microbial transglutaminase catalyzed the cross-linking of myofibrillar/soy protein isolate mixtures

MICROBIAL TRANSGLUTAMINASE CATALYZED THECROSS-LINKING OF MYOFIBRILLAR/SOY PROTEINISOLATE MIXTURESMIN YI HAN1,2, HAI ZHEN ZU1, XING LIAN XU1,3 and GUANG HONG ZHOU1

1National Center of Meat Quality and Safety Control, Synergetic Innovation Center of Food Safety and Nutrition, Nanjing Agricultural University,Nanjing 210095, China2College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang, China

3Corresponding author. College of FoodScience and Technology, Nanjing AgriculturalUniversity, Nanjing 210095, China.TEL: +862584395939;FAX: +862584395939;EMAIL: [email protected]

Received for Publication February 22, 2014Accepted for Publication June 2, 2014

doi:10.1111/jfpp.12316

ABSTRACT

The effects of temperature and ionic strength on the cross-linking of myofibrillarprotein isolate (MPI)/soy protein isolates (SPI) catalyzed by microbialtransglutaminase (MTGase) were studied. The SPI treated with MTGase formed asubstantial amount of cross-linking at temperatures ≥50C. All the SPI constituentsexcept the basic subunits (B) of glycinin were cross-linked and formed polymers.For MPI/SPI mixtures heated with MTGase, the actin band was gradually reducedwithin the temperature range from 20 to 90C. The addition of KCl graduallyreduced the protein band changes and the myosin heavy chain and actin bandsbecame less noticeable detected by the sodium dodecyl sulfate–polyacrylamide gelelectrophoresis. The rheological results indicated that the treatment of MPI withMTGase significantly improved the elasticity of the MPI gels, irrespective of theincubation time. MTGase treatment significantly enhanced the gel properties ofthe mixed MPI/SPI protein gels.

PRACTICAL APPLICATIONS

Myofibrillar proteins are the main contributors imparting textural attributes andfunctional properties to muscle foods. Defining the performance of myofibrillarproteins during heat-induced gelation is beneficial in maintaining quality anddeveloping processed meat products and processes. A better understanding of thegelation properties of protein (muscle and nonmuscle) in the presence or absenceof microbial transglutaminase, would contribute to improving its industrial utili-zation and the quality of meat products.

INTRODUCTION

Soy protein isolate (SPI) are widely used in ground or emul-sified muscle products because of their specific properties asbinders and emulsifiers (Pietrasik and Li-Chan 2002). To adegree, the functionality of SPI depends on their interactionwith muscle proteins (Mansour and Khalil 1999). SPIinclude two major globular fractions, glycinin andβ-conglycinin, which are remarkably resistant to denatur-ation (Petruccelli and Anon 1995; Feng and Xiong 2002). Inaddition, none of the major soy globulins react with muscleproteins under normal meat processing conditions (tem-

perature 65–73C, pH 5.5–6.0, ionic strength 0.1–0.6), whichis considered an important deficiency for their functionality(McCord et al. 1998).

In order to improve food proteins, many researchers haveshown great interest in the microbial transglutaminase(MTGase) cross-linking reaction. MTGase is an enzymethat catalyzes an acyl-transfer reaction between theγ-carboxyamide group of a peptide or protein-boundglutaminyl residues and primary amines. The MTGase pro-duced by Streptomyces bacteria is widely used in food pro-cessing (Taguchi and Momose 2002). The addition ofMTGase to food products can improve their quality. Nielsen

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(1995), Dickinson (1997), Kuraishi et al. (1998) and Motokiand Seguro (1998) identified the products of the MTGasereaction, i.e., ε-(γ-glutamyl) lysyl cross-links between foodproteins, and cross-linking different food proteins withMTGase such as milk proteins, egg proteins, raw myofibrilpreparation, myosin and soy protein isolates were reported(Motoki and Seguro 1998; Guo et al. 2013; Stangierski et al.2013). Patented method of producing gluten-free bread andtreating pale soft exudative meat with transglutaminasewere also issued (Milkowski and Sosnicki 1999; Diowkszet al. 2013). Recently, Zhong et al. (2013) demonstrated thefeasibility of using sequential preheating and MTGase pre-treatments to improve stability of whey protein isolate atpH 7.0 during thermal sterilization.

