Exploring Potential Bioactive Peptides in Fermented Bactrian Camel’s Milk and Mare’s Milk Made by Mongolian Nomads

To date, bioactive proteins and peptides from minor livestock milks and their fermented products have been scarcely reported. In Mongolia, nomads are commonly rearing five livestock animal species (i.e., cow, camel, goat, horse, and sheep) for milking and other purposes. In this study, we analyzed the peptide composition in fermented milks of Bactrian camels (Camelus bactrianus) and horses, produced by Mongolian nomads for self-consumption. Peptides from skimmed fermented milks were separated by ultrafiltration and reverse-phase high-performance liquid chromatography. Then, their amino acid sequences were determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Consequently, eleven peptides were identified in the fermented camel’s milk including four from β-casein (β-CN), three from αs1-CN, and two from both κ-CN and lactophorin. On the other hand, twenty-four peptides were identified in the fermented mare’s milk including nineteen from β-CN, three from αs1-CN, and one from both κ-CN and αs2-CN. According to previous reports on the bioactivities of milk-derived peptides, antibacterial and antihypertensive activities were promising in both the fermented camel’s milk and mare’s milk. In addition, potential antioxidant activity was conjectured in the fermented camel’s milk. Further investigations are currently needed to clarify the potential role of immunomodulatory peptides in the two fermented milks.


Introduction
The Camelus genus includes two domesticated species of camels: Dromedary camel (Camelus dromedaries, one-humped) and Bactrian camel (Camelus bactrianus, two-humped). According to statistics from the Food and Agriculture Organization (FAO), in 2018, the total population of Dromedary and Bactrian camels and available worldwide camel milk production yield were 35.8 million heads and 3.15 million tons (http://www.fao.org/faostat/en/#home). More than 75% of camel milk is assumed to be consumed by nomads as raw milk and processed dairy products. In 2018, camel milk production in Mongolia was 114,830 tons, accounting for 3.6% of the available camel milk production in the world. For the nomads living in the Gobi region of Mongolia, camels are multipurpose livestock animals, e.g., they supply milk, meat, wool, and hide as well as transportation. Because nomads consume greater quantities of Bactrian camel milk than milk from other domestic animals, they have established Sigma-Aldrich (St. Louis, MI, USA). Buffer solutions for the RP-HPLC were filtered through a 0.2 µm membrane filter from Millipore prior to use. Peptide calibration standard II (1-3 kDa) used for calibration of the MALDI TOF-MS, MALDI target plate, and α-cyano-4-hydroxycinnamic acid (α-CHCA) were purchased from Bruker Daltonik GmbH (Bremen, Germany). The HPLC system was a JASCO (Tokyo, Japan) and equipped with a PU-2089 pump, UV 2075 detector, MX 2080-32 dynamic mixer, and a CO-8020 column oven. The TSK gel ODS-80Ts (0.46 × 25 cm) column was from Tosoh Co. (Tokyo, Japan). All other chemicals were of analytical grade.

Milk Samples
Fermented Camel's milk was produced from September 2015 to April 2016, and fermented mare's milk from August to October 2016. Approximately 500 mL each of Bactrian camel's fermented milks were collected from 3 different herders: two herders near Hamriin hiid, Ulaanbadrakh soum, Dornogovi aimag and one herder in Dalanzadgad soum, Umnugovi aimag in Mongolia on 16 October 2016. Approximately 500 mL each of the mare's fermented milk were also collected from 3 herders located in a narrow region, 3-5 km apart from each other, in Adaatsag soum, Dundgovi aimag, Mongolia, on 2 October 2016. The numbers of milked animals in the herds ranged from 10-20 and 15-24 heads for camels and horses, respectively, at the time of sampling. It was confirmed that both the fermented camel's milk and mare's milk were free from contamination of milks derived from other livestock animals via investigation with the nomads who made them. All samples were collected and stored independently in sterile containers and kept at 4 • C in cooling boxes during transport (3 h at maximum) from the farms to the State Central Veterinary Laboratory in Ulaanbaatar and kept at −30 • C until frozen transport to Japan.

