, was obtained from Eurofood International. Beef hearts were purchased chilled from specialized stores. All chemicals used are of analytical grade. 2.2. Proximate composition and pH The moisture, protein and ash contents of the MPCBH were determined according to standard AOAC (1995) methods. The crude fat content was determined by AOAC (1991) method 991.36. All determinations were made in duplicate. pH measurements were made according to AOAC (1984). Ten grams of sample was homogenized with 90 ml distilled water for 2 min using the Braun mixer. The obtained mixture was filtered and the pH of the filtrate determined by means of a Hanna digital pH-meter. 2.3. Sample preparation The myofibrillar protein concentrate was obtained according to Ionescu, Aprodu, Zara, and Porneala (2006). The wet precipitate obtained after the final washing with phosphate with addition of 0.01% propyl gallate, as antioxidant, and centrifugation, was designated the myofibrillar protein concentrate of beef heart (MPCBH) and was characterized. To avoid protein denaturation during the entire washing cycle, the temperature of the dispersion was maintained below 10 C. The protein batter was obtained from wet MPCBH by mixing it with a 0.2 g mixture of guar and xanthan gum / 100 g proteins. Homogenization was performed in a Braun mixer for 2 min, with temperature control to avoid raising the temperature. In order to study the effect of transglutaminase concentration and setting time on the functional properties of MPCBH, many tests were performed using different enzyme concentrations (0.0, 0.5, 0.10, 0.20, 0.30, and 0.40 MTGase/100 g protein), and different setting times (30, 60 and 90 min) at 35 C. After adding the enzyme followed by 1 min homogenization, the samples were maintained in a water bath at 35 C for the given
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time; afterwards they were cooled at 20 C in a water bath. An aliquot of each sample was used to study the emulsifying properties of the raw batters, while the rest was placed in test tubes and subjected to thermal treatment (the temperature was increased at 1 C/ min) to achieve 70 C in the thermal centre of the sample, a temperature that was maintained for 10 min. The thermally treated samples were cooled with a mixture of water and ice to room temperature, and the obtained gels were stored overnight at 4 C. 2.4. Water losses and water-holding capacity (WHC) evaluation The quantity of the expressed water after thermal treatment was determined using the method described by Nishimoto, Hasimoto, Seki, Chimura, Toyoda, and Arai (1987), which was slightly modified. The water losses after thermal treatment were established by carefully separating the protein gel from the liquid expressed during cooking. The separated gel was weighed after all the liquid was absorbed by filter paper. The water losses during thermal treatment, Pt, are given by P t ¼ ws
ws wg 100; ws
where ws is the water content of the protein suspension, g; and wg is the water content of the gel, g. In order to determine the capacity of the gels to retain water, aliquots of the gel were placed on double layer filter papers in centrifuge tubes, weighed and centrifuged at 704g, for 10 min. After centrifugation, the gel was carefully separated, weighed and dried in an oven at 100 C for 24 h (Wang & Xiong, 1998). The water-holding capacity (WHC) of the proteins was estimated as WHC ¼
wg0 wg1 100; wg0
where wg0 is the water content of the gel before centrifugation, g; and wg1 is the water content of the gel after centrifugation, g. 2.5. Solubility of myofibrillar proteins in SDS The protein solubility in SDS (sodium dodecyl sulphate) was evaluated by the method proposed by Nishimoto et al. (1987) which was slightly modified. Gel (0.8 g) was homogenized with 15 ml of SDS solution (2% SDS, 8 M urea, 2% mercaptoethanol and 20 mM Tris–HCl; pH 8.0) using an high speed Ultraturax agitator for 3 min. The agitator was then washed in 10 ml distilled water, and the homogenized samples were transferred to glass containers, which, after closing, were maintained at room temperature, for 24 h, with occasional stirring. After 24 h, the samples were centrifuged at 1252g for 20 min, and the proteins soluble in SDS were determined in the resulting supernatant by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951).
2.6. Emulsion preparation To prepare the emulsions, the MPCBH was first dispersed in phosphate buffer with NaCl 0.6 M, pH 6.0 using the Braun mixer for 3 min. A model system consisting of 15 g protein suspension in saline solution (0.3% protein, w/v) and 60 mL sunflower oil (1:1, v/v) was used to test the ability of MPCBH to form and stabilize emulsions. The oil was added gradually, and the emulsion was obtained by continuous stirring for 3 min using a Braun mixer. The emulsion was divided into two parts: one for apparent viscosity evaluation, and the other for stability estimation. 2.7. Apparent viscosity evaluation of the MPCBH -based emulsions The apparent viscosity was evaluated by means of a RHEOTEST-2 type rotating viscosimeter manufactured by VEB-MEDINGEN, Germany. Due to the medium viscosity of the MPCBH-based emulsions the coaxial cylinder device S3 was used and 50 g of emulsion was tested. The working frequency was 50 Hz and the shear rate (_c) was varied within the range 0.1667–145.8 s1. The shear stress (sr ) was calculated starting from the value of a, which is the output value of the viscosimeter, using the relationship sr ¼ z a; where z is the device constant corresponding to the working domain I or II and to the cylinder type (dyn/cm2 div. scale). The apparent viscosity (g) of the MPCBH-based emulsions was calculated as g¼
sr 100ðPa sÞ: c_
2.8. Emulsion stability Twenty grams of emulsion was placed in a glass test tube with inner diameter of 15 mm and height of 120 mm. The sample was heated at 1 C/min to achieve 70 C in the thermal center of the product, and this temperature was maintained for 10 min. Afterwards the sample was cooled to 24 C in an ice bath and stored overnight at 4 C. Thermal destabilization, was determined through the creaming index defined as: (emulsion height/initial emulsion height · 100). 2.9. Statistical analysis Statistical analysis of the results was performed using Sigma Plot 2001/Statistics Date software. Five experimental batches were used for each test and the results reported as mean values. Experimental data were fitted using Table Curve 2D software and the regression equations were established based on statistical criteria (r2, Fit Standard Error or F Statistic).
