Free Access
Issue
Dairy Sci. Technol.
Volume 90, Number 1, January–February 2010
Page(s) 111 - 117
DOI https://doi.org/10.1051/dst/2009048
Published online 17 December 2009

© INRA, EDP Sciences, 2009

1. INTRODUCTION

Yak (Bos grunniens) milk has a high content of total solids (16.9–17.9%), proteins (4.9–5.9%) and fat (5.5–7.5%) [13, 14]. These values are significantly higher than those of cow (Bos taurus) and goat (Capra hircus) milk, but relatively similar to those of buffalo (Bubalus bubalis) and ewe (Ovis aries) milk [13]. Like in most of the other mammalian milks, proteins of yak milk mainly consist of the four individual caseins (αs1-CN, αs2-CN, β-CN and κ-CN) and the major whey proteins (α-lactalbumin, β-lactoglobulin, serum albumin (SA), lactoferrin and immunoglobulins) [11]. Although the polymorphism of yak milk proteins has been studied in the past [3] and more recently [10], a very few literature is available, and the results on the average content of each individual protein in bulk yak milk are often contradictory [8, 11]. In recent years, there has been a significant increase in the yak population in China, despite the difficulties in their husbandry. These cattle are used to living in an extreme climatic environment such as in Tibet and the surrounding areas. Due to the increase in yak milk production, a deeper knowledge and understanding of the biochemical compounds of this product is required in order to develop novel products other than the more traditional ones [14]: butter, fermented skimmed and whole milk, and Qula (a kind of traditional acid curd). Moreover, to fully realize the nutritional value of yak milk, all the usual transformation technologies used in the production of consumer milks, yoghurts, cheese, powders, etc., cannot be optimized without this knowledge. Hence, the objective of this study was to analyze the protein composition of Maiwa yak milk using RP-HPLC, and using purified individual caseins and whey proteins of known genotypes as standards.

2. MATERIALS AND METHODS

2.1. Yak milk samples

Twenty-four individual Maiwa yak milk samples, representative of a complete milking of the animals that were in the middle of their lactation period, were collected from the Sichuan province in China. After addition of sodium azide (0.4 g·L−1), the milk samples were immediately frozen at −20 °C and transported to the laboratory. Total protein contents were analyzed by the Kjeldahl method [4].

2.2. Sample preparation

Standard bovine caseins and whey proteins were purchased from Sigma: 50 mg κ-CN (lot C-0406, purity > 80%), 125 mg αs-CN (lot C-6780, purity > 85%), 50 mg β-CN (lot C-6950, purity > 90%), 10 mg α-La (lot L-5385 type I, purity > 85%), 15 mg β-LgB (lot L-8005), 10 mg β-LgA (lot L-7880) and 50 mg BSA were dissolved in 5 mL buffer solution (8 mol·L−1 urea, 165 mmol·L−1 Tris, 44 mmol·L−1 sodium citrate and 0.3% (v/v) β-mercaptoethanol).

Samples were thawed using flowing tap water prior to analysis. The fat was removed by centrifugation at 1000× g for 10 min at 4 °C. One milliliter of skimmed milk was dissolved in 4 mL of buffer solution. The diluted samples were filtered through a 0.45 μm cellulose membrane (A-FIT Biosciences Ltd., Beijing, China) and directly analyzed. All samples were analyzed in triplicate.

2.3. RP-HPLC of milk samples

Reversed-phase HPLC analyses were performed on a Jupiter C4 column (250 mm × 4.6 mm, 300 Å sized pores, 5 μm sized particles; Phenomenex, Torrance, CA, USA). The detection wavelength used was 220 nm. The HPLC equipment consisted of an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump (Agilent 1100 Series, G1311A). A variable wavelength ultraviolet detector (Agilent 1100 Series, GB15 A/B, DAD) was used. The equipment was controlled by the Agilent ChemStation for LC Systems software that controlled solvent gradient, data acquisition and data processing. The analyses were carried out by applying a binary gradient profile of the mobile phase. Solvent A was ultrapure water containing 0.1% (v/v) trifluoroacetic acid (TFA), and solvent B was HPLC-grade acetonitrile containing 0.1% (v/v) TFA (concentration of acetonitrile ≥ 99.9%). The gradient elution program was run at a constant flow rate of 1.0 mL·min−1 and was set as follows: 0–40 min linear gradient from 30% B to 50% B; 40–42 min linear gradient from 50% B to 100% B; 42–43 min isocratic elution 100% B; 43–46 min linear gradient from 100% B to 30% B, followed by a 5-min isocratic elution at the initial conditions. The total duration of a single run, including column equilibration, was 60 min [1]. The temperature of the column was kept at 30 °C.

Casein contents were determined by summation of the data obtained through RP-HPLC for the four individual caseins after verification that these values were in agreement with those obtained by classical precipitation at pH 4.6.

3. RESULTS AND DISCUSSION

3.1. Identification of caseins and whey proteins in yak milk

A characteristic RP-HPLC profile of yak milk proteins is presented in Figure 1. All proteins were efficiently separated. Comparison with the profile obtained with standard cow milk proteins allowed the identification and quantification of the four individual caseins and the main whey proteins, β-LgA, β-LgB, α-La and BSA. As it had been previously shown for cow κ-CN, the Maiwa yak casein chromatogram shows three or four peaks for this casein indicating not only the genetic variation described on the gene CSN3 by Prinzenberg et al. [10], but also several possible degrees of glycosylation [12]. To quantify the κ-CN, the three or four peaks (Fig. 1) were summed to give the total κ-casein value indicated in Table I. Because cow αs1-CN and αs2-CN were not available as individual proteins, the corresponding values for homologous yak caseins were determined from the αs-CN calibration obtained by applying the 4:1 proportion known for bovine milk [1]. Regarding β-CN, 15 individual Maiwa yak milk samples showed only one β-casein peak but for nine other Maiwa yak milk samples, two β-casein fractions were partially separated. These results indicate that a polymorphism of this individual casein is likely. However, this hypothesis disagrees with Mao et al. [7] who reported that β-casein was monomorphic but is in agreement with the hypothesis of Prinzenberg et al. [10]. As for most of the mammalian milks, yak milk caseins show a complex qualitative genetic polymorphism. Further research must be done to improve knowledge in this field for many purposes such as possible relationships with milk production [11], heat stability, and manufacturing properties [13].

thumbnail Figure 1.