Peptide Purification
Each of the fermented camel's milk and mare's milk samples (100 mL) was mixed with 1 mL of the protease inhibitor cocktail for mammals and defatted by centrifugation at 500× g for 10 min at 20 • C. The fat and cells were removed and then a supernatant as a whey was centrifuged again at the same condition to remove residual cream. Residual caseins and insoluble compounds were removed by centrifugation at 26,900× g for 60 min at 4 • C. The supernatant, namely, acid whey, was subjected to ultrafiltration at 4 • C using the Centriprep YM-3 (nominal molecular weight limit (NMWL) 3000). Membrane permeate was lyophilized and dissolved in 0.1% of TFA at 80 mg/mL concentration. A 100 µL aliquot of membrane permeate was injected into the ODS-80Ts column (0.46 × 25 cm), pre-equilibrated with 0.1% TFA, and connected to the HPLC system. After 5 min of static flow with 100% solvent A (0.1% TFA in water), elution was performed by a linear gradient from 0% to 50% solvent B (0.1% TFA in ACN) for 90 min at a flow rate of 1.0 mL/min and at 30 • C. The eluent was monitored by an ultraviolet detector at the wavelength of 214 nm. Eluted peptide fractions were collected manually, concentrated by rotary and vacuum evaporation, and lyophilized. The lyophilized peptides were dissolved in 50 µL of 0.1% TFA and stored at −20 • C until used.

Mass Spectrometry
The peptide solution was desalted using ZipTip C18 pipette tips (Millipore, Bedford, MA, USA) according to the manufacturer's instructions. A 1.0 µL aliquot of the desalted peptide solution and an equal volume of 10 mg/mL of α-CHCA saturated in 0.1% TFA/ACN (2:1, v/v) were mixed, and 1.0 µL of the mixture was loaded on a target plate (MTP 384 target plate ground steel T F, Bruker, Bremen, Germany). After the solvent dried, the target plate was mounted in an AutoflexII TOF/TOF mass spectrometer (Bruker, Bremen, Germany). Mass spectra were obtained using the pre-installed method, RP_1-3kDa (a reflector positive ion mode optimized to the mass range of 1-3 kDa). Peptide calibration standard II was used as an external mass calibrant. The acquired spectra were statistically analyzed using Flexanalysis 2.0 software (Bruker, Bremen, Germany). Amino acid sequences of the peptides were determined by fragmentation analysis of MALDI-generated ions using a technique of LIFT-TOF/TOF MS (MS/MS). The mass list of fragment ions was searched using the BioTools 3.0 interface (Bruker, Bremen, Germany) connected to the Mascot search engine [20]. In brief, other Mammalia were selected as taxonomy, and either MSDB, SwissProt, or NCBInr was chosen as a database (http://www.matrixscience.com/index.html). None of the fixed modifications were selected, but varied settings of the enzyme, variable modifications, peptide tolerance (±50-250 ppm), and MS/MS tolerance (±0.5-1.0 Da) were tested. Other settings were used as default. Peptides were identified only when their probability based Mowse scores showed statistical significance. In addition, amino acid sequence similarity was analyzed using protein BLAST interface at National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/).

N-Terminal Sequence Analysis
N-terminal amino acid sequences of the peptides were determined by the Edman degradation method [21] and following HPLC separation of phenylthiohydantoin derivatives of amino acids using the Procise 492 HPLC system (PerkinElmer, Waltham, MA, USA) at the Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University.

Peptide Profiles in the Fermented Milks Analyzed by RP-HPLC
Low molecular weight fractions (< kDa) prepared from fermented camel's milk and mare's milk using the Centriprep YM-3 were further separated with RP-HPLC. Fermented camel's milk and mare's milk showed distinct chromatograms with the RP-HPLC as shown in Figures 1 and 2. Ten to eleven major peaks and a number of minor peaks were found in three fermented camel's milks (C1, C2, and C3 in Figure 1). On the other hand, eleven to twenty-two major peaks (22 peaks for H1, 15 peaks for H2, and 11 peaks for H3) and plenty of minor peaks were observed in the fermented mare's milk (H1, H2, and H3 in Figure 2). No peaks appeared in all chromatograms after 75 min to the end of elution; hence, chromatograms after 75 or 80 min of elution were omitted in Figures 1 and 2. Consequently, C3 and H1, which showed the highest number of major peaks among camel and mare fermented milks, respectively, were selected as representatives for further fractionation and identification of peptides. In total, 33 peaks, of which 11 peaks from C3 (numbered from C3-1 to C3-11 according to their elution order) and 22 peaks from H1 (numbered from H1-1 to H1-22 according to their elution order) were manually collected and then analyzed by MALDI TOF-MS/MS.