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3.1. Chemical analysis of the MPCBH The results of chemical analysis showed that MPCBH is made up of 88.26 ± 1.23 % water; 10.12 ± 1.1% protein; 0.5 ± 0.34% fat and 0.54 ± 0.02% ash. The pH value of the MPCBH was 6.01. 3.2. Influence of the MTGase concentration and setting time on the degree of hydration The efficiency of using MTGase to improve the hydration properties of the myofibrillar proteins was tested by determining (i) water losses during the thermal gelling process of the protein systems based on MPCBH and (ii) WHC of the proteins after centrifugation. The influence of the MTGase concentration on the degree of hydration of the MPCBH was estimated by setting with enzyme at different concentrations for 60 min at 35 C. The results (Fig. 1) indicated that increasing the MTGase level from 0.05 to 0.3 g/100 g protein improved the WHC of the protein concentrate, due to the formation of cross-links among the polypeptide chains of the myofibrillar protein. Water losses after thermal treatment and centrifugation decreased in all cases on increasing the level of the added enzyme up to 0.3 g/100 g. The highest water losses were achieved in MPCBH without added enzyme, and the lowest in MPCBH with 0.3% MTGase. A further increase in enzyme concentration, to 0.4 g/100 g proteins led to a worsening of the WHC, which may be explained by enzyme competition for substrate. Our observations agree with Motoki, Nio, and Takinami (1984) and Kuraishi, Sakamoto, and Soeda (1996) who found that MTGase improves the WHC of proteins either by increasing the ability to swell and bind water, or by improving the ability to form a gel lattice. In order to estimate the influence of setting time on the degree of hydration, MPCBH was incubated with MTGase
3.3. Influence of the MTGase concentration and setting time on the aggregation degree Formation of protein aggregates, by inter- and intramolecular interactions mediated by MTGase, was estimated by determining the solubility of the gel proteins in SDS. The results indicated that production of e-(c-glutamil)lysine di-peptides was directly linked to the level of MTGase present in the reaction mixture. By adding MTGase, the solubility of MPCBH in SDS was substantially reduced (Fig. 3), due to the enzymatically catalyzed formation of covalent bonds between glutamine and lysine residues. The highest solubility in SDS was obtained for the MPCBH without enzyme, followed by the MPCBH with 0.05% MTGase. The decrease of protein solubility in SDS with increasing MTGase concentration confirms the participation of the enzyme in the polymerization process of the myofibrillar proteins of MPCBH. The influence of the setting time on protein aggregation was also estimated. Four setting periods were tested and the results showed that setting time influenced gel stability on thermal treatment and the solubility of proteins in SDS (Fig. 4). The MTGase effect on the myofibrillar proteins, reflected in the protein lattice strength was more marked at longer setting times.
72 14 69 12
10
66 0
0.1
0.2
0.3
0.4
MTG-ase concentration, g/100 g proteins Fig. 1. The influence of MTGase concentration on gel water losses after thermal treatment (black full line) and centrifugation (grey full line) and on WHC (black broken line).
Water losses, g/100 g gel
16
WHC, g/100 g gel
75
18
Water losses, g/100 g gel
(0.3 g /100 g proteins) for different times at 35 C. Increasing the setting time from 30 to 90 min, caused a decrease in water losses after thermal treatment and centrifugation (Fig. 2) and consequently an improvement of the WHC of the protein matrix; a further increase in setting time worsened the functional properties. On the basis of these results, an MTGase concentration of 0.3 g MTGase/100 g proteins and a setting time at 35 C of 60 min, were the optimal parameters for WHC improvement and are recommended to be used for increasing the yield of processed meat products and developing adequate sensorial and textural characteristics (Kuraishi et al., 1996).
21
76
18
73
15
70
12
WHC, g/100 g gel
3. Results and discussions
281
67 0
20
40
60
80
Setting time, min Fig. 2. The influence of setting time on gel water losses after thermal treatment (black full line) and centrifugation (grey full line) and on WHC (black broken line).
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32
7
30
6
Apparent viscosity, Pa.s
Soluble proteins in SDS, g/100 g d.s.
