Journal of Dairy Science
Volume 93, Issue 3 , Pages 841-848, March 2010

Characterization of wine rennet and its kinetics by gel electrophoresis

Technical Center, Beijing Sanyuan Foods Co. Ltd., Beijing 100085, P. R. China

Received 8 May 2009; accepted 27 November 2009.

Article Outline

Abstract 

The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln4-His5, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17, and Phe24-Phe25 at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu15-Tyr16. A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40°C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.

Key words: wine rennet, characterization, kinetics, gel electrophoresis

 

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Introduction 

Cheese making is a long and expensive process; consequently, there is an economic impetus toward shortening the time required, particularly the duration of ripening. Hydrolysis of caseins is the most important proteolytic event during cheese production and is caused chiefly by rennet, indigenous milk enzymes, and microbial enzymes derived from starter and nonstarter bacteria (Azarnia et al., 2006). Calf rennet (chymosin) is considered the best milk-clotting enzyme for cheese manufacturers, but it possesses a relatively low proteolytic activity and is easily inactivated during the cooking and stretching of cheeses (such as Swiss and Mozzarella), resulting in a long ripening time. On the other hand, a worldwide shortage of chymosin has developed in recent years largely because of an increase in cheese production. Coagulants from other sources, such as plant and microbial coagulants, have been used as calf rennet substitutes to produce cheese in some countries (Poza et al., 2004). Enzyme preparations of fungal origin show a considerably greater degree of chemostability or higher proteolytic activity compared with calf chymosin, which is a disadvantage for the quality of final product. A mixture of purified fungal rennets may provide preparations that are superior to calf rennet with respect to faster ripening and flavor development (Garg and Johri, 1994).

Glutinous rice wine, the culture of glutinous rice with commercial starter (chiu-yao), contains a rennet-like enzyme and alcoholic flavor and has been used exclusively as a coagulant for Chinese Royal cheese. The rennet-like protease from glutinous rice wine, namely wine rennet, was isolated and purified, and its biochemical and substrate characterized (Jiang et al., 2006, 2007; Xue et al., 2008). However, few data exist regarding its use as a rennet substitute or as an accelerator; even the kinetic behavior on the substrate of κ-casein has not been established definitively. The aim of this study was to further describe its characteristics, including the N-sequence, glycosylation state, specific cleavage sites on oxidized insulin B chain, and the kinetic parameters relative to the early action on bovine casein components.

Studies on dynamics of rennet toward natural caseins or synthetic peptides go back about 30 yr. Various methods, including HPLC, high-performance gel-permeation chromatography, automated ninhydrin, and spectrophotometry, have been developed to analyze hydrolysis (Visser et al., 1980; Carles and Martin, 1985; Carles and Ribadeau Dumas, 1985; Visser and Rollema, 1986; Vreeman et al., 1986). Ninhydrin and spectrophotometry are not direct assay methods for quantifying a specific hydrolysis fragment, whereas HPLC and high-performance gel-permeation chromatography require special equipment and sophisticated manipulation. As shown in Gaiaschi et al. (2000, 2001), SDS-PAGE may be a useful tool for quantitative analyze of the caseins and their fragments. In this work, urea-SDS/PAGE and densitometry were used to measure directly the kinetics of wine rennet on bovine casein components. In addition, we compared the dynamic behavior of rennets from Mucor miehei and calf under identical reaction conditions.

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Materials and Methods 

Enzyme Preparation 

Wine rennet, a purified freeze-dried powder from the laboratory of Technical Center of Beijing Sanyuan Foods Co. Ltd. (Beijing, China), was dissolved in 20mM sodium phosphate buffer, pH 6.5, to a concentration of 0.25g/L, corresponding to 6.9μM when the relative molecular mass (Mr) was taken to be 36,000Da (Jiang et al., 2007). Homogeneity of the enzyme preparation was examined by SDS-PAGE and N-terminal analysis. Chymosin and Mucor rennet from Mucor miehei were products of Sigma-Aldrich (St. Louis, MO) and were dissolved in the same buffer as wine rennet at a concentration of 5.95μM for calf rennet (Mr of 35,700Da; Vreeman et al., 1986), and 5.4μM for Mucor rennet (Mr of 39,000Da, according to the SDS-PAGE analysis).

