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All issues Volume 90 / No 5 (September–October 2010) Dairy Sci. Technol., 90 5 (2010) 601-609 Full HTML
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Issue
Dairy Sci. Technol.
Volume 90, Number 5, September–October 2010
Page(s) 601 - 609
DOI https://doi.org/10.1051/dst/2010022
Published online 28 May 2010

© INRA, EDP Sciences, 2010

1. INTRODUCTION

Yak can survive in temperatures as low as −40 °C and elevation ranging from 3000 to 5000 m, but cannot stand at temperatures above 25 °C and normal atmospheric pressure. Statistical data showed that China has 14 million yaks and accounts for 95% of total number of the world [15]. The dominating yak species in China are Maiwa yak, Zhongdian yak and Gannan yak; they feed themselves on the native forages all year round without any supplementation. This cold climate and high elevation-loving animal produces milk with a higher fat (6–10%) and protein (5.5%) content when compared to bovine milk [11].

In all types of milks, fat droplets are surrounded by a membrane named milk fat globule membrane (MFGM). MFGM mainly comprises proteins and phospholipids. There are over 40 identified different proteins, ranging in molecular weight from 15 to 240 kg·mol−1, among which at least six are glycoproteins [7]. The major proteins have molecular weights of 155, 67, 50 and 49 kg·mol−1, identified as xanthine oxidase (XO), butyrophilin (BUT), Periodic Acid Schiff 6 (PAS 6) and Periodic Acid Schiff 7 (PAS 7), respectively [6]. Moreover, it is assumed that some of the MFGM proteins possess specific nutritional properties [5]. As such, MFGM material and MFGM components have been isolated and characterized as valuable ingredients for application into new food products. However, further work is needed on the quantification of the different MFGM components in various dairy products and on the optimization of food-grade downstream extraction and production processes from milk or from its derivatives.

In contrast to the extensive work done on bovine milk, to our knowledge, no information is available on protein composition of yak MFGM. The aim of this study was therefore to detail protein composition of yak MFGM.

2. MATERIALS AND METHODS

2.1. Materials

Yak milk samples produced by three pure yak breeds were collected in autumn, the detailed information of which is given in Table I. The cream was separated according to the method of Ye et al. [13]. The cream was washed with a solution of KCl (1.5 g·L−1) within 3 h of collection and then the cream was held at 4 °C overnight before further processing and analysis.

Table I.

Sample information.

All the chemicals used were of analytical grade obtained from China Chemicals Reagent Co. (Harbin, China) or Sigma Chemical Co. (St. Louis, MO, USA) unless specified otherwise.

2.2. Determination of average fat globule size and specific surface area of whole yak milk

Fat globule size distribution was determined by a Laser Diffraction Particle Size Analyzer (Dandong Bettersize Instruments, Ltd., Liaoning, China). The casein micelles were dissociated by diluting the sample to 1/1000 in EDTA buffered saline (EDTA, 35 mmol·L−1, pH 7.0) prior to measurement. The refractive indices used were 1.458 and 1.460 for milk fat at 633 and 466 nm, respectively, and the refractive index was 1.33 for water [9]. Standard parameters were calculated by the software: the volumic average diameter (where v i is the volume of globules in a size class of average diameter, d i), The volume-surface average diameter and the specific fat surface area , where ρ is the milk fat density.

2.3. Determination of washed cream protein content

The total protein content of cream, washed with the solution of KCl (1.5 g·L−1), was determined using the Kjeldahl method (AOAC, 1974) by determining total nitrogen and multiplying it by a factor of 6.38.

2.4. Isolation of milk fat globule membrane material

Samples for MFGM were prepared from 1 L of fresh unpasteurized cream using the acidification method reported by Kanno and Kim [3]. The cream was washed three times with three volumes of Milli-Q water (38 °C) in a cream separator. The washed cream was allowed to crystallize for 4 h at 4 °C before churning through the use of a mixer. The resulting butter and buttermilk were separated using a sieve. The buttermilk was adjusted to pH 4.8 using hydrochloric acid (1 mol·L−1) in order to allow MFGM to precipitate out. The precipitated MFGM was centrifuged at 10 000× g, and MFGM pellet and supernatant were collected. The pH of both resuspended MFGM pellet (in water) and supernatant was adjusted to 6.8 using sodium hydroxide (1 mol·L−1). The resuspended MFGM pellet and supernatant were freeze dried and stored at −80 °C before analysis.