Combining nonmuscle proteins with MTGase appears toprovide a useful approach to improve protein functions,especially the gelation properties. Favorable effect ofMTGase on gelation of myofibrillar protein from chickenwas found by Ramirez-Suarez and Xiong (2003). A betterunderstanding of the gelation properties of protein (muscleand nonmuscle) in the presence or absence of MTGasewould contribute to improving its industrial utilization andthe quality of meat products. However, little informationexists on the reaction conditions between myofibrillarprotein isolate (MPI) from beef and SPI catalyzed byMTGase. Therefore, we evaluated the effect of MTGase onthe dynamic rheological and gelling properties of mixedMPI from beef and SPI under various conditions.

MATERIALS AND METHODS

Preparation of Samples

Beef (Longissimus dorsi) were purchased from a localgrocery store. Then the beef were cut into pieces andground through a precooled meat mincer machine (HKS-332, Jiaxing Ribon Machine Engineering Co., Ltd., Jiaxing,China) using a 4.5 mm plate. The ground beef were storedat −20C until needed for MPI preparation. MPI wereextracted following the procedure of Busch et al. (1972).After thawing at 4C, minced beef was homogenized at 4Cfor 20 s in a Waring blender (Waring Products Division,Dynamics Corp. of America, New Hartford, CT) with sixvolumes (v/w) of extraction buffer (20 mmol/L Tris–HCl,pH 7.0, 5 mmol/L ethylene diamine tetraacetic acid[EDTA]). After centrifugation at 1,000 × g for 10 min(Beckman Avanti J-E centrifuge, Beckman Coulter, Fuller-ton, CA) at 4C, the pellets were suspended in the extractionbuffer and this procedure was repeated five times. After thelast centrifugation, the pellets were suspended in fivevolumes (v/w) of extraction buffer and homogenized in aWaring blender for 25 s. To remove the connective tissue,

the homogenate was filtered through a 20-mesh nylon net,and then centrifuged at 1,000 × g for 10 min (BeckmanAvanti J-E centrifuge, Beckman Coulter) and washed withbuffer. Then, the pellets were suspended in 100 mmol/L KCland centrifuged under the same conditions. All the isola-tions were conducted at 4C. Protein content of final pellet(about 8%) during the preparation of MPI was determinedby the Biuret method with bovine serum albumin as a stan-dard (Gornall et al. 1949). Commercial SPIs were purchasedfrom Sun Biology (pH 7.0, protein content of 89.14%;Nantong, China). The MTGase was supplied by Yiming(Taizhou, China) as a mixture (99% maltodextrin and 1%MTGase) with MTGase activity of approximately 100units/g. The enzyme concentration reported here is thecommercial concentration, and all chemicals used were atleast reagent grade.

Electrophoretic Analysis

Experiments were performed with two to three replicationsto examine the effect of temperature and ionic strength onMTGase-catalyzed MPI/SPI mixture cross-linking. About2 g SPI-alone (2.0% protein 0.6 mol/L KCl in 50 mmol/LK2HPO4, pH 7.0), MPI-alone (2.0% protein, 0.6 mol/L KClin 50 mmol/L K2HPO4, pH 7.0) and MPI/SPI mixtures (1:1ratio, 4.0% total protein, 0.6 mol/L KCl in 50 mmol/LK2HPO4, pH 7.0) were treated and incubated for 0 h with0.5% MTGase. All the solutions were heated in a thermo-statically controlled water bath (HH42, Changzhou GuohuaElectric Appliance Co., Ltd., Changzhou, China) from 20 to90C at a rate of 1C/min to elucidate the dynamic, progres-sive protein changes during the heat-induced gel networkstructure formation. Samples were taken for measurementat 20, 30, 40, 50, 60, 70, 80 and 90C (no incubation). Toexamine the effects of ionic strength, (0, 0.15, 0.45,0.60 mol/L KCl), the MPI/SPI mixtures (1:1 ratio, 0.15%total protein, 50 mmol/L K2HPO4, pH 7.0) were incubatedwith 0.2% MTGase preparation at 4C in a refrigerator(HYC-940, Haier Group, Qingdao, China) and analyzedafter 0, 5, 15, 30, 60, 120 and 240 min. The protein changescaused by possible cross-linking reactions in all the control(no MTGase) and enzyme-treated samples were analyzedusing sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS–PAGE).

