Camel milk protein and application thereof

Abstract

The invention discloses camel milk protein and application thereof. The invention relates to camel milk protein. The camel milk protein comprises a functional protein combination. The camel milk protein is derived from naturally-fermented camel milk, by comparing the change of protein properties and structures before and after natural fermentation, a proteomics technology is adopted, and evolution rules of protein types, content, functions and metabolic pathways are systematically analyzed, so that clear functional protein composition is obtained; the polypeptide has antibacterial, anti-inflammatory and metabolic regulation potentials, functional characteristics are changed from basic metabolism to immune defense, specific enrichment of ribosome and muscle cytoskeleton pathways and the like, and the polypeptide can be applied to food and dairy products.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to a camel milk protein, and more specifically to a camel milk protein with a clearly defined functional protein composition and its applications. Background Technology

[0002] In traditional pastoral areas, fresh camel milk is difficult to preserve for long periods due to its perishable nature. Local herders utilize natural fermentation techniques to transform it into a uniquely flavored fermented camel milk beverage with a longer shelf life, considered a treasure of their traditional culinary culture. Kazakh medicine believes that fermented camel milk is warming in nature and sweet and sour in taste, possessing effects such as regulating internal organs, strengthening the body, replenishing blood and bones, invigorating the stomach and reducing inflammation, and promoting restful sleep. It can be used to treat or assist in the treatment of respiratory and digestive system diseases, as well as kidney disease, physical weakness, anemia, diabetes, and osteoporosis. Modern research shows that the fermentation process produces antibacterial organic acids and bioactive peptides in camel milk, significantly enhancing its health benefits, such as regulating intestinal flora and boosting immunity.

[0003] Currently, research on naturally fermented camel milk mainly focuses on metagenomics and culture omics, aiming to elucidate the succession patterns of microbial communities. These studies have identified *Lactobacillus* genus (…). Lactobacillus ), yeast ( Kazachstania humilis The study also utilized metabolomics techniques (such as SAFE, SDE, HS-SPME-GC / MS) to characterize systemic metabolites, identifying characteristic aroma-active compounds in naturally fermented camel milk and providing a scientific basis for the quality evaluation of camel milk products.

[0004] Natural fermentation can remodel the protein composition of camel milk through microbial metabolism, thereby significantly enhancing its nutritional value. However, the regulatory effects of natural fermentation on changes in the structure, composition, and function of the camel milk proteome are currently unclear. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a camel milk protein with a clearly defined functional protein composition, offering a technical pathway for developing high-value-added fermented camel milk products and upgrading the industrialization of specialty dairy products from border regions.

[0006] On one hand, the present invention provides a camel milk protein comprising the following functional protein combination: phosphatidylcholine transporter ABCB4 subtype X4, prothymocyte α, myelin basic protein, serine protease inhibitor Kazal type 1, apoptosis protease activator 1, polyunsaturated fatty acid lipoxygenase ALOX15B, transductionin-like enhancer protein 6, nucleophosphorus protein, vascular permeability factor, procadherin α-1-like, centrosome protein 290 kDa subtype X5, calcineurin B subunit type 1, serum alkaline protease inhibitor, DNA-dependent metalloproteinase SPRTN subtype X3, and serine hydroxymethyltransferase.

[0007] In some embodiments, the camel milk protein is obtained by the following method: using fresh camel milk as a substrate, adding pre-fermented milk as a natural starter, and carrying out solid-state fermentation in a constant temperature environment of 25±1℃, stirring three times a day for 48-72 hours to obtain naturally fermented camel milk; transferring the naturally fermented camel milk to a centrifuge tube, centrifuging at 4℃, scraping off the upper fat layer after centrifugation, and repeating the centrifugation operation until the fat layer is completely removed to obtain camel milk protein.

[0008] In some embodiments, the pre-fermented milk is made from fresh camel milk through conventional lactic acid fermentation by L. bulgaricus and Streptococcus thermophilus.