282
28 26 24 22
Blank 0.05% MTGase 0.1% MTGase 0.2% MTGase 0.4%MTGase
5 4 3 2 1
20 0
0.1
0.2
0.3
0
0.4
0
MTG-ase concentration, g/100 g proteins
20
30
40
50
Shear rate, s-1
Fig. 3. The influence of MTGase concentration on protein solubility in SDS.
Fig. 5. The influence of MTGase concentration on the apparent viscosity of the MPCHB-based emulsions.
35
6
Apparent viscosity, Pa.s
Soluble protein in SDS, g/100 g d.s.
10
30
25
20 0
20
40
60
80
100
Setting time, min Fig. 4. The influence of setting time on protein solubility in SDS.
Figs. 3 and 4 indicate nonlinear correlations between protein soluble in SDS and the enzyme level or setting time. We can partially explain these results as at a setting temperature of 35 C factors conditioning the denaturation and aggregation of protein during the gelling process are important. 3.4. Emulsifying properties The effect of MTGase on emulsifying properties of MPCBH was determined by evaluating the rheological behaviour of the emulsions and estimating emulsion stability on thermal treatment. Again different enzyme concentrations were used and the results are shown in Fig. 5 as apparent viscosity vs. shear rate. The MTGase catalyzed cross-linking of the myofibrillar proteins’ chains led to increased apparent viscosity of the MPCBH-based emulsions over the entire range of tested shear rates. The enhancement in viscosity was directly correlated with enzyme concentration. The influence of setting time on the rheological behavior of the MPCBH-based emulsions is presented in Fig. 6. For all shear rates, the apparent viscosity of the emulsions of MPCBH treated with 0.3 g MTGase/100 g proteins, decreased on increasing the setting time from 0 to 90 min.
Blank 30 min. 60 min. 90 min.
5 4 3 2 1 0 0
10
20
30
40
50
Shear rate, s-1 Fig. 6. The influence of setting time on the apparent viscosity of the MPCHB-based emulsions.
In all cases, the apparent viscosity vs. shear rate curves indicated decreasing emulsion viscosity with increasing shear rate up to a constant value, which corresponds to the point where the destruction rate of the structure match the reformation rate of the protein aggregates. This behavior is specific to pseudo-plastic fluids, with time-dependent structural viscosity. The viscoelastic behavior of the MPCBH-based emulsions was mathematically modeled as g ¼ a þ b cc ; where a represents the flowing threshold, b is the consistency index, and c the power index. The regression curves describe the nonlinear dependence of apparent viscosity (g) on the shear rate (_c), while the regression coefficients (r2) with values exceeding 0.9 indicate the continuous and uniform dissociation of the protein aggregates and the weakly linked fatty acid molecules of sunflower oil followed by the arrangement of these molecules along the flowing direction under the action of the shear forces. The thermal stability of the MPCBH-based emulsions was evaluated by determining the creaming index. The
A. Ionescu et al. / Meat Science 79 (2008) 278–284
Creaming index, %
100 98 96 94 92 90 Blank
30 min
60 min
90 min
Setting time Fig. 7. The influence of setting time on the stability of the MPCBH-based emulsions on thermal treatment.
emulsions based on MPCBH set with MTGase were less stable compared to the sample without MTGase. As expected, the setting time of the MPCBH with 0.3 g MTGase/100 g proteins before emulsifications was reflected in the stability of the emulsions on thermal treatment (Fig. 7). The results, in terms of emulsions stability agree with those of Motoki et al. (1984) who studied the influence of transglutaminase on the functional properties of casein. They explained differences between the stability of the emulsions based on native proteins and polymerized ones on the lower solubility of the cross-linked proteins. The increase in the number of cross-links determines the hydrophilic/hydrophobic balance of the emulsifier and the protein stabilizer by reducing the number of hydrophilic groups, which determines the separation of part of the aqueous phase of the emulsion. 4. Conclusions Activa TG-1 MTGase catalyzed the cross-linking of the myofibrillar proteins from beef heart and improved their functional properties. Gel properties were influenced by the enzyme level and setting time. The results suggest that 0.3 g MTGase/100 g proteins and a setting time of 60 min at 35 C are optimum to improve hydration and aggregation properties of the MPCBH. The enzymatic catalyzed cross-linking of the myofibrillar proteins’ chains influenced the viscoelastic behavior of the MPCBH-based emulsions, the apparent viscosity being directly correlated with enzyme concentration and setting time. Emulsion stability on thermal treatment was inversely correlated with the setting conditions. References AOAC (1984). Official methods of analysis (11th ed.). Washington, DC: Association of Official Analytical Chemists. AOAC (1991). Official methods of analysis (15th ed.). Washington, DC: Association of Official Analytical Chemists.