Substrate Solutions 

Oxidized insulin B chain (sequencing grade) and bovine casein components (αS1-, β-, and κ-casein) were obtained from Sigma-Aldrich. The solution of insulin B chain (0.5mg/mL) and casein components (1.5mmol/L) were freshly prepared in 0.02M sodium phosphate buffer, pH 6.5. For kinetic measurements, concentrations of 6.8 to 341μM κ-casein (Mr of 19,000Da), and 3.6 to 181μM αS1-casein (Mr of 25,000Da) and β-casein (Mr of 24,000Da) were used. The protein concentrations of enzyme and substrate stock solutions were determined by using the micro-bicinchoninic acid method of Pierce Biochemicals (Rockford, IL) in which BSA (10 to 200μg/mL) served as the reference.

N-Terminal Sequence Determination 

The enzyme purified by SDS-PAGE (stacking gel, 4% acrylamide; running gel, 12% acrylamide) was transferred to polyvinylidene fluoride (PVDF) membrane. The N-terminal sequence was determined at the Life Science department, University of Beijing (China). A search for similarity of sequence was performed using the Blast algorithm in the National Center for Biotechnology Information (NCBI) protein bank.

Deglycosylation Analysis 

Deglycosylation of wine rennet was separately performed using peptide-N-glycosidase F (PNGase F) and o-glycosidase (Sigma-Aldrich) according to the manufacturer's instructions. For N-glycosylation analysis, 20μg of purified wine rennet diluted in 20μL of twice-distilled H2O was boiled for 10min in 1× glycoprotein denaturing buffer (0.5% SDS, 1% β-mercaptoethanol) to fully expose all glycosylation sites, and then incubated with 1 unit of PNGase F in 1× G7 reaction buffer (50mmol/L sodium phosphate, pH 7.5) supplemented with 1% Nonidet P-40 at 37°C for 2h. For the analysis of o-glycosylation, 100μg of purified wine rennet was diluted in 13μL of deionized water and mixed with 4μL of 5× reaction buffer (250mM sodium phosphate, pH 5.0). After the mixture was incubated at 37°C for 1h, 2μL of o-glycosidase was added and incubated at 37°C for 1 to 3h. The untreated and treated wine rennets were analyzed by 12% SDS-PAGE.

Cleavage Specificities Toward Oxidized Insulin B Chain 

Hydrolysis of Insulin B Chain 

One microgram of each enzyme was added to 100-μL solutions of oxidized insulin B chain (0.5mg/mL) prepared in the previous step. The mixtures were incubated at 37°C for 3h at pH 6.5. Then, 1 drop of concentrated NH4OH was added to inactivate the enzyme. To prepare the samples for HPLC-MS analysis, the hydrolysate was filtered through 0.22-μm filters.

HPLC-MS Analysis 

The untreated insulin B chain and the rennet-treated products were analyzed by HPLC on an Agilent 1100 system (Agilent Technologies, Santa Clara, CA) with a diode array detector using a Zorbax Eclipse XDB-C18 reverse phase column (5μm, 4.6 × 150mm, Agilent Technologies). The peptides were eluted with a linear gradient of acetonitrile (5–40% in 40min) in 0.1% formic acid at a flow rate of 0.8mL/min. The effluents were monitored by measuring the absorbance at 215nm and directly subjected to molecular mass determination using a HPLC-coupled Esquire 6000 ion trap mass spectrometer (Bruker, Bremen, Germany) operating in the positive electrospray mode, with a potential of 4kV applied to the electrospray needle. Full scanning analysis was performed in the range of 50 to 3,000m/z. The HPLC-MS spectra were analyzed by using the Biotools software (Bruker) for comparison with the variations.

Determination of the Kinetic Parameters 

Enzyme–Casein Reaction 

Caseins were digested with rennet (50:1, vol/vol) at 40°C for 120s, and the reactions were stopped immediately by mixing (1:1, vol/vol) with sample buffer containing mercaptoethanol (0.2M) and urea (8.0M). Duplicates were prepared and examined for each substrate concentration.