Chromatograms of standard bovine milk proteins and skimmed yak milk (1, skimmed yak milk and 2, standard bovine milk). Column: Phenomenex Jupiter C4 (250 mm × 4.6 mm, 300-Å pores, 5 μm particles); UV, λ = 220 nm; mobile phase: water containing 0.1% TFA/acetonitrile containing 0.1% TFA.

Table I.

Mean concentration of casein fractions in Maiwa yak milk.

3.2. Protein distribution in yak milk

Table I summarizes the data of individual and total casein determined on the 24 samples of collected yak milks. Total casein content of yak milk (40.2 g·L−1 on average) is 1.5 times the concentration of cow milk [13] and 11 times that of human milk [6]. Besides having high levels of casein that had been reported previously [13], the results of this study indicate that there is also a high proportion of β-CN (more than 45% on average and ranging from 37.18% to 51.16%), which, to our knowledge, has not previously been described in the literature. This high level of β-CN is thought to result in a smooth and soft coagulum in the stomach, which is easily digested by the enzymes of the intestinal tract. This perhaps explains why yak milk is usually given, after dilution, to babies by Tibetan nomads to complement breast milk [14]. On the other hand, it would be interesting to determine the sequence of yak β-CN to elucidate whether the bioactive peptides β-CN 1–25, 60–65, 177–183, etc. are present and have the same physiological abilities as their homologous sequences in human and cow β-CN [5]. Also, the high proportion of β-CN and, consequently, the lower proportion of αs-CN (about 40% for yak milk vs. 49% for bovine milk) together with a small increase of κ-CN (15% compared to 12% for bovine milk) will influence the usefulness of yak milk for cheese making (renneting time, final firmness of the curd, whey drainage, etc.). More research is needed for an in-depth characterization of yak casein micelles properties, such as size distribution, ξ potential and mineralization.

Table II summarizes the results of whey protein determinations. The few data available in the literature concerning whey proteins in yak milk strongly limit a discussion of these data. Indeed, Ochirkhuyag et al. [9] have identified the amino acid composition of the main whey proteins, β-lactoglobulin and α-lactalbumin, but they did not give the content of these proteins. However, comparison with cow whey proteins shows that the proportion of total β-Lg in total proteins appears to be in the same range in yak milk. On average, there were a higher proportion of SA in yak milk compared to cow milk, but the individual variations were high. The value found for α-La is puzzling because it is not in line with the usual relationship between lactose and α-La contents in mammalian milks [2]. Indeed, α-La is half of the lactose synthetase enzyme [2] and its content increases with lactose concentration. In the collected samples of yak milk, the lactose concentrations (50.1–59.2 g·L−1) were higher than that of bovine milk.

Table II.

Mean concentration of whey protein fractions in Maiwa yak milk.

4. CONCLUSION

In conclusion, as previously mentioned for the casein group, there is a huge lack of knowledge on the whey proteins of yak milk. Further in-depth research is required both for nutritional purposes (major and minor whey proteins are now known to play an essential physiological role in immune defense, brain development, bone mineralization, etc. [5]) and for a better and complete utilization of milk yak components by the dairy industry. Also, the acquired knowledge would allow the development of new diversified beneficial products adapted to the needs of people living in extreme climatic environments.

Acknowledgments

This study was founded by National Natural Science Foundation of China and by the society ISDP. Both organizations are warmly thanked for their help.

References

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  10. Prinzenberg E.M., Jianlin H., Erhardt G., Genetic variation in the κ-casein gene (CSN3) of the Chinese yak (Bos grunniens) and phylogenetic analysis of CNS3 sequences in the genius Bos, J. Dairy Sci. 91 (2008) 1198–1203. [CrossRef] [PubMed] (In the text)
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  12. Swaisgood H.F., Chemistry of caseins, in: Fox P.F. (Ed.), Advanced Dairy Chemistry, Vol. 1: Proteins, Elsevier Publishers, New York, USA, 1992, pp. 63–110. (In the text)
  13. Walstra P., Geurts T.J., Noomen A., Jellema A., van Boekel M.A.J.S., Dairy Technology, Publisher Marcel Dekker, New York, USA, 1999. (In the text)
  14. Wiener G., Jianlin H., Ruijun L., The yak, Regional Office for Asia and the Pacific, Food and Agriculture Organisation of the United Nations, 2003. (In the text)

All Tables

Table I.

Mean concentration of casein fractions in Maiwa yak milk.

Table II.

Mean concentration of whey protein fractions in Maiwa yak milk.

All Figures

thumbnail Figure 1.

Chromatograms of standard bovine milk proteins and skimmed yak milk (1, skimmed yak milk and 2, standard bovine milk). Column: Phenomenex Jupiter C4 (250 mm × 4.6 mm, 300-Å pores, 5 μm particles); UV, λ = 220 nm; mobile phase: water containing 0.1% TFA/acetonitrile containing 0.1% TFA.

In the text