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AOAC (1995). Official methods of analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Carrascal, J. R., & Regenstein, J. (2002). Emulsion stability and water uptake ability of chicken breast muscle proteins as affected by microbial transglutaminase. Journal of food science, 67(2), 734–739. Christensen, B. M., Sørensen, E. S., Højrup, P., Petersen, T. E., & Ramussen, L. K. (1996). Localization of potential transglutaminase cross-linking sites in bovine caseins. Journal of Agriculture and Food Chemistry, 44, 1943–1947. Desmond, E. M., & Kenny, T. A. (1998). Preparation of surimi-like extract from beef hearts and its utilization in frankfurters. Meat Science, 50(1), 81–89. Ionescu, A., Aprodu, I., Zara, M., & Porneala, L. (2006). Estimation of some functional properties of myofibrillar protein concentrate from beef heart. Journal Scientific Study& Research-Chemistry & Chemical Engineering, Biotechnology, Food Industry, 7, 1–10. Ionescu, A., Aprodu, I., Zara, M., Vasile, A., & Gurau, G. (2003). The obtaining and characterization of the mechanically deboned chicken meat myofibrillar protein concentrates. The Annals of University ‘‘Dunarea de Jos’’ of Galati, Fascicle IV-Food Technology, 128–132. Jiang, S. T., Hsieh, J. F., Ho, M. L., & Chung, Y. C. (2000). Microbial tranglutaminase affects gel proprieties of gold threadfin-bream and Pollack Surimi. Journal of Food Science, 65(4), 694–699. Kato, A., Wada, T., Kobayashi, K., Seguro, K., & Motoki, M. (1991). Characterization of ovomucin-food protein conjugates prepared through the transglutaminase reaction. Agricultural and Biological Chemistry, 55, 1027–1031. Kuraishi, C., Sakamoto, J., & Soeda, T. (1996). The usefulness of transglutaminase for food processing. In Biotechnology for improved food and flavors. ASC Symposium Series 637 (Vol 637, pp. 29–38), Washington, DC. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological and Chemistry, 193(1), 256–257. Matsumura, Y., Chanyongvorakul, Y., Kumazawa, Y., Ohtsuka, T., & Mori, T. (1996). Enhanced susceptibility to transglutaminase reaction of- lactalbumin in the molten globule state. Biochimica et Biophysica Acta, 1292, 69–76. McKeith, F. K., Bechetel, P. J., Novakofski, J., Park, S., & Arnold, J. S. (1988). Characteristics of surimi-like material from beef, pork meat, and beef byproducts. Proceedings International Congress of Meat Science and Technology, 34, 325–326. Motoki, M., Nio, N., & Takinami, K. (1984). Functional properties of food proteins polymerized by transglutaminase. Agriculture and Biological Chemistry, 48, 1257–1261. Muguruma, M., Sakamoto, K., Numata, M., Yamada, H., & Nakamura, T. (1990). The effect of microbial transglutaminase on gelation of Myosin B, Myosin and Actin. Nippon Shokuhin Kogyo Gakkaishi, 37, 446–449. Nishimoto, S., Hasimoto, A., Seki, N., Chimura, I., Toyoda, K., & Arai, C. (1987). Influencing factors on changes in myosin heavy chain jelly strength of salted meat paste from Alaska pollack during setting. Nippon Suisan Gakkaishi, 53, 2011–2020. Nonaka, M., Tanaka, H., Okiyama, A., Motoki, M., Ando, H., Umeda, K., et al. (1989). Polymerization of several proteins by Ca2+independent transglutaminase derived from microorganisms. Agricultural and Biological Chemistry, 53, 2619–2623. Nonaka, M., Toiguchi, S., Sakamoto, H., Kawajiri, H., Soeda, T., & Motoki, M. (1994). Changes caused by microbial transglutaminase on physical properties of thermally induced soy protein gels. Food Hydrocolloids, 8, 1–8. Sakamoto, H., Kumazawa, Y., & Motoki, M. (1994). Strength of protein gels prepared with microbial transglutaminase as related to reaction conditions. Journal of Food Science, 59, 866–871. Seguro, K., Kumazawa, Y., Ohtsuka, T., Toiguchi, S., & Motoki, M. (1995). Microbial transglutaminase and e-(c-glutamyl)lysine cross-link effects on elastic properties of kamaboko gels. Journal of Food Science, 60, 305–311.
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MEAT SCIENCE Meat Science 79 (2008) 278–284 www.elsevier.com/locate/meatsci
The effects of transglutaminase on the functional properties of the myofibrillar protein concentrate obtained from beef heart Aurelia Ionescu a, Iuliana Aprodu a
b,* ,
Aura Daraba˘ a, Lenutßa Porneala˘
a
Department of Biochemistry, ‘‘Dunarea de Jos’’ University, 111 Domneasca Street, 800201 Galati, Romania b Department of Biomechanics, Politecnico di Milano, P.za L. da Vinci, 32, 20133 Milano, Italy Received 26 July 2007; received in revised form 25 September 2007; accepted 26 September 2007
Abstract The aim of the present study was to evaluate the effect of bacterial transglutaminase on the functional properties of the myofibrillar protein concentrate from beef heart. The degrees of hydration and aggregation and emulsifying properties were studied. The degree of polymerization of the myofibrillar proteins depended on the enzyme concentration and setting time; the best results in terms of functional properties were obtained with 0.3 g transglutaminase/100 g protein with 60 min setting at 35 C. This investigation confirms that transglutaminase may be used for the production of myofibrillar protein aggregates with enhanced functional properties. 2007 Elsevier Ltd. All rights reserved. Keywords: Myofibrillar proteins; Bacterial transglutaminase; Functional properties; Rheological behavior
1. Introduction Myofibrillar proteins play an important role when processing meat, being responsible for the formation of cohesive structures and a firm texture following thermal treatment (Xiong, 1997). Functional behavior of myofibrillar proteins is influenced by their ability to form viscous gels via protein–protein interactions, to retain water and, in the case of emulsions, to form resistant films on the surface of the fat droplets. The properties of processed meat products such as, tenderness, juiciness and perception of flavors during mastication are based on these functional properties of the proteins. In order to improve the functional properties of meats, processors are currently using a wide range of vegetable additives, especially soy protein derivates. Often, consumers reject these additives as being chemically modified or as being obtained from genetically modified plants. *
Corresponding author. E-mail address: [email protected] (I. Aprodu).