Electrophoresis Analysis 

The kinetic behavior of wine rennet on different casein components was analyzed by the modified urea-PAGE method of Andrews (1983), which was performed with 16.5% acrylamide gels (Sigma) and 0.1% SDS using a Mini-Protean Tetra electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The peptides formed during the enzyme reaction were quantified with a gel scanner and the Image Master 1D software (ver. 4.0; Tanon GIS 2010, Shanghai Tanon Science & Technology Co. Ltd., Shanghai, China). To calculate the amount of casein-derived fragments in each sample, a calibration curve was generated by plotting the known value of caseins loaded onto the gel versus the corresponding average density of pixels across the band length and integrating over the band width with Image Master software and assuming that the fragments had approximately the same affinity for Coomassie Blue. Every sample was analyzed at least 3 times, and the coefficient of variation was always <5%.

Calculation of Kinetic Parameters 

The initial velocity of the enzyme reaction was calculated using the amount of casein-derived fragments quantified by gel analyses with time. Initial rates (υ) and substrate concentrations ([S]) were used to construct Lineweaver-Burk plots of 1/υ against 1/[S]. From the linear plots, the kinetic parameters Michaelis constant (Km), catalytic turnover number (κcat), and proteolytic coefficient (κcat/Km) were calculated.

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Results and Discussion 

N-Terminal Sequence and Sequence Alignment 

The homogeneity of wine rennet determined by SDS-PAGE under denaturing and native conditions showed that it was a single polypeptide chain and in a molecular monomeric state because it migrated as a single band at the same rate (Figure 1). The N-terminal sequencing confirmed that the polypeptide comprised 15 amino acids, as shown in Table 1.

Table 1. The N-terminal amino acid sequence determination of wine rennet and the NCBI Blast search results
ProteinSourceCompared sequenceHomology1 (%)
Wine rennetChiu-yao (commercial starter)1GTGSVPVTDYE/QNDVE15—
Rhizopuspepsin-2 precursorRhizopus niveus69GTGSVPVTDYYNDIE8386
Rhizopuspepsin-2Rhizopus chinensis2GVGTVPMTDYGNDVE1673
Mucor rennetMucor bacilliformis1GTGTVPVTDDGNDIE1573
Mucor rennetMucor pusillus71GSVDTPGLYDFDLEE8520
Calf chymosinBos taurus60EVASVPLTNYLDSQY7340
EndothiapepsinCryphonectria parasitica90STGSATTTPIDSLDDA10533

1Comparison with sequences in National Center for Biotechnology Information database.

The microorganisms involved in the fermentation of glutinous rice were isolated and identified, and included mainly mold, yeast, and bacterium; the milk-clotting enzyme may be produced by the mold (Liu et al., 2001; Teng, 2005). Results of sequence analysis by the NCBI Blast algorithm showed that the N-terminal peptide sequence of the wine rennet conformed to that of the rhizopuspepsin from Rhizopus fungus, and the homology was 86 and 73% with Rhizopus niveus (direct submission) and Rhizopus chinensis (Takahashi, 1988), respectively (Table 1). Sequence identities with calf chymosin (Hidaka et al., 1986), Mucor rennin from Mucor pusillus (direct submission), and endothiapepsin from Cryphonectria parasitica (Barkholt, 1987) were <40%. However, the percentage of sequence identity between wine rennet and Mucor bacilliformis protease, a potentially better substitute of the calf rennet (Machalinski et al., 2006), was up to 73%. This finding confirmed that the wine rennet was produced from the mold strain.

Glycosylation Analysis 

It has been reported that the amino acid sequence of Mucor rennet contains possible N-glycosylation sites, and the enzyme from M. miehei has a high carbohydrate content (Rickert and McBride-Warren, 1974; Aikawa et al., 1990). In contrast, rennets such as rhizopuspepsin from R. niveus are not glycosylated as is the case for bovine chymosin (Costa et al., 1997). The glycosylation state of wine rennet was determined with PNGase F (N-glycosylation) and o-glycosidase (o-glycosylation), and results are shown in Figure 2. No changes in molecular weight between the digested and undigested rennet suggest that this protease is not glycosylated during the fermentation of commercial starter. We also confirmed that wine rennet is a member of the rhizopuspepsin group.