The SDS–PAGE was performed using Bio-Rad verticalslab gel apparatus (Bio-Rad Laboratories, Inc., Hercules,CA) with a 12.5% acrylamide gradient separating gel and4% acrylamide stacking gel (Laemmli 1970). Briefly, treatedprotein samples were mixed with SDS–PAGE sample buffer(4% SDS, 20% glycerol, 0.125 mol/L Tris–HCl, 10%β-mercaptoethanol and 0.5% bromphenol blue [pH 6.87])in a 1:1 (v/v) ratio, and dissolved by heating in boiling waterfor 3 min. Aliquots of 20 μg of protein per lane were loaded

MTG CATALYZED CROSS-LINKING PROTEIN MIXTURES M.Y. HAN ET AL.

Journal of Food Processing and Preservation •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.2

onto the acrylamide gel. Electrophoresis was first run at80 V for about 60 min, followed by 120 V for 2 h. The gelswere stained with 0.1% Coomassie brilliant blue R-250 inmethanol : acetic acid : distilled water (5:1:4 by volume) forabout 60 min. The distaining was performed in the samesolution except the Coomassie brilliant blue R-250 for 12 h.Broad Range Protein MW Markers, purchased fromPromega (Madison, WI), was used as molecular weightstandards for SDS–PAGE. The molecular weight of sepa-rated bands was calculated using FR-980 Biological Electro-phoresis Image Analysis System (Shanghai FURI Scienceand Technology Co., Ltd., Shanghai, China).

Rheological Measurement

MPI-alone (2.0% protein, 0.6 mol/L KCl in 50 mmol/LK2HPO4, pH 7.0) and MPI/SPI mixtures (1:1 ratio, 4.0%total protein, 0.6 mol/L KCl in 50 mmol/L K2HPO4, pH 7.0)were incubated with 0.5% MTGase for 0, 30, 120 and240 min at 4C in a refrigerator (HYC-940, Haier Group,Qingdao, China). The MTGase-treated samples were sub-jected to dynamic rheological testing using an AR1000 rhe-ometer (TA Instruments Ltd., Crawley, UK) equipped withtwo parallel plates (40 mm diameter) with a gap width of0.5 mm (Xiong and Blanchard 1994). Each sample (0.5 g)were loaded in the rheometer, equilibrated at 20C for10 min and heated from 20 to 80C at 1C/min. Excesssample was trimmed off and a thin layer of silicone oil wasapplied to the exposed free edges to block moisture. Thestorage (G′) and loss (G″) moduli were obtained at a fixedfrequency of 0.1 Hz with a maximum strain of 0.02. Theelasticity and viscosity of the gels are indicated by thestorage (G′) and loss (G″) moduli, respectively.

Statistical Analysis

A one-way analysis of variance was performed on quantita-tive gelation parameters (G′, G″) to test the significancebetween treatments using the Statistical Analysis Systemprogram (SAS 8.12, SAS Institute Inc., Cary, NC). A confi-dence level of 5% was used to compare means (P < 0.05).When significance was detected between treatments, themean values were compared using Duncan’s multiple rangetest.

RESULTS AND DISCUSSION

SDS–PAGE Analysis of MTGase-TreatedMPI/SPI Mixtures at Different Temperaturesand Ionic Strengths

Effect of Temperature. The electrophoretic changes forSPI-alone and with MTGase treatment are shown in Fig. 1.Treatment of SPI with MTGase produced various changes

with temperature compared with without MTGase: a heavyband with a molecular weight exceeding 310 kDa occurredat temperatures ≥30C; all the SPI components disappearedprogressively, except the basic glycinin subunit (B) and the100 kDa protein band, at temperatures ≥50C. The 100 kDaprotein band might be the dimer of tropomyosin inducedby MTGase (Chanarat and Benjakul 2012). We attributedthe disappearance of all the SPI components to their cross-linking into polymers that were too large to enter the

FIG. 1. SDS–PAGE OF SOY PROTEIN ISOLATE (2.0%, 0.6 MOL/L KCl IN50 MMOL/L K2HPO4, pH 7.0) TREATED WITHOUT (A) OR WITH 0.5%MICROBIAL TRANSGLUTAMINASE (B) AFTER HEATING AT DIFFERENTTEMPERATURESLane MW = high molecular protein molecular weight marker (in kDa);α′, α, β = subunits of β-conglycinin; A1, 2,3,4 and B = acidic and basicsubunits of glycinin.