0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.09.011
Nowadays, interest in developing new technologies to obtain functional extracts, ingredients and additives from slaughter-house by-products, is increasing (McKeith, Bechetel, Novakofski, Park, & Arnold, 1988; Srinivasan, Xiong, & Decker, 1996; Wan, Xiong, & Decker, 1993). Due to their functional properties, myofibrillar protein concentrates obtained from mechanically deboned poultry meat and beef heart can be used in various meat products (Desmond & Kenny, 1998; Ionescu, Aprodu, Zara, & Porneala, 2006; Ionescu, Aprodu, Zara, Vasile, & Gurau, 2003). Functional properties of proteins, such as gelling capacity, gel strength, viscosity, thermal stability and waterholding capacity are directly related with the proteins’ composition and structure. The use of enzymatically catalyzed reactions to modify protein structure is an important way of improving their functional properties (Sakamoto, Kumazawa, & Motoki, 1994; Seguro, Kumazawa, Ohtsuka, Toiguchi, & Motoki, 1995; Seki et al., 1990). One of the most studied enzymes used for structural modification of myofibrillar proteins is bacterial transglutaminase. Transglutaminase (TGase; EC 2.3.2.13) is an enzyme widely spread in nature; in animal organisms it is localized
A. Ionescu et al. / Meat Science 79 (2008) 278–284
in liver, muscular tissue and body fluids. Transglutaminase catalyses the intra- and intermolecular transverse crosslinking of proteins by an acyl transfer reaction between glutamine (Glu) and lysine (Lys) residues. The structure of the proteins is stabilized by the strong covalent (e-(c-Glu)Lys) cross-links between the peptide chains. Jiang, Hsieh, Ho, and Chung (2000) proved, through SDS–PAGE analysis, that bands corresponding to H-meromyosin disappeared and bands corresponding to cross-linked polymers appeared. The affinity of TGase for different types of protein depends on the distribution of Glu residues as well on the secondary and tertiary structures of the proteins (Matsumura, Chanyongvorakul, Kumazawa, Ohtsuka, & Mori, 1996). Casein, soy proteins, conalbumin, rabbit and carp myosin, beef actin and myosin, ovomucin are examples of proteins which are suitable substrates for TGase (Christensen, Sørensen, Højrup, Petersen, & Ramussen, 1996; Kato, Wada, Kobayashi, Seguro, & Motoki, 1991; Muguruma, Sakamoto, Numata, Yamada, & Nakamura, 1990; Nonaka et al., 1989; Nonaka, Toiguchi, Sakamoto, Kawajiri, Soeda & Motoki, 1994; Sakamoto et al., 1994; Tanaka, Nonaka, & Motoki, 1990). Moreover, Carrascal and Regenstein (2002) found the effect of microbial TGase on the functional properties of chicken meat proteins. They report an enhancement of emulsion stability and water uptake of chicken muscle proteins treated with TGase. Our study was focused on bacterial TGase obtained by industrial fermentation of Streptoverticillium mobarense, which is commercialized under the name Activa TG, WM, and MP. The Activa products offer various possibilities for the processed food industry (dairy products, meat products, fish and surimi based foods) by (i) standardizing the texture and water-holding capacity, (ii) improving gel consistency and increasing the firmness, elasticity and juiciness of hams, salamis and products based on concentrates of myofibrillar proteins, (iii) improving the nutritive value of the raw materials of low biological value, and (iv) substituting polyphosphates in hams and salamis while maintaining the taste and flavor. There is little information about the interaction of MTGase with myofibrillar proteins from beef heart. We may expect some variations regarding the interaction between MTGase and myofibrillar proteins from beef heart with respect to those ones reported for chicken muscle proteins, due to differences in the structural organization within the fibers. Myosin of the red fibers, which are the main fibers of cardiac muscle, generally form shorter filaments than the myosin of white muscular fibers. Xiong (1997) found that at protein concentrations of 4–10 mg/ ml, the consistency index of breast poultry protein suspensions (white fibers) was higher than one of poultry thighs (red fibers). The objectives of the present paper were to obtain a functional ingredient based on beef heart myofibrillar pro-