The importance of glycosylation is not well known in the aspartyl-protease family. Aikawa et al. (1990) found that, besides the effect on secretion from cells, glycosylation caused distinct modulation of the enzyme properties including milk-clotting and proteolytic activities, resulting in a lower ratio of clotting activity and proteolytic activity, which was disadvantageous for cheese-making. It has been proposed that glycosylation leads to higher thermostability by stabilizing molecular conformation (Costa et al., 1997), and thus cannot be inactivated at normal pasteurization temperatures, which leads to problems in processing of cheese whey (Garg and Johri, 1994). The deglycosylation analysis suggests that wine rennet may be superior to Mucor rennet as a chymosin substitute.

Identification of Specificity Sites 

According to Garg and Johri (1994), rennets hydrolyze peptide bonds flanked by bulky hydrophobic amino acids, and calf chymosin, Mucor rennin, and endothiapepsin from C. parasitica preferentially split aromatic and hydrophobic amino acid as evidenced by bond specificity on the oxidized insulin B chain. To determine the cleavage specificity of wine rennet, the product of enzyme-digested insulin B chain was analyzed by HPLC-MS (Figure 3). Seven fractions were separated on HPLC (Figure 3A), each of which was further identified from its molecular mass scanning by MS (Figure 3B). The fragments a, b, c, and g in Figure 2A corresponded to peptides Phe25-Ala30 (725.7Da), Leu17-Phe24 (928.01Da), His5-Leu15 (1,602.3Da), Phe1-Leu15 (1,715.3Da), Phe1-Tyr16 (1,877.2Da), and Leu15-Ala30 (1,911.4Da), respectively (Figure 3B). For fraction d, peptide mapping indicated that it was a mixture of fragments Leu17-Ala30 (1,634.6Da) and Tyr16-Phe24 (1,043.1Da), because the molecular mass calculated from the amino acid sequence was consistent with the observed mass, 818.5Da ([M+2H]2+) and 1,091.3Da ([M+H]+). The signal peak with a mass of 874.7Da ([M+4H]4+) determined by MS was the fraction h in HPLC and matched the sequence of untreated insulin B chain. The investigation of HPLC-MS spectra revealed that there were 5 cleavage sites on the B-chain of insulin at pH 6.5 by wine rennet, including Gln4-His5, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17, and Phe24-Phe25 (shown in Figure 4). Because of the higher frequency of peptides starting or ending with Tyr16, Leu17, and Leu15 compared with that of other peptides, it was suggested that Tyr16-Leu17 was the susceptible target attacked at pH 6.5, followed by Leu15-Tyr16.

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  • Figure 3. 

    Analysis of wine protease digest from oxidized insulin B chain by HPLC-mass spectrometry; A) HPLC analysis, where a to h indicate HPLC fractions; B) mass spectrometry analysis.

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  • Figure 4. 

    Summary of the specificity of the rennet-like protease from rice wine and compared with other sources on the oxidized insulin B chain at pH 6.5 and 37°C. Wine=the rennet-like enzyme from glutinous rice wine; Calf=calf chymosin; Mucor=aspartic protease from Mucor miehei; Rhizopus=aspartic protease from Rhizopus chinensis (Athaudaa and Takahashia, 2002).

Furthermore, the cleavage specificity of calf and Mucor rennet toward insulin B chain were observed under the same conditions. As shown in Figure 4, wine rennet showed a specificity similar to that of acid proteases from M. miehei and R. chinensis (Athaudaa and Takahashia, 2002), but different from chymosin. Although the fungal rennet had the same cleavage and preferential sites as wine rennet, about 100% of insulin B chain was hydrolyzed (not showed the HPLC results) by Mucor rennet; and the major sites by rhizopuspepsin was Phe24-Phe25 (Athaudaa and Takahashia, 2002). For chymosin, a large portion of substrate was not degraded, but 3 distinct cleavage sites, Glu13-Ala14, Leu15-Tyr16 and Leu17-Val18, were identified. The cleavage at Leu17-Val18 was the major site and not observed for wine or Mucor rennets. It was noted that the sites of the insulin B chain attacked by the rennets were different from those in previous reports (Garg and Johri, 1994). This may be related to the difference of the pH used for digestion in this study, which has a marked effect on the cleavage specificity for aspartic protease (Athaudaa and Takahashia, 2002).