M.Y. HAN ET AL. MTG CATALYZED CROSS-LINKING PROTEIN MIXTURES


separating gel (Lakemond et al. 2000). Treatment of theMTGase-catalyzed MPI resulted in the disappearance orreduction of all the MPI components (Fig. 2), which wasconsistent with the result of Xiong’s group (Ramirez-Suarezand Xiong 2002, 2003).

Heated MPI/SPI mixtures without MTGase were readilydissolved in the SDS/β-mercaptoethanol solution which wasobserved during the preparation of SDS–PAGE samples;and fundamentally an identical electrophoretic pattern wasproduced by samples heated to various temperatures(Fig. 3a). After adding MTGase, all the SPI componentsexcept the basic subunit (B) vanished; however, the changesin low temperature (20–30C) are minor, which indicatedthe compact globular structures of the 11S and 7S soybean

proteins make them rather poor substrates for MTGase,despite their relatively high glutamine contents (Larre et al.1992) and heat-treated soybean glycinins are more suscep-tible to enzyme polymerization than native glycininsbecause the surface lysine and glutamine residues ofglycinin increase with heating (Kieliszek and Misiewicz2014). The changes of SPI were accompanied by the suddendisappearance of the myosin heavy chain (MHC), tropo-myosin and troponin at temperatures ≥50C, which may beascribed to myosin denaturation (∼40–60C) (Bertram et al.2006), this may indicate denatured myosin is susceptible to

FIG. 2. SDS–PAGE OF MYOFIBRILLAR PROTEIN ISOLATE (2.0%,0.6 MOL/L KCl IN 50 MMOL/L K2HPO4, pH 7.0) TREATED WITHOUT(A) OR WITHOUT (B) 0.5% MICROBIAL TRANSGLUTAMINASE AFTERHEATING AT DIFFERENT TEMPERATURESLane MW = molecular weight marker (in kDa); MHC = myosin heavychain; LC1 = light chain 1; LC2 = light chain 2.

FIG. 3. SDS–PAGE OF MYOFIBRILLAR PROTEIN ISOLATE/SOY PROTEINISOLATE (1:1 RATIO, 4.0% TOTAL PROTEIN, 0.6 MOL/L KCl IN50 MMOL/L K2HPO4, pH 7.0) MIXTURE TREATED WITHOUT (A) ORWITH (B) 0.5% MICROBIAL TRANSGLUTAMINASE AFTER HEATING ATDIFFERENT TEMPERATURESLane MW = molecular weight marker (in kDa); MHC = myosin heavychain; α′, α, β = subunits of β-conglycinin; A1, 2,3,4 and B = acidic andbasic subunits of glycinin.



cross-link with actin or SPI catalyzed by MTGase. Moreover,the actin band density was reduced gradually, which sug-gested that MHC appeared to be a preferable substrate forcross-linking induced by MTGase compared with actin(Chanarat and Benjakul 2012, 2013). Kishi et al. (1991)reported MHC supplies over 50% of all cross-linkagesunder optimal conditions. Troponin T, MHC oligomers andproteins of 80 and 55 kDa account for the other 50%. Inturn, actin, tropomyosin, troponin I, troponin C and titin

(β-connectin) are weak substrates for transglutaminase.Chanarat and Benjakul (2012) demonstrated the changes oftroponin and believed the troponin also served as substratefor cross-linking reaction induced by MTGase. Similarobservations were reported by Nakahara et al. (1999), whorevealed that MTGase preferentially cross-linked connectin,followed by MHC, troponin T and actin, respectively. Incontrast, no changes were found in the content of actinobtained from beef (Dondero et al. 2006), chicken meat (An

FIG. 4. SDS–PAGE OF 0.2% MICROBIAL TRANSGLUTAMINASE (MTGASE)-MEDIATED 0 MOL/L (A), 0.15 MOL/L (B), 0.45 MOL/L (C), 0.6 MOL/L (D)KCl MYOFIBRILLAR PROTEIN ISOLATE/SOY PROTEIN ISOLATE (1:1 RATIO, 0.15% TOTAL PROTEIN, 50 MMOL/L K2HPO4, pH 7.0) INCUBATION FORVARIOUS TIMES AT 4CLane MW = molecular weight marker (in kDa); Con = the samples without MTGase; MHC = myosin heavy chain; α′, α, β = subunits ofβ-conglycinin; A1,2,3,4 and B = acidic and basic subunits of glycinin.