[0009] In some embodiments, the fresh camel milk is preferably sourced from Altay, Tacheng, and Urumqi.

[0010] In some embodiments, the centrifugation conditions are centrifugation at 4000 g for 20 min.

[0011] In some embodiments, the camel milk protein has an average particle size of 614.80±61.45 nm and a zeta potential of -8.9±5.4 mV.

[0012] In some embodiments, the fluorescence intensity of the camel milk protein's surface hydrophobicity is 7323.44±1032.12, and the free thiol content is 17.23±2.81 μmol / g.

[0013] In some embodiments, the relative content of β-sheet and β-turn of the camel milk protein is lower than that of fresh camel milk.

[0014] In some embodiments, the camel milk protein exhibits upregulated protein expression compared to fresh camel milk. The upregulated proteins include one or more of the following: antimicrobial peptide-7-like protein, antimicrobial peptide-2-like isoform X2, CXC motif chemokine, CC motif chemokine, CD59 glycoprotein, α-lactalbumin, κ-casein, whey acid protein, aminopeptidase, superoxide dismutase (copper-zinc type), E3 ubiquitin ligase, mucin-4, mucin-15, clathrin light chain, β-2-glycoprotein 1, ferritin, properin, CD44 antigen, insulin-like growth factor II, and protein disulfide isomerase. In some embodiments, the camel milk protein, compared to fresh camel milk, includes one or more of the following functional properties: a) Possesses antibacterial, anti-inflammatory, and metabolic regulatory potential; b) Functional characteristics shifted from basal metabolism to immune defense; c) Specific enrichment of ribosomes and muscle cytoskeleton pathways.

[0015] On one hand, the present invention provides a method for preparing camel milk protein as described in claim 1, the method comprising: using fresh camel milk as a substrate, adding pre-fermented milk as a natural starter, carrying out solid-state fermentation in a constant temperature environment of 25±1℃, stirring at regular intervals three times a day, and fermenting for 48-72 hours to obtain naturally fermented camel milk; transferring the naturally fermented camel milk to a centrifuge tube, centrifuging at 4℃, scraping off the upper fat layer after centrifugation, repeating the centrifugation operation until the fat layer is completely removed, thereby obtaining camel milk protein.

[0016] In some embodiments, the pre-fermented milk is made from fresh camel milk through conventional lactic acid fermentation by L. bulgaricus and Streptococcus thermophilus.

[0017] In some embodiments, the fresh camel milk is preferably sourced from Altay, Tacheng, and Urumqi.

[0018] In some embodiments, the centrifugation conditions are centrifugation at 4000 g for 20 min.

[0019] On the other hand, the present invention provides an application of camel milk protein, wherein the camel milk protein is used as a food or dairy product in one or more of the following situations: a) Possesses antibacterial, anti-inflammatory, and metabolic regulatory potential; b) Functional characteristics shifted from basal metabolism to immune defense; c) Specific enrichment of ribosomes and muscle cytoskeleton pathways; The camel milk protein is as described in any of the previous schemes.

[0020] The beneficial effects of this invention are: This invention focuses on naturally fermented camel milk. By comparing the changes in protein properties and structure before and after natural fermentation, and using proteomics technology, it systematically analyzes the evolution of protein types, contents, functions, and metabolic pathways. It elucidates the changes in milk protein structure and composition driven by microorganisms, providing theoretical and technical support for the development of high-value-added fermented camel milk products and the industrial upgrading of dairy products with border characteristics. Attached Figure Description