Kinetic Properties 

The wine rennet, an exclusive coagulant for Chinese Royal cheese, exhibits caseinolytic activity toward bovine κ-casein and other casein components (Jiang et al., 2007). However, there are no reliable data about the kinetic properties related to the reaction of wine rennet on caseins. In this paper, the experiments determined the catalytic parameters of wine rennet on the main caseins by gel electrophoresis. The proteolytic fragments are shown in Figure 5, and the profiles of digestion products obtained by chymosin and Mucor rennet were run in parallel. As detected by Gaiaschi et al. (2000, 2001) for proteolytic fragments derived from αS1- and β-casein, the bands defined as α1 and β1 in Figure 5 corresponded to the αS1-casein fragment (24–199) and the β-casein fragment (29–209 and 30–209), respectively. The band marked κ1 was para-κ-casein (1–105) for chymosin and Mucor rennet (Shammet et al., 1992) or para*-κ-casein (1–94) for wine rennet (Jiang et al., 2007). The amount of product associated with bands α1, κ1, and β1 at each substrate concentration were determined by densitometry. Figure 6 shows the linear Lineweaver-Burk plots of 1/υ against 1/[S], from which the dynamic parameters were determined and incorporated in Table 2.

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  • Figure 5. 

    Urea-SDS/PAGE patterns of bovine casein digestion by different rennets at 40°C and pH 6.5. Mk=molecular weight standard; αS, β, κ=αS1-, β-, and κ-casein; w=wine rennet; c=chymosin; m=Mucor rennet; α1, β1, and κ1=the products derived from αS1-, β-, and κ-casein.

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  • Figure 6. 

    Lineweaver-Burk plots for the cleavage of κ-casein (A), αS1-casein (B), and β-casein (C) by wine, calf, and Mucor rennets at pH 6.5 and 40°C. υ = initial rate; [S]=substrate concentration.

Table 2. Kinetic parameters1 of rennet action on bovine caseins
SubstratePeptideWine rennetCalf rennetMucor rennet
Km (mM)κcat (s−1)κcat/Km (mM/s)Km (mM)κcat (s−1)κcat/Km (mM/s)Km (mM)κcat (s−1)κcat/Km (mM/s)
κ-CN1–1052(1–94)30.043±0.0025.9±0.12135.60.206±0.00133.1±1.1160.70.131±0.00616.7±0.13127.3
β-CN29–20930.064±0.0021.28±0.0320.10.277±0.0046.78±0.2724.40.179±0.0052.0±0.0611.2
αS1-CN24–19930.929±0.02513.0±0.3714.00.215±0.0033.4±0.00415.90.455±0.0106.0±0.0913.2

1Km=Michaelis constant; κcat=catalytic turnover number; and κcat/Km=proteolytic coefficient.

2The AA positions in casein ± errors.

3The para*-κ-casein for wine rennet.

As expected, different kinetic behaviors were observed for the 3 proteases on each substrate. With regard to the wine rennet, the Km was 15 to 22 times higher for αS1-casein (0.929±0.025mM) than for β-casein and κ-casein (0.064±0.007mM and 0.043±0.002mM, respectively), whereas the κcat/Km value of κ-casein (135.6mM/s) was about 7- to 10-fold greater than that of β- and αS1-casein (Table 2). The results suggested that wine rennet hydrolysis of κ-casein compared with αS1- and β-casein showed a much higher affinity and catalytic efficiency during the initial reaction, which was an important property for rennet at the primary phase of milk clotting (Garg and Johri, 1994). The results in Table 2 indicate similar phenomena for the other 2 enzymes. Consequently, for all bovine caseins, κ-casein showed an optimum substrate property to the 3 rennets as reflected by the parameter κcat/Km.

