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α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak β-LG had 2 variants, β-LG A (P02754) and β-LG E (L8J1Z0). Both domestic yak and wild yak milk contained β-LG E, but it was absent in bovine milk. The amino acid at site 158 of β-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and β-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and β-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.
). Almost 20% of the total protein of milk is whey protein, and α-LA and β-LG are the 2 main whey proteins, accounting for 20% and 50% of the total, respectively (
Bovine α-LA is a globular protein with a molecular mass of approximately 14.2 kDa, which comprises 123 AA. The α-LA protein structure contains 4 disulfide bonds (Cys6-Cys120, Cys61-Cys77, Cys73-Cys91, and Cys28-Cys111) and does not have free thiol groups (
in: Fox P.F. Uniacke-Lowe T. McSweeney P.L.H. O'Mahony J.A. Dairy Chemistry and Biochemistry. 2nd ed. Springer International Publishing,
New York, NY2015: 145-239
). In addition, α-LA is a binding protein and has 2 Ca2+ binding sites in its secondary structure. Protein structure stabilization of α-LA increases with cations bound to the Ca2+ sites (
). The protein is uniquely expressed in the lactating mammary gland and is present in the milk of all major mammals.
β-Lactoglobulin is a globular protein with a molecular mass of 18.3 kDa that belongs to the lipocalin family. The β-LG monomer is a single polypeptide chain consisting of 162 AA, and its AA sequence was reported in 1972 (
). Each monomer contains 2 disulfide bonds, one at Cys66-Cys160 and one at Cys106-Cys119, and a free thiol group at Cys121. The free thiol group is buried in the protein structure at pH < 7.5 (
). β-Lactoglobulin has also been found in the milk of monogastric animals, but it is nonexistent in human milk, which has α-LA as the dominant whey protein (
in: Park Y.W. Haenlein G.F. Milk and Dairy Products in Human Nutrition: Production, Composition and Health. John Wiley & Sons,
Hoboken, NJ2013: 477-497
The AA sequences of whey protein and casein have been studied in different species and regions. A previous study revealed that the main whey protein components of yak and camel milk are almost identical to those of bovine milk, but differences exist between the physicochemical properties of whey proteins (
). Therefore, structural and functional differences exist between milk from different species, and these differences affect the heat stability of whey proteins and milk aggregation (
). During thermal treatment, the aggregation rate of α-LA is lower than that of β-LG because β-LG has a free thiol group, which participates in intermolecular disulfide bond or thiol-disulfide exchange reactions (
). Therefore, under thermal treatment, whey protein and casein aggregates of milk from a particular species may have protein structures that are distinct from those that existed before treatment (
China has 15 million yaks, accounting for approximately 95% of yaks worldwide; yak milk is ranked third in economic importance behind bovine and buffalo milk (
in: Fox P.F. Uniacke-Lowe T. McSweeney P.L.H. O'Mahony J.A. Dairy Chemistry and Biochemistry. 2nd ed. Springer International Publishing,
New York, NY2015: 145-239
). In yak milk, the total protein (52.5 g/L), whey protein (10.29 g/L), and casein (40.21 g/L) amounts were all found to be higher than those in bovine milk. In yak milk, whey protein is approximately 20% of the total milk protein (
Detailed information on formation and characteristics of α-LA and β-LG are needed. Therefore, this study aimed to investigate the characteristics, structure, and functional and thermal properties of yak α-LA and β-LG to facilitate yak milk utilization.
MATERIALS AND METHODS
Collection of Yak Milk
Raw yak milk samples were collected from Aba County of Sichuan Province and chilled at 4°C for subsequent experiments. Skim milk was obtained from fresh yak milk by centrifugation at 5,000 × g for 30 min at 4°C to remove the cream layer (Beckman L8-80M ultracentrifuge and Beckman 50.2 Ti rotor; Beckman Instruments Inc., Palo Alto, CA).