[0021] Figure 1 Changes in particle size of camel milk protein before and after natural fermentation Figure 2 Changes in the zeta potential of camel milk protein before and after natural fermentation Figure 3 Infrared spectra of camel milk protein before and after natural fermentation Figure 4 Analysis results of camel milk protein amide I band before and after natural fermentation Figure 5 Changes in the hydrophobicity of camel milk protein surface before and after fermentation Figure 6 The content of free sulfhydryl groups in natural camel milk protein Figure 7 Electrophoretic patterns of camel milk proteins before and after natural fermentation (Note: M: Marker; 1-6: naturally fermented camel milk; 7-12: unfermented camel milk) Figure 8 Identification of protein content in camel milk before and after natural fermentation Figure 9 PCA diagram of camel milk proteins Figure 10 Volcano plot of differentially expressed proteins in camel milk before and after natural fermentation Figure 11 GO enrichment analysis of the first 5 significantly upregulated (A) and significantly downregulated (B) milk proteins Figure 12 KEGG enrichment chords of milk proteins before and after natural fermentation of camel milk: upregulation (A) and downregulation (B) of the top 10 significantly differentially expressed metabolic pathways. Detailed Implementation

[0022] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0023] Experimental samples This study systematically collected regionally representative camel milk samples, including six independent biological replicates (n=6) of raw camel milk (R1-R6) and their corresponding naturally fermented camel milk (F1-F6). The samples were geographically distributed across three regions: Altay, Tacheng, and Urumqi, with two pastures selected from each region as sampling sites. Each pasture represented one biological replicate. Following standard sampling procedures, one sample each of fresh camel milk and its traditionally naturally fermented product was collected from each pasture (see Table 1), for a total of 12 samples. Immediately after collection, the samples were placed in portable iceboxes (4°C) for cryopreservation and transported to the laboratory. They were then aliquoted and stored at -80°C until subsequent analysis to ensure the integrity of the biomolecules.

[0024] The natural fermentation process is a local traditional method: using fresh camel milk as a base, adding pre-fermented milk as a natural starter, and carrying out solid-state fermentation in a constant temperature environment of 25±1℃, with timed stirring three times a day using sterilized wooden sticks, the entire process taking 48-72 hours. The pre-fermented milk is made from fresh camel milk through conventional lactic acid fermentation under the action of L. bulgaricus and Streptococcus thermophilus.

[0025] Table 1: Summary of Sampling Information Experimental reagents and instruments The main reagents and instruments used in this study are shown in Tables 2 and 3.

[0026] Table 2 Main Reagents Table 3: Main Instruments Sample pretreatment: Take 10 mL of fresh camel milk and naturally fermented camel milk samples respectively, transfer them to 15 mL centrifuge tubes, and centrifuge at 4000 g for 20 min at 4℃. After centrifugation, use a sterile scraper to remove the upper fat layer, and repeat the centrifugation operation until the fat layer is removed. Vortex the skim milk samples to mix them, aliquot them into EP tubes, and store them in an ultra-low temperature freezer at -80℃ for long-term use.

[0027] Particle size and zeta potential determination: Transfer 500 μL of the test solution and add 4.5 mL of distilled water to dilute it at a volume ratio of 1:10. After thorough mixing using a vortex mixer, the particle size of the camel milk protein particles was characterized using a dynamic light scattering instrument.

[0028] The instrument parameters were set as follows: the temperature control system of the detection cell was maintained at 25.0℃, the sample refractive index was set to 1.59, and the refractive index of the dispersion medium (deionized water) was set to 1.33. All samples were subjected to three parallel experiments, and the average value was taken as the final result. Under the same conditions, the zeta potential of camel milk protein was further measured.

[0029] SDS-PAGE analysis: First, prepare a 12% separating gel and a 5% stacking gel system. Then, load the sample containing 20 µg of protein with 5×SDS loading buffer (composition: 250 mM Tris-HCl pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5%). β The sample was mixed with mercaptoethanol at a volume ratio of 4:1. After the protein was denatured by water bath treatment at 100℃ for 5 min, it was briefly centrifuged (12,000×g, 1 min) and 7 μL was loaded onto the sample. An equal volume of protein marker was set as a reference.