The present studies also revealed some differences in kinetic properties among rennets from wine (Rhizopus species), calf stomach, and M. miehei. The differences between the wine rennet and Mucor rennet were smaller than those between these 2 rennets and calf rennet. Analysis of Km indicated that the microorganism (Rhizopus and Mucor) enzymes acted preferentially on κ-casein, followed by β-casein and finally αS1-casein, whereas the calf rennet acted preferentially on κ-casein, followed by αS1-casein and then β-casein. Results in Table 2 show that κcat (and thus Vmax) of chymosin on κ-casein was almost 2-fold greater than that of Mucor rennet and almost 6-fold greater than that of wine rennet. Nevertheless, calf chymosin had a similar proteolytic coefficient (κcat/Km) to the latter 2 rennets because of a higher Km value. In the case of β-casein, the kinetic parameters (except the Km of wine rennet) on the Lys28-Lys29 bond were of the same order of magnitude for the 3 enzymes in this study, although the Km for wine rennet was about 4.3 and 2.7 times lower than that for chymosin and Mucor rennet, respectively (Table 2). For αS1-casein, wine rennet had a higher κcat value, which indicated that wine rennet should enhance the proteolysis of αS1-casein during the enzymatic phase of cheese making, although it had lower affinity as implied by higher values of Km; all rennets exhibited almost the same κcat/Km values. Moreover, the urea-PAGE profiles of the hydrolysis of αS1- and β-casein by wine rennet showed several other fragments that cannot be observed in digestions by calf rennet or Mucor rennet (Figure 5). Overall, the wine rennet exhibited significant milk-clotting activity on κ-casein and caseinolytic activity on αS1- and β-caseins.

It was noted that the kinetic parameters for chymosin on κ- and αS1-caseins in this study were different from those found previously (Hill et al., 1974; Carles and Martin, 1985; Carles and Ribadeau Dumas, 1985; Vreeman et al., 1986). This fact was mainly related to the difference of substrate and the content of active enzyme. The heterogeneity of bovine caseins has a certain extent of influence on kinetics of the reaction (Vreeman et al., 1986). For κ-casein, in addition to the amino acid replacements, the amounts of carbohydrate and phosphate group were different in each genetic variant. The characterization of bovine caseins employed in present experiment was not determined. The isolated κ-casein mainly exists in solutions with an Mr of 600,000 (30 monomers) and a diameter of 23nm, whereas at low concentrations (<25μM), only monomers (Mr 19,000) are present. The wide difference in size of κ-casein particle could influence the kinetic parameters (Vreeman et al., 1986). Under our working conditions, κ-casein concentrations were lower compared with those employed in previous studies (Hill et al., 1974; Carles and Martin, 1985; Carles and Ribadeau Dumas, 1985). The enzyme concentrations used to calculate the initial velocity of the reaction of wine rennet on bovine caseins in this paper were the content of whole protein, not of the active enzyme. Therefore, different results were obtained from our experiment.

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Conclusions 

In summary, wine rennet is a monomeric and nonglycosylated acid protease produced by molds of the genus Rhizopus. The HPLC-mass spectra revealed that wine rennet resulted in 2 major split bonds, Tyr16-Leu17 and Leu15-Tyr16, and 3 other cleavage sites, Gln4-His5, Ala14-Leu15, and Phe24-Phe25 on the insulin B chain at pH 6.5, which was different from calf chymosin under the same conditions. The results of kinetics showed that the 3 studied rennets had a similar coagulant efficiency, reflected by the proteolytic coefficient for κ-casein, but the reaction rates were distinct from each other. The catalytic characterization of wine rennet showed a higher caseinolytic activity on αS1- and β-casein than the Mucor enzyme. In other words, wine rennet purified from glutinous rice wine showed significant milk-clotting ability and caseinolytic activity; therefore, it would be a good assistant for calf rennet or as an accelerator used in the process of cheese making.

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Acknowledgments 

We thank Wang Yunhai (Beijing University of Technology) for his help in performing HPLC-MS spectra and anonymous reviewers for their insightful comments. This work was funded by the Research and Development Program of China (2006BAD04013) from the National Department of Science and Technology.

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PII: S0022-0302(10)00050-0

doi:10.3168/jds.2009-2364

Journal of Dairy Science
Volume 93, Issue 3 , Pages 841-848, March 2010

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