Separation of Yak α-LA and β-LG
The pH of skim yak milk was adjusted to 4.6 with 0.5 mol/L of HCl and stored at 4°C for 30 min. Yak milk was centrifuged at 5,000 × g for 30 min at 4°C to remove casein protein. The yak whey protein solution underwent ultrafiltration at 4°C with 50,000-Da membranes. The separation was performed according to the method of
. Yak milk was treated with sodium citrate (150 × 10−3 mol/L), acidified to pH 3.9 using 6 mol/L of citric acid, incubated at 35°C for 45 min, and then centrifuged at 10,000 × g for 30 min at 4°C to precipitate α-LA. The α-LA was resolubilized with 0.1 mol/L of CaCl2 and centrifuged at 10,000 × g for 20 min at 4°C, and the precipitate was discarded. The supernatant from the whey protein was washed twice with 7% NaCl and centrifuged at 10,000 × g for 30 min at 4°C. The supernatant was dialyzed (24 h, 4°C) against distilled water using a 3,000-molecular weight cutoff cellulose ester dialysis bag. The dialysate was lyophilized for approximately 48 h at −40°C to a constant weight, and α-LA and β-LG were separated.
Gel Electrophoresis
Sodium dodecyl sulfate PAGE was performed with a 12% separating polyacrylamide gel and a 5% stacking polyacrylamide gel with a Bio-Rad (Hercules, CA) electrophoresis system. Fifteen microliters of yak α-LA and β-LG was loaded in the gel, and the run time was 80 min at a constant voltage of 100 V with Tris-glycine-SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). Afterward, the gel was washed with deionized water, then stained with Coomassie Brilliant Blue R-250 in a 5:4:1 solution of methanol-water-acetic acid for 1 h, and then destained for 24 h with destaining solution composed of 12% methanol and 7% acetic acid in deionized H2O (
). The isolation of α-LA and β-LG was performed on a Jupiter C4 column (250 mm × 4.6 mm, 300-Å pores, 5-mm particles; Phenomenex, Torrance, CA). The separation of β-LG and α-LA fractions, composition analysis, purity, and yield determinations were performed in duplicate.
MALDI-TOF MS Analysis
Yak whey protein molecular weights and protein identities were determined using MALDI-TOF MS. Samples were analyzed by a MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA). These analyses were performed according to the methods described by
. Samples were spotted on a target with 1 μL of prepared matrix solution. The final protein concentration was 0.1 to 10 μm, and sinapinic acid was used as the matrix. Signals from 100 to 200 laser shots were summed per mass spectrum. The mass accuracy was estimated as ±0.5 Da.
In-Gel Digestion and Tandem Mass Spectrometry Analysis
The SDS-PAGE gel bands of yak α-LA and β-LG were cut into pieces and bleached in a solution consisting of 25 mmol/L of NH4HCO3 in 50 mL/100 mL of acetonitrile. When the gels were completely destained, the supernatant was removed, and the gels were dehydrated in 100 mL/100 mL of acetonitrile for 15 min to dry the gel. Then, the gels were placed in 10 μL of digestion buffer containing 50 mmol/L of NH4HCO3 and 10 mg/mL trypsin at 4°C for 20 min. The supernatant was then removed, and 10 μL of digestion buffer (trypsin free) was added to rehydrate the gels overnight at 37°C. The enzymatic peptides were extracted with a solution of 50 mL/100 mL of acetonitrile with 2 mL/100 mL of formic acid before further concentration by drying (
The quantitative bioanalysis of protein peptides obtained from the gels was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein online desalinization was conducted with a C18 trapping cartridge (300 nm × 5 mm; Agilent, Santa Clara, CA), and the flow rate was 30 μL/min; the protein solution from the milk was applied to a C18 RP column (75 μm × 15 cm, Agilent) using an acetonitrile gradient of 5 to 40 mL/100 mL containing 0.1 mL/100 mL of formic acid with a flow rate of 200 μL/min to separate the protein peptides. These peptides were detected by electrospray ionization quadrupole time-of-flight mass spectrometry (Bruker Daltonics, Hamburg, Germany). The mass spectrometer was operated in positive mode. The source voltage was 2.0 kV, and the nitrogen gas flow rate was 6.0 L/h with a voltage of 30 V (
). The sequences of the peptides were analyzed with Bruker data analysis software (Bruker Daltonics Germany). Protein identification and BLAST were accomplished with data from the National Center for Biotechnology Information (NCBI) databases. All LC-MS/MS experiments were conducted and analyzed in triplicate.