Peptide Analysis by MALDI TOF-MS/MS
The results of the peptide analysis on the 11 peaks separated from the fermented camel's milk, C3, are summarized in Figure 3 and Table 1. Two lactophorin-derived peptides, 76 HQNQNPK 83 and R 75 RHQNQNPK 83 , were identified in peaks C3-3 and C3-4, respectively (Figures S1 and S2). Two α s1 -CN-derived peptides, T 65 RNEPTEDH 73 and D 64 TRNEPTEDH 73 , were found in peak C3-5 ( Figure S3), and another α s1 -CN-derived peptide, R 16 PKYPLR 22 , was in peak C3-10 ( Figure S7). In total, four β-CN-derived peptides were identified. Among them, two peptides, H 221 PVPQP 226 and P 212 VPDPVRGL 220 , were found in peak C3-8 ( Figure S5). Another two β-CN-derived peptides, V 194 PYPQR 199 and Q 210 EPVPDPVR 218 , were found in peaks C3-9 and C3-11, respectively (Figures S6 and S8). Moreover, the N-terminal glutamine residue in the latter peptides had a possibility of being pyroglutamylated, which gave 17 Da a smaller molecular mass than the unmodified peptide. A κ-CN-derived peptide, R 110 PRPRPS 116 , was found in peaks C3-6 ( Figure S4) and C3-7 (data not shown). Another κ-CN-derived peptide, P 104 PTVERPARNRHD 116 , was found in peak C3-9, but MS/MS analysis of the latter part was incomplete ( Figure S6). Amino acid sequence of κ-CN-derived peptide P 104 PTVERRPRPRPS 116 at the same position was reported in dromedary camel [22], but its molecular mass was 1543.870, which was slightly higher than what we detected; therefore, unknown mutations or post-translational modifications may occur in the peptides isolated from the fermented Bactrian camel's milk. Despite successful analysis of IRIPV in peaks C3-1 and C3-2, NLRLPV and HLLQPF in peak C3-3, and NNASHNGNNSAPI in peak C3-8, their origins were not assigned to camel milk proteins and remain unknown.   Location of the identified peptides in their mother proteins. The peptides identified in this study are indicated by underlines. The NCBI reference numbers of the mother proteins are indicated in the parentheses. The numbers present at the left-most of the column indicate the number of amino acid residues that started from the N-terminal methionine of the precursors. Dotted lines indicate sequence inconsistency between the identified peptide and the mother protein used as reference, probably due to the presence of genetic variants.  Figure 3).