[0030] The electrophoresis procedure employed a two-stage voltage control: an initial voltage of 80 V was set, and the voltage was increased to 120 V until electrophoresis was complete when the indicator migrated to the interface between the two gel layers. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 for 30 minutes, followed by destaining with a destaining solution (containing a mixture of 10% acetic acid and 40% methanol) under shaking conditions, with the destaining solution being replaced three times until the background became transparent. Finally, the gel was scanned and the data analyzed using a gel imaging system.

[0031] Secondary structure determination: Weigh out the camel milk freeze-dried powder sample and grind it thoroughly with potassium bromide powder at a mass ratio of 1:100. Press the mixture into transparent sheets. Detection was performed using a Fourier transform infrared spectrometer with parameters set to a scanning range of 4000-400 cm⁻¹. Signal acquisition involved averaging 64 scans, and the resolution was set to 8 cm⁻¹. To ensure data reliability, three parallel pellets were prepared for each sample for repeated testing.

[0032] Surface hydrophobicity measurement: Camel milk protein samples were serially diluted with freshly prepared phosphate-buffered saline (0.01 mol / L PBS, pH 7.0) to prepare a 0.1 mg / mL working solution. 4.00 mL of the protein solution was accurately measured, and 20 μL of the ANS fluorescent probe (8 mmol / L) was added. After thorough mixing by vortexing, the solution was incubated at 25°C for 15 min in the dark. Surface hydrophobicity was detected using fluorescence spectroscopy, with the detection wavelength set at 390 nm for excitation and 500 nm for emission, and the excitation and emission slit widths both set to 5 nm. All experiments were performed in triplicate for data validation.

[0033] Determination of free thiol groups: Transfer 500 μL of the sample to be tested, and add 5 mL of Tris-Gly buffer (containing 0.086 mol / L Tris, 0.09 mol / L glycine, 0.004 mol / L EDTA, and 8 mol / L urea) and 20 μL of Ellman's reagent (DTNB concentration: 8 mg / 2 mL Tris-Gly buffer). Vortex thoroughly to mix, and incubate at room temperature in the dark for 15 min. Measure the absorbance at 412 nm using a UV-Vis spectrophotometer, with the mixture without DTNB as a blank control. The free thiol content is calculated using the following formula: (1) Where: A412—the absorbance of the sample at 412 nm; D—Dilution factor; A blank —The absorbance of the sample blank at 412 nm; C – Sample solids concentration (mg / mL).

[0034] Protein enzymatic hydrolysis: Add 40 mM DTT to the sample and vortex at 37°C and 600 rpm for 1.5 h. After the sample cools to room temperature, add 20 mM IAA to block the reduced cysteine ​​residues and incubate in the dark for 30 min. Transfer the sample to a 10 kDa molecular weight cutoff Microcon ultrafiltration tube and wash three times with 100 μL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) and twice with 25 mM NH4HCO3 buffer. Add trypsin at a trypsin-to-protein ratio of 1:50 and incubate overnight (15-18 h) at 37°C. Collect the digestion product through the filtrate. Use C... 18 The peptides were desalted using a solid-phase extraction column, concentrated by vacuum centrifugation, and then reconstituted in 40 μL of a solution containing 0.1% (v / v) formic acid. The peptide concentration was determined by 280 nm UV spectrophotometry, and the peptides were calibrated by iRT for subsequent DIA experiments.

[0035] LC-MS / MS analysis: Sample peptides were analyzed in DIA mode using an Orbitrap Astral mass spectrometer coupled with a Vanquish Neo UHPLC system.

[0036] Mass spectrometry parameters are set as follows: MS 1 The full scan quality range is 380-980. m / z 120,000 (200) resolution m / z Automatic gain control (AGC) target 500%, maximum ion implantation time 50 ms; MS in DIA mode 2 The scan uses 299 independent windows, with an isolation window width of 2. m / z HCD collision energy 28 eV, AGC target 500%, maximum injection time 22 ms.