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teins and to determine the effect of MTGase on its functional properties. 2. Materials and methods 2.1. Materials Activa TG-1 MTGase, provided by Ajinomoto (Inc. Teanec, NJ, USA) was used. The enzymatic product is made up of 99% maltodextrin and 1% transglutaminase with a declared enzymatic activity of about 100 UE/g. The enzyme is active over large ranges of temperature (2– 60 C) and pH (5–8), and is inactivated at high temperatures, depending on the composition of the food. Lygamme EFI80, which is a mixture of 30% xanthan gum and 70% guar gum, was obtained from Eurofood International. Beef hearts were purchased chilled from specialized stores. All chemicals used are of analytical grade. 2.2. Proximate composition and pH The moisture, protein and ash contents of the MPCBH were determined according to standard AOAC (1995) methods. The crude fat content was determined by AOAC (1991) method 991.36. All determinations were made in duplicate. pH measurements were made according to AOAC (1984). Ten grams of sample was homogenized with 90 ml distilled water for 2 min using the Braun mixer. The obtained mixture was filtered and the pH of the filtrate determined by means of a Hanna digital pH-meter. 2.3. Sample preparation The myofibrillar protein concentrate was obtained according to Ionescu, Aprodu, Zara, and Porneala (2006). The wet precipitate obtained after the final washing with phosphate with addition of 0.01% propyl gallate, as antioxidant, and centrifugation, was designated the myofibrillar protein concentrate of beef heart (MPCBH) and was characterized. To avoid protein denaturation during the entire washing cycle, the temperature of the dispersion was maintained below 10 C. The protein batter was obtained from wet MPCBH by mixing it with a 0.2 g mixture of guar and xanthan gum / 100 g proteins. Homogenization was performed in a Braun mixer for 2 min, with temperature control to avoid raising the temperature. In order to study the effect of transglutaminase concentration and setting time on the functional properties of MPCBH, many tests were performed using different enzyme concentrations (0.0, 0.5, 0.10, 0.20, 0.30, and 0.40 MTGase/100 g protein), and different setting times (30, 60 and 90 min) at 35 C. After adding the enzyme followed by 1 min homogenization, the samples were maintained in a water bath at 35 C for the given
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time; afterwards they were cooled at 20 C in a water bath. An aliquot of each sample was used to study the emulsifying properties of the raw batters, while the rest was placed in test tubes and subjected to thermal treatment (the temperature was increased at 1 C/ min) to achieve 70 C in the thermal centre of the sample, a temperature that was maintained for 10 min. The thermally treated samples were cooled with a mixture of water and ice to room temperature, and the obtained gels were stored overnight at 4 C. 2.4. Water losses and water-holding capacity (WHC) evaluation The quantity of the expressed water after thermal treatment was determined using the method described by Nishimoto, Hasimoto, Seki, Chimura, Toyoda, and Arai (1987), which was slightly modified. The water losses after thermal treatment were established by carefully separating the protein gel from the liquid expressed during cooking. The separated gel was weighed after all the liquid was absorbed by filter paper. The water losses during thermal treatment, Pt, are given by P t ¼ ws
ws wg 100; ws
where ws is the water content of the protein suspension, g; and wg is the water content of the gel, g. In order to determine the capacity of the gels to retain water, aliquots of the gel were placed on double layer filter papers in centrifuge tubes, weighed and centrifuged at 704g, for 10 min. After centrifugation, the gel was carefully separated, weighed and dried in an oven at 100 C for 24 h (Wang & Xiong, 1998). The water-holding capacity (WHC) of the proteins was estimated as WHC ¼
wg0 wg1 100; wg0
where wg0 is the water content of the gel before centrifugation, g; and wg1 is the water content of the gel after centrifugation, g. 2.5. Solubility of myofibrillar proteins in SDS The protein solubility in SDS (sodium dodecyl sulphate) was evaluated by the method proposed by Nishimoto et al. (1987) which was slightly modified. Gel (0.8 g) was homogenized with 15 ml of SDS solution (2% SDS, 8 M urea, 2% mercaptoethanol and 20 mM Tris–HCl; pH 8.0) using an high speed Ultraturax agitator for 3 min. The agitator was then washed in 10 ml distilled water, and the homogenized samples were transferred to glass containers, which, after closing, were maintained at room temperature, for 24 h, with occasional stirring. After 24 h, the samples were centrifuged at 1252g for 20 min, and the proteins soluble in SDS were determined in the resulting supernatant by the Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951).