Discussion
This study aimed to provide basic information on the peptide profiles of fermented Bactrian camel's milk and mare's milk produced by Mongolian nomads. The RP-HPLC chromatograms of low molecular fractions (< kDa) of the three individual fermented camel's milks (C1, C2, and C3) showed a lower number of major peaks compared to those of the fermented mare's milks (H1, H2, and H3) (Figures 1 and 2). Among C1, C2, and C3, a high similarity of the peak profiles was observed between C1 and C2 (collected from two herders near Hamriin hiid, Ulaanbadrakh soum, Dornogovi aimag), and those were partially different from C3 (collected from one herder in Dalanzadgad soum, Umnugovi aimag). This may be partly due to the different individual caseins' polymorphism, although further investigation is required. Another possible reason for the similarity in the peptide profiles of C1 and C2 is that they shared common microbiota to a large extent, probably owing to intercommunion among the nomads living in the area. Although H1, H2, and H3 were collected in the same area, Adaatsag soum, Dundgovi aimag, their RP-HPLC chromatograms were largely different, probably reflecting the unique microbiota in each of them. It is controversial that different microbiota could provide similar peptide profiles when the same milk material and similar manufacturing process were applied for the production of fermented milk products. Parallel analyses on the microbiota and the peptide profile using the same fermented milk sample should provide a clue on this issue.
In this study, 11 and 24 peptides were identified in the fermented camel's milk C3 and the fermented mare's milk H1, respectively. A high number of unidentified peptides could arise from presence of unknown post-translational modifications in their mother proteins. Peptides originated from caseins were dominant in both C3 and H1. Especially, a variety of β-CN-derived peptides accounted for the majority of the identified peptides due to the high abundance of β-CN (45-65% of total caseins) in camel or mare milks [14,33]. The most specific feature of peptides found in the fermented camel's milk C3 was the presence of κ-CNand lactophorin-derived peptides ( Figure 1 and Table 1). The κ-CN-derived peptide, R 110 PRPRPS 116 , could be aligned using protein BLAST with a C-terminal region of bovine para-κ-CN HPHPHLS, which is known to be produced by chymosin treatment during cheese manufacturing [34]. This part of camel's κ-CN is in the proximity of the chymosin cleavage site [35]. To date, no health-promoting effect has been reported for this peptide, in contrast to glycomacropeptide, which is the counterpart to the para-κ-CN in the chymosin hydrolysate of κ-CN. Bovine lactophorin and its N-and C-terminal truncated variant (f3-135) are known as proteose-peptone component 5 [36] and GlyCAM-1 [37], respectively. On the other hand, the lactophorin-derived peptides, R 75 RHQNQNPK 83 and R 76 HQNQNPK 83 , found in this study were embedded in the middle part of mature lactophorin. Ibrahim et al. [38] recently found two lactophorin-derived peptides, E 113 NTMRETMDFLKSLF 127 and A 79 TTLEGKLVEL 89 , in pepsin-digested camel milk whey hydrolysates that confer tolerance against H 2 O 2 -induced oxidative stress to yeast cells. The biological significance of the lactophorin-derived peptides found in the fermented camel's milk in this study remains to be elucidated. One α 2 -CN-derived peptide, K 16 HNMEHR 22 , was characteristically found in fermented mare's milk H1, but its bioactivity also remains unclear.
Antioxidant activity could be expected to come from a β-CN-derived peptide V 194 PYPQR 199 found in the fermented camel's milk C3, owing to the high sequence similarity to AVPYPQR, which has been reported as an antioxidant peptide derived from bovine β-CN [26]. Similar human β-CN-derived peptides, VPYPQ, QVVPYPQ, and PYPQ, have also been reported as antioxidant peptides [42,43]. Recently, three antioxidant peptides, RLDGQGRPRVWLGR, TPDNIDIWLGGIAEPQVKR, and VAYSDDGENWTEYRDQGAVEGK, have been newly found in Bactrian camel's milk hydrolysates prepared by the action of several proteolytic enzymes, including trypsin, pepsin, alcalase, and papain [44]; however, we could not find any similar peptides to the three antioxidant peptides in our experiment.
It is evidenced that casein-derived peptides, produced by the action of digestive enzymes, such as trypsin, pepsin, and chymosin, can exhibit immunomodulatory activities [45]. In this study, no immunomodulative peptides identical to those which have already been found in bovine CN-derived peptides were found. Only one peptide, Q 210 EPVPDPVR, was found in fermented camel's milk fraction C3-11, as being similar (77.8% amino acid sequence identity) to a bovine β-CN derived peptide, Y 208 QEPVLGPVR 217 , which showed stimulatory activity against human peripheral blood lymphocytes [29]. Therefore, further exploration of immunomodulative peptides in Mongolian fermented milks and functional characterization of such potential bioactive peptides should be performed. Finally, it should be stressed that the peptide analysis was performed with just one sample each of fermented camel's milk and mare's milk, and not in a quantitative manner in this study; hence, further investigations are needed.

Conclusions
Variations in peptides (<3000 Da) in Bactrian fermented camel's milk and mare's milk made by Mongolian nomads were partially illustrated in this study. It was confirmed that such traditional fermented milks were certainly attractive sources of bioactive peptides. Our results suggest that the presence of antihypertensive and antipathogenic peptides should be promising in Mongolian fermented camel's milk and mare's milk. In addition, the presence of antioxidant and antidiabetic peptides are likely in the fermented camel's milk and mare's milk, respectively. However, further investigation is needed to confirm the presence of immunomodulative peptides in the fermented milks. Moreover, further studies should be required for demonstrating the biological activities of the identified peptides in this study.