[0037] DIA data were analyzed using DIA-NN (v1.8.1), with the following key parameters set: Enzyme digestion parameter was trypsin / P (specific cleavage), with a maximum allowed missed cleavage count of 1; fixed modifications included cysteine ​​carboxymethylation (C), and dynamic modifications included methionine oxidation (M), protein N-terminal acetylation (Acetyl), and deamidation (N / Q). The UniProt database (Camelus bactrianus) was used for searching, with precursor and fragment ion mass error tolerances set at ±10 ppm and ±0.02 Da, respectively. Protein identification confidence was ≥99% (both peptide and protein level FDR ≤1%).

[0038] Statistical and bioinformatics analysis: Independent samples t-tests were performed using SPSS 25 statistical software (IBM, USA) to compare the differences between naturally fermented and unfermented camel milk groups. Results are expressed as mean ± standard deviation, and the significance threshold was set at ( ). P <0.05). Data visualization was performed using Origin 2021 (OriginLab, USA).

[0039] The differentially expressed proteins were selected based on FC > 1.5 or < 0.67. Further GO functional annotation and KEGG metabolic pathway analysis were performed on the differentially expressed proteins using the DAVID bioinformatics database (v6.8, https: / / david.ncifcrf.gov). Venn diagrams, volcano diagrams, and visualization of GO and KEGG pathway enrichment analyses were achieved using the MicroBioInformatics online analysis platform (https: / / www.bioinformatics.com.cn).

[0040] Example 1: Natural fermentation regulates the particle size distribution and potential of camel milk proteins. The particle size of milk proteins reflects the degree of aggregation or dissociation of milk protein molecules. Particle size affects the stability of the system; the larger the particle size, the worse the stability of the solution system. Before natural fermentation, the particle size distribution of camel milk proteins is relatively concentrated. Figure 1 The average particle size was 322.91 ± 3.12 nm. After natural fermentation, the particle size distribution changed significantly, with the average particle size increasing to 614.80 ± 61.45 nm, indicating that camel milk proteins aggregated during fermentation, forming larger particles. This may be due to the effects of organic acids, enzymes, and other substances produced by microbial metabolism during fermentation on protein molecules, leading to enhanced interactions between protein molecules and thus promoting protein aggregation. This increase in particle size may affect the stability of camel milk proteins, reducing their stability in solution and making them more prone to precipitation or stratification.

[0041] Zeta potential is a key indicator for measuring the surface charge characteristics of colloidal particles. Its value reflects the strength of electrostatic repulsion between particles and directly affects the stability of the dispersion system. The charge characteristics of the milk protein system change significantly after fermentation. Figure 2 The zeta potential before fermentation was -20.76±3.32 mV, and the zeta potential after fermentation became -8.9±5.4 mV, with a significant decrease in absolute value. P <0.05). This potential change directly weakens the electrostatic repulsion between particles in the milk protein dispersion system, affecting the colloidal stability of the system, thereby inducing protein aggregation and the formation of a three-dimensional gel network structure, ultimately manifesting as the enhanced viscoelasticity and altered rheological properties characteristic of fermented dairy products. This series of processes, through the synergistic effect of multiple factors such as charge regulation, conformational rearrangement, and intermolecular interactions, provides a theoretical basis for regulating the structural properties of fermented dairy products.

[0042] Example 2: Natural fermentation regulates the structure of camel milk proteins Fourier transform infrared spectroscopy can be used to characterize the structural features of proteins. Figure 3 Infrared spectra of unfermented and naturally fermented camel milk are shown. Differences exist between the two in certain wavenumber regions, indicating that natural fermentation alters the protein structure of camel milk. The original spectra of the two milk samples were processed using Peakfit 4.12 software, and the amide bands were analyzed. Figure 4 The secondary structure and corresponding relative content of each milk sample were obtained (Table 4). The analysis results showed that the secondary structure of camel milk proteins differed before and after natural fermentation. The relative contents of β-sheets and β-turns in fresh camel milk were higher than those in naturally fermented camel milk, while the relative contents of random coils were lower. This indicates that natural fermentation affected the secondary structure of camel milk proteins, leading to the unfolding or rearrangement of their molecular structure, thereby affecting the interaction between proteins and the surrounding medium.