et al. 1996) and fish (Lantto et al. 2007; Chanarat andBenjakul 2013). An intramolecular association of myosin oractin treated with MTGase perhaps existed at temperaturesof 20 to 90C. Therefore, the interactions between MPI andSPI in the mixed proteins contributed to the Gln–Lys cross-linking of heteropolymers or aggregates.

Effect of Ionic Strength. The following protein changeswere seen in the mixed MPI/SPI incubated with MTGasewith 0 mol/L KCl (Fig. 4a): an instant reduction in MHCand the disappearance of actin (45 kDa) within 5 min; anincrease in MHC and the reappearance of actin after120 min; the emergence of a new protein band at about31 kDa at the beginning of incubation, and the disappear-ance of this band by 120 min; and a protein band at about310 kDa seen at the top of the gel at the end of incubation(120–240 min). In the mixed protein system, the initialchange in bands was complete by 5–15 min, while all theessential MPI components reoccurred after 120 min. Theseresults showed that the intramolecular interaction catalyzedby the enzyme in the MPI/SPI mixture was reversible. Ittook all the MPI components 5 h to recover when the MPI-alone was treated with MTGase (Ramirez-Suarez andXiong 2002), and 2 h to recover when the MPI/SPI mixturewas treated. Therefore, the presence of SPI probablyaccelerated the reaction. Folk (1969) also noted thatthe transglutaminase-catalyzed reaction was reversible. AsRamirez-Suarez et al. (2001) indicated, the reversibilityresulted from the reversible cross-linking activity ofMTGase, to a large extent.

Electrophoretic analysis of the mixed MPI/SPI with0.15 mol/L KCl (Fig. 4b) produced results similar to thoseseen in Fig. 4a. The protein changes gradually diminished atan increased ionic strength. The changes in the MHC andactin bands became less noticeable. At 0.45 and 0.60 mol/LKCl, the mixed proteins bands of β-conglycinin (α′, α) van-ished progressively, while the intensity of the band for theacid subunits (A1,2,3,4) gradually decreased in 120 to 240 min(Fig. 4c,d), suggesting that the effect of the increased ionicstrength on SPIs was more pronounced, and that differentenzyme reactions with ionic strength were probably attrib-utable to protein changes in either the MTGase or MPIprotein substrates. Changes of SPI in the MPI/SPI mixturewere more obvious in high salt concentration especially forlonger MTGase incubation time, as band intensity ofβ-conglycinin (α′, α) and acidic subunits (A1,2,3,4) ofglycinin decreased remarkably at the end of incubation,which is in agreement with Ramirez-Suarez and Xiong(2003). At low ionic strength, the acid subunits were moreaccessible to MTGase as they were outside the glycinincomplex (Marcone et al. 1998). A high salt concentrationaltered the soy globulin complex and enhanced the suscepti-bility of the reactive groups (Lys–NH2 and Gln–CONH2) to

MTGase (Lakemond et al. 2000). However, there is no cor-responding appreciable change in MPI which may suggestthat there were minimal MPI/SPI interactions at high ionicstrength conditions (Ramirez-Suarez and Xiong 2003).

Dynamic Rheological Properties

The heat-induced rheological changes in G′ and G″ in MPIswith or without MTGase treatment are shown in Fig. 5.MPI without MTGase reached a maximum storagemodulus (G′ = 55.26 Pa) and a maximum loss modulus(G″ = 18.21 Pa) at about 60C. However, MPI with MTGasereached a maximum storage modulus (G′ = 345.7 Pa) and amaximum loss modulus (G″ = 50.5 Pa) at about 60C.Therefore, the treatment of MPI with MTGase significantlyimproved the gelation (P < 0.05). Results obtained from thisexperiment are consistent with those presented in availableliteratures (Dondero et al. 2006; Chanarat and Benjakul2013; Jiang and Xiong 2013). However, both G′ and G″decreased sharply with increasing temperature. The reason