Fourier Transform Infrared Spectroscopy
Fourier transform infrared (FTIR) spectroscopy was carried out using a Perkin-Elmer FTIR spectrometer (Spectrum One B, PerkinElmer Inc., Waltham, MA). All sample spectra were acquired via the KBr pressed-disc method. Yak α-LA and β-LG (10%) were dissolved in water and lyophilized. Difference spectroscopy, second derivative, and curve fitting were used to analyze the FTIR measurements. OriginPro 8.5 software (OriginLab Corporation, Northampton, MA) was used to analyze the data.
Differential Scanning Calorimetry
Solutions of yak α-LA and β-LG 20% (wt/vol) were dissolved in H2O. Fifteen microliters of each solution was placed in differential scanning calorimetry (DSC) pans. The samples were analyzed by DSC (TA 3000, Mettler Instrument Corporation, Greifensee, Switzerland). The scanned temperature was from 10°C to 100°C with a heating rate of 5°C/min. In each DSC run, an empty pan was used as a control. After heating, the sample was cooled to 20°C in the DSC equipment and heated under the same conditions to determine the degree of denaturation, which was performed according to the methods described by
As shown by the results in Figure 1, the purified yak α-LA and β-LG exhibited only 1 peak in RP-HPLC. In the RP-HPLC results, the elution times of yak α-LA and β-LG were 38.2 and 35.2 min, respectively, and the elution times were similar to those of bovine α-LA and β-LG, which were 38.5 min and 34.8 min, respectively. These results were in agreement with previous reports that described the characteristic RP-HPLC profile of yak milk proteins (
). The protein distribution characteristics of yak milk were similar to those of bovine milk. In yak milk, β-LG has 2 polymorphisms, β-LG A and β-LG B. The protein content of β-LG B was much greater than that of β-LG A, and the content of yak α-LA was significantly less than that of bovine origin (
). The purity of α-LA and β-LG was determined analytically by RP-HPLC; only 1 main peak was detected, and the sample purity was 90% or higher based on the HPLC trace.
Figure 1The HPLC chromatograms of purified yak (a) α-LA and (b) β-LG.
The MALDI-TOF MS patterns of yak α-LA and β-LG are shown in Figure 2. Yak α-LA had a single peak with an apparent molecular mass of 14,176.33 Da, and the molecular mass was close to that of bovine α-LA, which has a molecular mass of 14,200 Da (Figure 2A). The profile of β-LG had 2 peaks with a molecular mass of 18,197.28 Da and 18,270.30 Da (Figure 2B). The results showed that yak milk β-LG has 2 homologous isomers, but α-LA has only one.
Figure 2The MALDI-TOF MS spectra of yak (a) α-LA and (b) β-LG. Intens. = intensity (in arbitrary units).