[0043] Table 4. Effects of fermentation treatment on the infrared spectral characteristics of camel milk protein amide I band. To further understand the changes in protein structure during fermentation and their impact on functional properties, surface hydrophobicity was tested. The fluorescence intensity of the surface hydrophobicity before natural fermentation was 2056.45±356.32, and the fluorescence intensity after fermentation was 7323.44±1032.12, indicating a significant increase in surface hydrophobicity of camel milk protein after fermentation. Figure 5 )( P <0.05). This indicates that the conformation of camel milk protein molecules changes during natural fermentation, resulting in more hydrophobic amino acid residues being exposed on the protein surface.

[0044] Free sulfhydryl groups, as the active form of cysteine ​​residues in proteins, are key indicators for assessing redox state and structural stability. Their content determination can track disulfide bond dynamics and conformational evolution. The free sulfhydryl group content before natural fermentation was 9.86±3.63 μmol / g, while the free sulfhydryl group content in camel milk protein after fermentation was 17.23±2.81 μmol / g, showing a significant increase (P<0.05). Figure 6 This indicates that more thiol groups are exposed or formed during natural fermentation. Enzymes produced by microbial metabolism during fermentation may catalyze the reduction of disulfide bonds in proteins, and protein hydrolysis may also lead to polypeptide chain breakage, thereby exposing more thiol groups. The exposure of thiol groups can affect the redox properties, stability, and interactions with other molecules of proteins. This change in redox state not only affects protein structural stability but also significantly alters its emulsifying, gelling, and other functional properties.

[0045] Example 3: Natural fermentation regulates the composition of camel milk proteins. Electrophoretic analysis showed that naturally fermented camel milk and unfermented camel milk exhibited significant differences in their protein composition. Figure 7 Specifically, the fermentation group samples showed improvements in lactoferrin, serum albumin, and... α -Casein, β -casein and κ Significant changes were observed in characteristic bands such as casein, manifested as altered band migration rates or weakened intensity; while in the control group, the positions and intensities of each protein band remained relatively stable, without significant shifts or changes in intensity. Further analysis indicated that proteases produced by microbial metabolism during natural fermentation may cause structural modifications or release of functional peptides in some milk proteins through specific enzymatic hydrolysis.

[0046] Using DIA proteomics technology, we conducted an in-depth analysis of naturally fermented and unfermented camel milk, comprehensively revealing the differences in protein composition between the two types of camel milk. A total of 1671 proteins were identified, of which 963 were common to both types of camel milk, 653 were unique to unfermented camel milk, and 65 were unique to naturally fermented camel milk. Figure 8 These unique proteins may originate from microbial metabolites or products of host protein degradation. This finding fully reflects the remodeling effect of fermentation on the proteome.

[0047] In the comparison of presence and absence of differences, using the screening criteria of half or more of the data in one group not being null values ​​and all data in the other group being null values, 15 milk proteins were screened from naturally fermented camel milk (Table 5), while 158 were found in unfermented camel milk. The specific expressed proteins and low-quality proteins identified in naturally fermented camel milk revealed its complex bioactive functional network. Phosphatidylcholine transporter ABCB4 isoform X4 and serine protease inhibitor Kazal type 1 may be involved in lipid metabolism regulation and protease activity inhibition, thereby maintaining dairy product stability and inhibiting the growth of harmful microorganisms; prothymocyte prolactin... α The presence of apoptosis protease activator 1 (APP1) suggests potential immunomodulatory and cellular homeostasis functions, possibly enhancing the bioactivity of fermentation products by regulating inflammatory or apoptotic pathways. Degradation or modification of low-quality proteins such as the polyunsaturated fatty acid lipoxygenase ALOX15B may affect lipid oxidation processes, thereby altering flavor compound formation. Furthermore, molecules such as calcineurin B subunit 1 and centrosome proteins suggest the role of calcium signaling and cell cycle regulation in the dynamics of the fermentation microbial community. These findings collectively indicate that natural fermentation can enrich camel milk with functional components possessing antibacterial, anti-inflammatory, and metabolic regulatory potential, potentially providing a new perspective for studying the bioactivity and potential health benefits of camel milk.