FIG. 5. REPRESENTATIVE RHEOGRAMS (G′ [a] AND G″ [b]) OFHEAT-INDUCED GEL OF MYOFIBRILLAR PROTEIN ISOLATE (MPI, 2.0%PROTEIN, 0.6 MOL/L KCl IN 50 MMOL/L K2HPO4, pH 7.0) WITHOUTOR WITH 0.5% MICROBIAL TRANSGLUTAMINASE (MTGASE, NOINCUBATION)



for the decrease was suggested to involve rearrangement ofcross-linkage between polypeptides (Egelandsdal et al. 1986;Wan et al. 1993) and could be explored in the further study.Jiang and Xiong (2013) suggested that a 7-h incubationwith MTGase accentuated the gel-strengthening effect bythese SPI samples. The B subunit in of SPI was the maincomponent manifesting structure reinforcement in thosemixed gels. In contrast, the G′ of MPIs treated with MTGasestarted to rise gradually until the temperature arrived atabout 45C (Fig. 5), which suggested a softer gel formed(Mohtar et al. 2013), indicating that the cross-linking pro-teins could develop an elastic structure at lower tempera-tures compared with MPI without MTGase. Figure 6 showsthe values of G′ and G″ of the MPI/SPI mixtures treatedwith MTGase for different times. Compared with MPI

(without MTGase), the maximum G′ (42.26 Pa) and G″(17.14 Pa) of the mixtures without MTGase tended belower, which meant that nonmodified SPIs had a detrimen-tal effect on the gelation of muscle protein, decreasing theviscoelasticity by hindering the protein–protein interaction.The length of the incubation time with MTGase had a slighteffect on the gelation of the mixed proteins. The highest G′value (913.5 Pa) was found when the sample was treated at0 min. As the incubation time increased, G′ decreased,although the maximum G′ value at 240 min was higherthan that at 120 min. G″ was much less variable than G′ forall observed times. Overall, G″ was substantially lower thanG′. These results indicated that incubation time caused lessrheological modification of the samples than the treatmentwith MTGase. A possible explanation is that MTGase

FIG. 6. REPRESENTATIVE RHEOGRAMS (G′[a] AND G″ [b]) OF HEAT-INDUCED OFMYOFIBRILLAR PROTEIN ISOLATE (MPI)/SOYPROTEIN ISOLATE (SPI, 1:1 RATIO, 4.0%TOTAL PROTEIN, 0.6 MOL/L KCl IN50 MMOL/L K2HPO4, pH 7.0) MIXTUREAFTER INCUBATION AT 4C WITH 0.5%MICROBIAL TRANSGLUTAMINASE (MTGASE).THE TREATMENT SAMPLES WEREINCUBATED WITH MTGASE FOR 0, 30, 120AND 240 MIN. MPI/SPI REPRESENTSUNTREATED SAMPLE WITHOUT MTGASE



produced more intramolecular cross-linking during incuba-tion, so that when the proteins were heated, the less openedprotein structure had fewer available reactive sites fornetwork formation (Oakenfull et al. 1997). Therefore, incu-bation time had adverse effect on the viscoelastic propertiesof the MTGase-treated mixtures. Regarding the temperaturerange corresponding to the maximum G′ and G″ values, theoptimum temperature for the gelation of enzyme-inducedMPI and the mixed proteins was 60–65C, which was slightlyhigher than the optimum temperature (50C) for the cataly-sis of MTGase (Ajinomoto 1998).

CONCLUSIONS

MTGase cross-linked all the MPI components except actinat 60 to 90C. For MPI/SPI mixtures heated with MTGase,the actin band was reduced and the “intramolecular” asso-ciation of myosin and actin because of Gln–Lys cross-linking did not exist. In the mixed protein system, theprotein gelation characteristic changes reached a maximumin 5 to 15 min. The addition of KCl reduced the proteinchanges and the MHC and actin bands became less notice-able. MTGase treatment significantly enhanced the elasticityof the mixed MPI/SPI protein gels.

ACKNOWLEDGMENTS

This research was supported by the Natural Science Foun-dation of Hebei Province (Grant No. C2013208014) and theShandong Modern Agricultural Technology & IndustrySystem (SDAIT-13-011-11), China.

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microbial transglutaminase catalyzed the cross-linking of myofibrillar/soy protein isolate mixtures

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