The results of the tryptic peptide matches of yak α-LA and β-LG determined by LC-MS/MS are shown in Table 1, Table 2. The α-LA and β-LG SDS-PAGE bands were sufficient for peptide identification by LC-MS/MS. According to the number of retrieved high-scoring segment pairs and the total number of fragmented precursors, the threshold of unique spectra was set at ≥2. Thus, the unique spectra contained more than 2 identity indicators or extensive homology (P < 0.05). Yak α-LA and β-LG had 14 and 21 tryptic peptides and 6 and 15 unique spectra, respectively. The coverage percentages for yak α-LA and β-LG were 42.25% and 81.46%, respectively, as determined by LC-MS/MS. The missing sequences may have resulted from the inability to match the short tryptic peptides obtained from the in-gel digestion and the relatively few reports on the genomic information of the corresponding proteins in the NCBI databases (
As shown in Table 1, yak α-LA comprised 123 AA. Bovine α-LA has the same number of AA. The AA sequence of yak α-LA was found in NCBI databases (accession no. L8IIC8), which confirmed that the homologous isomers of yak α-LA were type B. However, yak β-LG had 2 homologous isomers, both of which comprised 162 AA (Table 2). The yak β-LG AA sequence was found in NCBI, which confirmed that the types were β-LG A (accession no. P02754) and β-LG E (accession no. L8JIZ0). The results were similar to those of previous research investigating the whey protein and casein variants, the composition, and the protein properties of yak milk (
Polymorphisme des lactoprotéines de bovinés népalais. I.—Mise en evidence, chez le yak, et caractérisation biochimique de deux nouveaux variants: β-lactoglobuline Dyak et caséine αs1E.
Homology analyses using BLAST were conducted to match the AA sequences of yak α-LA and β-LG with other representative proteins, and the results are shown in Figure 3, Figure 4. The AA sequence of yak α-LA was highly homologous with bovine and wild yak α-LA (Figure 3). The yak α-LA sequence had 123 AA with a molecular mass of 14,187.05 Da. The AA sequence and molecular mass were the same as those of wild yak (L8IIC8) listed in the NCBI databases. Until now, the NCBI database included 4 types of α-LA entries, all of which were for wild yaks. The accession L8IIC8 was the α-LA B variant, which had a different AA at site 71. In yak α-LA, the AA at this site was Asn (N), but it was Asp (D) for bovine and wild yak. Asn is uncharged, but Asp is negatively charged; therefore, Asp is more acidic and hydrophilic than Asn. Thus, the molecular mass and pI were both different for the different AA sequences. This result is in agreement with previous research that investigated AAs and variants in bovine and yak milk and bovine whey protein (
As shown in Figure 4, yak β-LG had 2 homologous isomers, types A and E, and both were composed of 162 AA. β-Lactoglobulin A was identical to bovine β-LG, with a molecular mass of 18,209.14 Da. The molecular mass of β-LG E was 18,281.21 Da, and site 158 in the β-LG E sequence was Gly (G). This site was the same in wild yak β-LG, but it was Glu (E) in bovine β-LG. Gly is uncharged, and Gln is negatively charged; therefore, Gly is a hydrophilic AA, and Glu is relatively acidic. The different AA sequences could make protein structure different, thus leading to differences in physicochemical properties.
The Secondary Structures of Yak α-LA and β-LG
Figure 5 shows the yak α-LA and β-LG secondary structural compositions. The yak α-LA secondary structures (Figure 5a) consisted of α-helix (1,650–1,660 cm−1), β-sheet (1,610–1,642 cm−1), β-turn (1,660–1,680 cm−1), β-antiparallel (1,680–1,700 cm−1), and random coil (1,642–1,650 cm−1) structures, and their contents were 18.6, 30.5, 26.5, 10.7, and 13.7%, respectively. Yak α-LA was the typical α-structure composition protein, and the α-helix content was less than that of bovine α-LA. The decrease of α-helix content could lead to a decrease in protein stability and a low denaturation temperature (Td).
Figure 5The Fourier transform infrared spectrograms and curve-fitting results of amide I band for yak (a) α-LA and (b) β-LG in the range of 1,600 to 1,700 cm−1.
As shown in Figure 5b, yak β-LG secondary structural contents were 15.3% α-helix (1,650–1,660 cm−1), 36.1% β-sheet (1,610–1,642 cm−1), 21.9% β-turn (1,660–1,680 cm−1), 16.2% β-antiparallel (1,680–1,700 cm−1), and 12.5% random coil (1,642–1,650 cm−1). The secondary structure of yak β-LG had more α-helix and β-sheet than that of bovine β-LG, so the protein structure of yak β-LG had higher stability. Therefore, the Td was higher than that of bovine β-LG. The FTIR spectra predicted that yak α-LA and β-LG would have secondary structural compositions similar to those of bovine whey protein, which accorded with yak and bovine species being homologous (
). The difference in AA sequence led to the secondary structures being slightly different, which could lead to differences in protein properties.