[0048] Table 5. Proteins present only in naturally fermented camel milk in comparisons of presence and absence of differences. To explore the differences in overall protein expression between naturally fermented and unfermented camel milk, principal component analysis (PCA) was first performed on the quantified proteins in all samples. Within the 95% confidence interval, the distributions of the two groups of samples did not overlap. Figure 9 Furthermore, the replicates within the same group showed good clustering; group F samples were mainly concentrated in the left region on the PC1 axis, while group R samples were mainly distributed in the right region. This indicates a significant difference in protein expression profiles between naturally fermented camel milk and unfermented camel milk.

[0049] To analyze proteins with differential expression among different groups, the experimental data were further screened for differential expression. A two-sample, two-tailed t-test was used for univariate statistical analysis to calculate p-values, and the FC value was combined with this to screen differentially expressed proteins. The distribution of proteins in the volcano plot generally presents a "V" shape. Figure 10 Based on the distribution characteristics of the volcano map, the screening criteria were set as follows: P <0.05, FC>1.5 indicates significantly upregulated expression; P <0.05 and FC <0.67 were considered significantly downregulated. The results showed that 507 differentially expressed proteins were identified from the two camel milk samples, of which 195 were... Significantly upregulated expression, 312 significantly downregulated expression (Table 6).

[0050] Table 6 Some upregulated proteins after natural fermentation Example 4: Natural fermentation regulates the function of camel milk protein Distribution of camel milk protein functional annotation GO enrichment analysis revealed that natural fermentation significantly altered the functional annotation distribution of camel milk proteins. Figure 11 At the microbial growth factor (MF) level, the upregulation of post-fermentation proteins in functions such as antimicrobial peptide-mediated humoral immune responses and neutrophil chemotaxis may be related to microbial metabolites (such as antimicrobial peptides secreted by lactic acid bacteria) or bioactive peptides generated from proteolysis during fermentation. Furthermore, the upregulation of the insulin-like growth factor receptor signaling pathway suggests that fermentation may enhance the interaction between milk proteins and growth factors by modifying their conformation, thereby affecting nutrient absorption or tissue repair. In contrast, the downregulation of GTPase activity and ATP-dependent protein folding function in pre-fermentation proteins may reflect energy metabolism reprogramming during fermentation, with microorganisms preferentially utilizing milk proteins as carbon sources, leading to a decrease in the host cell's basal metabolic activity. CCs results showed that post-fermentation proteins were more likely to be distributed in the extracellular space and multivesicular bodies, while pre-fermentation proteins were mainly located in the cytoplasm and endoplasmic reticulum lumen, possibly related to the breakdown and efflux of intracellular structural proteins (such as casein micelles). BP analysis showed that the activity of peptidase inhibitors and calcium ion binding of proteins were significantly enhanced after fermentation, suggesting that fermentation may extend the shelf life and improve texture properties by inhibiting protein degradation or chelating free calcium ions. The downregulation of the classical complement activation pathway, which enriches proteins before fermentation, may be related to the denaturation of heat-sensitive proteins or their degradation by microbial proteases during fermentation. This change may reduce the allergenicity of dairy products, but further verification is needed.