2.6. Emulsion preparation To prepare the emulsions, the MPCBH was first dispersed in phosphate buffer with NaCl 0.6 M, pH 6.0 using the Braun mixer for 3 min. A model system consisting of 15 g protein suspension in saline solution (0.3% protein, w/v) and 60 mL sunflower oil (1:1, v/v) was used to test the ability of MPCBH to form and stabilize emulsions. The oil was added gradually, and the emulsion was obtained by continuous stirring for 3 min using a Braun mixer. The emulsion was divided into two parts: one for apparent viscosity evaluation, and the other for stability estimation. 2.7. Apparent viscosity evaluation of the MPCBH -based emulsions The apparent viscosity was evaluated by means of a RHEOTEST-2 type rotating viscosimeter manufactured by VEB-MEDINGEN, Germany. Due to the medium viscosity of the MPCBH-based emulsions the coaxial cylinder device S3 was used and 50 g of emulsion was tested. The working frequency was 50 Hz and the shear rate (_c) was varied within the range 0.1667–145.8 s1. The shear stress (sr ) was calculated starting from the value of a, which is the output value of the viscosimeter, using the relationship sr ¼ z a; where z is the device constant corresponding to the working domain I or II and to the cylinder type (dyn/cm2 div. scale). The apparent viscosity (g) of the MPCBH-based emulsions was calculated as g¼
sr 100ðPa sÞ: c_
2.8. Emulsion stability Twenty grams of emulsion was placed in a glass test tube with inner diameter of 15 mm and height of 120 mm. The sample was heated at 1 C/min to achieve 70 C in the thermal center of the product, and this temperature was maintained for 10 min. Afterwards the sample was cooled to 24 C in an ice bath and stored overnight at 4 C. Thermal destabilization, was determined through the creaming index defined as: (emulsion height/initial emulsion height · 100). 2.9. Statistical analysis Statistical analysis of the results was performed using Sigma Plot 2001/Statistics Date software. Five experimental batches were used for each test and the results reported as mean values. Experimental data were fitted using Table Curve 2D software and the regression equations were established based on statistical criteria (r2, Fit Standard Error or F Statistic).
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3.1. Chemical analysis of the MPCBH The results of chemical analysis showed that MPCBH is made up of 88.26 ± 1.23 % water; 10.12 ± 1.1% protein; 0.5 ± 0.34% fat and 0.54 ± 0.02% ash. The pH value of the MPCBH was 6.01. 3.2. Influence of the MTGase concentration and setting time on the degree of hydration The efficiency of using MTGase to improve the hydration properties of the myofibrillar proteins was tested by determining (i) water losses during the thermal gelling process of the protein systems based on MPCBH and (ii) WHC of the proteins after centrifugation. The influence of the MTGase concentration on the degree of hydration of the MPCBH was estimated by setting with enzyme at different concentrations for 60 min at 35 C. The results (Fig. 1) indicated that increasing the MTGase level from 0.05 to 0.3 g/100 g protein improved the WHC of the protein concentrate, due to the formation of cross-links among the polypeptide chains of the myofibrillar protein. Water losses after thermal treatment and centrifugation decreased in all cases on increasing the level of the added enzyme up to 0.3 g/100 g. The highest water losses were achieved in MPCBH without added enzyme, and the lowest in MPCBH with 0.3% MTGase. A further increase in enzyme concentration, to 0.4 g/100 g proteins led to a worsening of the WHC, which may be explained by enzyme competition for substrate. Our observations agree with Motoki, Nio, and Takinami (1984) and Kuraishi, Sakamoto, and Soeda (1996) who found that MTGase improves the WHC of proteins either by increasing the ability to swell and bind water, or by improving the ability to form a gel lattice. In order to estimate the influence of setting time on the degree of hydration, MPCBH was incubated with MTGase
3.3. Influence of the MTGase concentration and setting time on the aggregation degree Formation of protein aggregates, by inter- and intramolecular interactions mediated by MTGase, was estimated by determining the solubility of the gel proteins in SDS. The results indicated that production of e-(c-glutamil)lysine di-peptides was directly linked to the level of MTGase present in the reaction mixture. By adding MTGase, the solubility of MPCBH in SDS was substantially reduced (Fig. 3), due to the enzymatically catalyzed formation of covalent bonds between glutamine and lysine residues. The highest solubility in SDS was obtained for the MPCBH without enzyme, followed by the MPCBH with 0.05% MTGase. The decrease of protein solubility in SDS with increasing MTGase concentration confirms the participation of the enzyme in the polymerization process of the myofibrillar proteins of MPCBH. The influence of the setting time on protein aggregation was also estimated. Four setting periods were tested and the results showed that setting time influenced gel stability on thermal treatment and the solubility of proteins in SDS (Fig. 4). The MTGase effect on the myofibrillar proteins, reflected in the protein lattice strength was more marked at longer setting times.
72 14 69 12
10
66 0
0.1
0.2
0.3
0.4
MTG-ase concentration, g/100 g proteins Fig. 1. The influence of MTGase concentration on gel water losses after thermal treatment (black full line) and centrifugation (grey full line) and on WHC (black broken line).
Water losses, g/100 g gel
16
WHC, g/100 g gel
75
18
Water losses, g/100 g gel
(0.3 g /100 g proteins) for different times at 35 C. Increasing the setting time from 30 to 90 min, caused a decrease in water losses after thermal treatment and centrifugation (Fig. 2) and consequently an improvement of the WHC of the protein matrix; a further increase in setting time worsened the functional properties. On the basis of these results, an MTGase concentration of 0.3 g MTGase/100 g proteins and a setting time at 35 C of 60 min, were the optimal parameters for WHC improvement and are recommended to be used for increasing the yield of processed meat products and developing adequate sensorial and textural characteristics (Kuraishi et al., 1996).