2.5. Electrophoresis

Protein composition of MFGM from washed creams was determined by SDS-PAGE. Samples were suspended in 0.5 mL of reducing buffer (6% Tris-0.5 mol·L−1, 10% glycerol, 5% β-mercaptoethanol, 2% SDS and 0.05% bromophenol blue). Samples were heated at 95 °C for 5 min and then centrifuged at 2500× g for 30 min in order to remove the fat from the sample. Supernatants (10 μL) were loaded onto 10% SDS-polyacrylamide gels. Molecular mass markers ranging from 30 to 200 kg·mol−1 (TransGen, Biotech, China) and milk proteins were run at 200 V. Protein bands were stained with a solution of Coomassie Brilliant Blue R-250. Gels were destained with a solution of methanol and glacial acetic acid at concentrations of 160 and 10 mL·L−1, respectively. Scanned images of the destained gels were analyzed using the ImageMaster software (Amersham Pharmacia Biotech, Newcastle upon Tyne, UK). The apparent molecular mass (Mw) of the bands on the SDS-PAGE was estimated from the mobility of proteins in the gel when compared with the mobility of the molecular mass markers. The gels were scanned using an Ultrascan XL laser densitometer, and the results were analyzed using an LKB 2400 GelScanXL software program (LKB Produkter AB, Bromma, Sweden) to obtain quantitative results.

2.6. Statistical analysis

Statistical analysis was performed using t test. A value of P ≤ 0.05 was considered significant. The results were achieved through the SPSS version 9.0 windows program (SPSS, Inc., 1998, Chicago, IL, USA).

3. RESULTS AND DISCUSSION

3.1. Characterization of fat globule from different yak breeds

The characterization of fat globule of three yak breeds is given in Table II. The size distributions of the fat globules from three yak breeds are presented in Figure 1. The volumic average diameter d 43 of Maiwa yak milk (4.34 μm) and Gannan yak milk (4.16 μm) was higher (P < 0.05) than that of Zhongdian yak milk (4.10 μm). The average specific fat surface area of Gannan yak milk (2.05 m2·mg−1) and Zhongdian yak milk (2.01 m2·mg−1) was higher (P < 0.05) than that of Maiwa yak milk (1.92 m2·mg−1). The total protein content of MFGM in Maiwa yak milk (11.12 ± 0.12 mg·g−1 fat) and Gannan yak milk (12.24 ± 0.09 mg·g−1 fat) was significantly higher (P < 0.05) than that of Zhongdian yak milk (9.72 ± 0.26 mg·g−1 fat). The values for total protein content of MFGM in Maiwa yak milk and Gannan yak milk were well similar to those found in bovine milk as reported by Patton and Huston [12] (10 mg·g−1 of fat globules) and Mulder and Walstra [10] (9 mg·g−1 of fat globules). The surface protein coverage in the fat globule of Maiwa yak milk (5.67 ± 0.07 mg·m−2) and Gannan yak milk (5.97 ± 0.08 mg·m−2) was higher (P < 0.05) than those of Zhongdian yak milk (4.81 ± 0.15 mg·m−2). The surface protein coverage in the milk fat globule of the three yak breeds was higher than that reported for bovine milk by Ye et al. [13] (1.85 mg·m−2) and lower than those indicated by Mulder and Walstra (9 mg·m−2) [10]. The higher surface protein coverage could be due to the combination of lower specific fat surface area and higher total protein of Maiwa yak, Gannan yak and Zhongdian yak milk fat globule which were different from those of bovine milk reported by Ye et al. [13] and Mulder and Walstra [10].

thumbnail Figure 1.

Particle size distribution of fat globule from Maiwa yak milk (…), Gannan yak milk (―) and Zhongdian yak milk (­­).
Table II.

Characteristics of yak milk fat globule from different yak breeds.

3.2. MFGM proteins of different yak breeds

Figure 2 illustrates the protein patterns of MFGM, isolated from Maiwa yak, Gannan yak and Zhongdian yak, determined by SDS-PAGE (10% acrylamide) under reducing conditions (Figs. 2A–2C). There were five major protein bands, ranging in molecular weight from 47 to 225 kg·mol−1. The apparent molecular mass of major bands on SDS-PAGE was estimated by comparison with the mobility of molecular weight standards. The approximate quantities of these proteins in the MFGM were determined by scanning the stained bands. The major bands corresponded to mucin 1 (MUC 1, Mw, 225.6 kg·mol−1), the largest protein of the MFGM. Many studies on cow’s milk have shown that the apparent molecular weight (Mw) of this protein ranges from 170 to 225 kg·mol−1 depending on the breed [6]. Molecular weight (Mw) of yak other MFGM proteins determined in this study was as follows: xanthine oxidase (Mw, 157.4 kg·mol−1), PAS III/IV (Mw, 78–98 kg·mol−1), butyrophilin (Mw, 67.5 kg·mol−1), PAS 6 (Mw, 50.2 kg·mol−1) and PAS 7 (Mw, 47.8 kg·mol−1). The major protein bands on SDS-PAGE (10% acrylamide) in MFGM of the three yak breeds showed similar electrophoretical patterns compared with those from MFGM of bovine milk described by Keenan and Dylewski [4] and Ye et al. [13], who reported that the major protein bands corresponded to mucin 1, xanthine oxidase, butyrophilin, PAS 6 and PAS 7. A faint band with molecular weight of 62 kg·mol−1 was also observed, near butyrophilin.