The Thermal Properties of Yak α-LA and β-LG
The thermogram (Figure 6a) of yak α-LA showed a single transition peak temperature (Td) at 51.4°C. The Td corresponded to holo-α-LA, and the enthalpy (ΔH) was 1.1 ± 0.1 J/g. The Td of yak α-LA in the second heating cycle was 52.1°C, with an enthalpy of 1.08 ± 0.1 J/g. The results indicated that heating cycles have little effect on the Td of yak α-LA, and the reversibility was almost 100% in the second heating cycle. The ΔH of yak α-LA was similar to that of bovine α-LA, but the Td was lower (
). The DSC thermograms in Figure 6b show that yak β-LG had a single transition peak at 80.9°C, and the ΔH was 2.71 ± 0.2 J/g. In the second heating cycle of the thermograms, the transition peak of yak β-LG disappeared, indicating that all yak β-LG was denatured during the first heating cycle and the denaturation transition was irreversible. The thermal characterization of yak β-LG was similar to that of bovine β-LG, which agreed with
These results can be attributed to the differences in the AA sequences of the different milk sources. In the yak α-LA sequence, the AA at site 61 was Asn, which is uncharged and less acidic than Asp. In the yak β-LG sequence, Gly is a hydrophilic and uncharged AA, while Glu is a negatively charged AA above pI.
So far, a large number of studies have shown that the aggregation of yak milk is slightly different from that of bovine milk (
). One of the reasons is the difference in AA sequence and protein secondary structure, which could affect the functionality of the protein. The Td of yak milk β-LG was higher than that of bovine milk β-LG. Because the particular protein structure of β-LG plays a dominant role in the protein aggregation in milk, the Td of β-LG could significantly affect the aggregation content and aggregation temperature. Thus, this may be the main reason for the difference in the thermal aggregation and milk properties (viscosity, turbidity, and particle size) between yak and bovine milk.
CONCLUSIONS
The peptide sequence data from α-LA and β-LG isolated from yak milk were obtained by LC-MS/MS analysis from in-gel tryptic digests. Yak α-LA and β-LG consisted of 123 and 164 AA, respectively. The AA sequences of yak α-LA and β-LG exhibited significant homology with bovine whey protein by BLAST. The AA sequences differed at site 71 in α-LA and site 162 in β-LG, which were Asp and Gly. The types of yak α-LA and β-LG were α-LA B and β-LG A and β-LG E. Furthermore, the secondary structural characters of yak whey proteins were different from bovine whey proteins. Yak α-LA had an unstable protein structure and lower Td than bovine α-LA. However, yak β-LG had a higher Td than bovine β-LG. These results indicate that yak whey proteins have different thermal properties, emulsification, and foamability than bovine milk. However, further investigation to understand the thermal properties and thermal aggregation relationship between yak α-LA and β-LG is important.
ACKNOWLEDGMENTS
The authors thank the “Young Talents” Project of Northeast Agricultural University, Harbin (No. 18QC51), for providing financial support. The authors declare that they have no conflicts of interest.
REFERENCES
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Alli I.
Separation and characterization of β-lactoglobulin and α-lactalbumin from whey and whey protein preparations.
in: Park Y.W. Haenlein G.F. Milk and Dairy Products in Human Nutrition: Production, Composition and Health. John Wiley & Sons,
Hoboken, NJ2013: 477-497
in: Fox P.F. Uniacke-Lowe T. McSweeney P.L.H. O'Mahony J.A. Dairy Chemistry and Biochemistry. 2nd ed. Springer International Publishing,
New York, NY2015: 145-239
Polymorphisme des lactoprotéines de bovinés népalais. I.—Mise en evidence, chez le yak, et caractérisation biochimique de deux nouveaux variants: β-lactoglobuline Dyak et caséine αs1E.
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