21
76
18
73
15
70
12
WHC, g/100 g gel
3. Results and discussions
281
67 0
20
40
60
80
Setting time, min Fig. 2. The influence of setting time on gel water losses after thermal treatment (black full line) and centrifugation (grey full line) and on WHC (black broken line).
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32
7
30
6
Apparent viscosity, Pa.s
Soluble proteins in SDS, g/100 g d.s.
282
28 26 24 22
Blank 0.05% MTGase 0.1% MTGase 0.2% MTGase 0.4%MTGase
5 4 3 2 1
20 0
0.1
0.2
0.3
0
0.4
0
MTG-ase concentration, g/100 g proteins
20
30
40
50
Shear rate, s-1
Fig. 3. The influence of MTGase concentration on protein solubility in SDS.
Fig. 5. The influence of MTGase concentration on the apparent viscosity of the MPCHB-based emulsions.
35
6
Apparent viscosity, Pa.s
Soluble protein in SDS, g/100 g d.s.
10
30
25
20 0
20
40
60
80
100
Setting time, min Fig. 4. The influence of setting time on protein solubility in SDS.
Figs. 3 and 4 indicate nonlinear correlations between protein soluble in SDS and the enzyme level or setting time. We can partially explain these results as at a setting temperature of 35 C factors conditioning the denaturation and aggregation of protein during the gelling process are important. 3.4. Emulsifying properties The effect of MTGase on emulsifying properties of MPCBH was determined by evaluating the rheological behaviour of the emulsions and estimating emulsion stability on thermal treatment. Again different enzyme concentrations were used and the results are shown in Fig. 5 as apparent viscosity vs. shear rate. The MTGase catalyzed cross-linking of the myofibrillar proteins’ chains led to increased apparent viscosity of the MPCBH-based emulsions over the entire range of tested shear rates. The enhancement in viscosity was directly correlated with enzyme concentration. The influence of setting time on the rheological behavior of the MPCBH-based emulsions is presented in Fig. 6. For all shear rates, the apparent viscosity of the emulsions of MPCBH treated with 0.3 g MTGase/100 g proteins, decreased on increasing the setting time from 0 to 90 min.
Blank 30 min. 60 min. 90 min.
5 4 3 2 1 0 0
10
20
30
40
50
Shear rate, s-1 Fig. 6. The influence of setting time on the apparent viscosity of the MPCHB-based emulsions.
In all cases, the apparent viscosity vs. shear rate curves indicated decreasing emulsion viscosity with increasing shear rate up to a constant value, which corresponds to the point where the destruction rate of the structure match the reformation rate of the protein aggregates. This behavior is specific to pseudo-plastic fluids, with time-dependent structural viscosity. The viscoelastic behavior of the MPCBH-based emulsions was mathematically modeled as g ¼ a þ b cc ; where a represents the flowing threshold, b is the consistency index, and c the power index. The regression curves describe the nonlinear dependence of apparent viscosity (g) on the shear rate (_c), while the regression coefficients (r2) with values exceeding 0.9 indicate the continuous and uniform dissociation of the protein aggregates and the weakly linked fatty acid molecules of sunflower oil followed by the arrangement of these molecules along the flowing direction under the action of the shear forces. The thermal stability of the MPCBH-based emulsions was evaluated by determining the creaming index. The
A. Ionescu et al. / Meat Science 79 (2008) 278–284
Creaming index, %
100 98 96 94 92 90 Blank
30 min
60 min
90 min
Setting time Fig. 7. The influence of setting time on the stability of the MPCBH-based emulsions on thermal treatment.
emulsions based on MPCBH set with MTGase were less stable compared to the sample without MTGase. As expected, the setting time of the MPCBH with 0.3 g MTGase/100 g proteins before emulsifications was reflected in the stability of the emulsions on thermal treatment (Fig. 7). The results, in terms of emulsions stability agree with those of Motoki et al. (1984) who studied the influence of transglutaminase on the functional properties of casein. They explained differences between the stability of the emulsions based on native proteins and polymerized ones on the lower solubility of the cross-linked proteins. The increase in the number of cross-links determines the hydrophilic/hydrophobic balance of the emulsifier and the protein stabilizer by reducing the number of hydrophilic groups, which determines the separation of part of the aqueous phase of the emulsion. 4. Conclusions Activa TG-1 MTGase catalyzed the cross-linking of the myofibrillar proteins from beef heart and improved their functional properties. Gel properties were influenced by the enzyme level and setting time. The results suggest that 0.3 g MTGase/100 g proteins and a setting time of 60 min at 35 C are optimum to improve hydration and aggregation properties of the MPCBH. The enzymatic catalyzed cross-linking of the myofibrillar proteins’ chains influenced the viscoelastic behavior of the MPCBH-based emulsions, the apparent viscosity being directly correlated with enzyme concentration and setting time. Emulsion stability on thermal treatment was inversely correlated with the setting conditions. References AOAC (1984). Official methods of analysis (11th ed.). Washington, DC: Association of Official Analytical Chemists. AOAC (1991). Official methods of analysis (15th ed.). Washington, DC: Association of Official Analytical Chemists.
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