thumbnail Figure 2.

SDS-PAGE patterns (10% acrylamide gel), obtained under reducing conditions, of MFGM material from fresh whole yak milk. MFGM from Maiwa yak milk (A); MFGM from Gannan yak milk (B) and MFGM from Zhongdian yak milk (C). Membrane proteins are named according to Mather [6].

There were no qualitative electrophoretical differences observed between the MFGM of Maiwa yak, Gannan yak and Zhongdian yak milks (Figs. 2A–2C). However, there were quantitative differences (P < 0.05) in the percentage of some of the protein components among the milk of three yak breeds (Tab. III). There were five major protein bands on SDS-PAGE in the MFGM of three yak breeds. There were no differences observed (P > 0.05) in mucin 1, xanthine oxidase, PAS 6 and PAS 7 in the MFGM of the three yak breeds, but the percentages of mucin 1 and butyrophilin were significantly higher (P < 0.05) in Maiwa yak and Gannan yak MFGM than in Zhongdian yak MFGM. These values could be due to a higher content of total MFGM proteins in Maiwa yak and Gannan yak milks. To our knowledge, there was no report describing difference between breeds for both bovine MUC1 and butyrophilin. On the other hand, Ye et al. [13] have reported differences in xanthine oxidase, butyrophilin and total MFGM proteins according to the stage of lactation and cow’s feeding. It must also be pointed out that the percentages reported in Table III are based on staining intensity, which is known to be varying with protein species and consequently, giving enhancement or lowering of the observed differences. The proportion (Tab. III) observed between major proteins (70%) and minor proteins (30%) was similar for all milks. Xanthine oxidase and butyrophilin, respectively, accounted for 14% and 18–22% of the MFGM proteins in three yak breeds. In cow’s milk, concentration of xanthine oxidase and butyrophilin varies within the lactation period, but they were present in constant molar proportions of about 1:4 [8, 14]. On the other hand, the relative proportion of these proteins shows interspecies differences, for example, in human milk, the level of xanthine oxidase is much higher than that of butyrophilin [1, 2] and in goat’s milk, proportion is 1:1 [14]. Our results showed a proportion of about 1:2 between these two proteins. No significant variation (P > 0.05) was observed among Maiwa, Zhongdian and Gannan yak milks for PAS 6 and PAS 7. The observed faint bands of Periodic Acid Schiff III (PAS III) and Periodic Acid Schiff IV (PAS IV) may be due to the loose association of these proteins to MFGM which maybe has led to a removal from the membrane during washing with salt solutions.

Table III.

Protein components in the MFGM obtained from different yak breeds.

4. CONCLUSION

In the present study, breeds variations in fat globule membrane protein components were observed among three yak breeds. A significant difference (P < 0.05) was found in the percentage of mucin 1 and butyrophilin of the MFGM components among the milk of the three yak breeds. Based on these data, there were some differences in fat globule characterization and protein components of MFGM among different yak breeds.

Acknowledgments

This work was funded by National Nature Science Foundation of China (No. 30871953). The authors thank New Hope Dairy Holding Co. Ltd. for milk samples and the Southwest University for Nationalities of Sichuan for laboratory analyses.

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All Tables

Table I.

Sample information.

In the text
Table II.

Characteristics of yak milk fat globule from different yak breeds.

In the text
Table III.

Protein components in the MFGM obtained from different yak breeds.

In the text

All Figures

thumbnail Figure 1.

Particle size distribution of fat globule from Maiwa yak milk (…), Gannan yak milk (―) and Zhongdian yak milk (­­).
In the text
thumbnail Figure 2.

SDS-PAGE patterns (10% acrylamide gel), obtained under reducing conditions, of MFGM material from fresh whole yak milk. MFGM from Maiwa yak milk (A); MFGM from Gannan yak milk (B) and MFGM from Zhongdian yak milk (C). Membrane proteins are named according to Mather [6].
In the text