[0051] Regulating the core functional properties of camel milk proteins KEGG enrichment analysis showed that natural fermentation significantly altered the core functional properties of camel milk proteins through metabolic reprogramming. Figure 12The sustained enrichment of proteins in immune-related pathways such as complement and coagulation cascades, lysosomes, and endocytosis after fermentation suggests that fermentation may enhance the bioactivity of dairy products by activating host defense mechanisms. The enrichment in ribosome and muscle cytoskeleton pathways suggests that microbial proteases may selectively degrade structural proteins during fermentation, leading to protein aggregation and conformational remodeling. Consistent with this, physicochemical analysis showed increased protein particle size, decreased absolute zeta potential, and increased surface hydrophobicity after fermentation, further supporting protein structure unfolding and exposure of the hydrophobic core. Secondary structure analysis indicated that after fermentation, β... - The decrease in folding and β-turn angles, and the increase in the proportion of random coils, indicate a reduction in protein orderliness and a shift towards a flexible conformation. This may promote the exposure of functional sites, but it may also weaken its thermal stability. The significant enrichment of proteins in basal metabolic pathways such as carbon metabolism, glycolysis / gluconeogenesis, and endoplasmic reticulum protein processing before fermentation reflects that their original functions were primarily energy supply and maintenance of structural integrity. The disappearance of these pathways after fermentation corroborates that microbial metabolic competition leads to a redistribution of carbon sources in the host protein, with milk proteins being broken down into smaller peptides for microbial use, thereby triggering structural unfolding and functional transformation. Notably, the co-enrichment of complement and coagulation cascades before and after fermentation may stem from the retention of some protease-resistant components.

[0052] In summary, this invention demonstrates that natural fermentation significantly alters the physicochemical properties, structural characteristics, and composition of camel milk proteins. The average particle size of the milk proteins increased from 322.91 nm to 614.80 nm after fermentation. P The absolute value of the zeta potential was <0.05, indicating a significant decrease, suggesting enhanced protein aggregation and decreased colloidal stability. This is closely related to the enhanced intermolecular interactions induced by microbial metabolites. Electrophoresis and infrared spectroscopy further confirmed that fermentation led to structural modifications or degradation of major components such as lactoferrin and casein, manifested as weakened characteristic band intensity and secondary structure reorganization. β - Folding and β - The proportion of turns decreased, while the proportion of random coils increased significantly, suggesting a decrease in protein order and a tendency towards a flexible conformation. The surface hydrophobicity and free thiol content increased simultaneously. P <0.05 indicates the exposure of the hydrophobic core and the dynamic rearrangement of disulfide bonds, which may enhance its emulsification and gelation properties through redox regulation, but may also accelerate aggregation and precipitation.

[0053] Proteomics analysis identified 1671 proteins, including 65 unique proteins found in naturally fermented camel milk (such as phosphatidylcholine transporter ABCB4 and prothymocytes). αCamel milk proteins play a crucial role in lipid metabolism regulation, immune response, and cell cycle regulation. GO and KEGG enrichment analyses of 507 differentially expressed proteins (195 upregulated and 312 downregulated) revealed a significant shift in the functional properties of fermented milk proteins from basal metabolism (e.g., carbon metabolism, glycolysis) to immune defense (e.g., complement and coagulation cascades, lysosomes). The complement pathway was continuously enriched both before and after fermentation, potentially activating alternative pathways through microbial metabolites, thereby enhancing antibacterial and antiviral functions. Furthermore, the specific enrichment of ribosome and muscle cytoskeleton pathways suggests that the degradation of structural proteins may release functional peptides. This study systematically reveals the multidimensional mechanisms by which natural fermentation regulates the properties of camel milk proteins, providing a theoretical basis and target support for the development of functional fermented dairy products.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

Claims

1. A camel milk protein, characterized in that, The camel milk protein comprises the following functional protein combination: phosphatidylcholine transporter ABCB4 subtype X4, prothymocyte α, myelin basic protein, serine protease inhibitor Kazal type 1, apoptosis protease activator 1, polyunsaturated fatty acid lipoxygenase ALOX15B, transduction-like enhancer protein 6, nucleophosphorus protein, vascular permeability factor, procadherin α-1-like, centrosome protein 290 kDa subtype X5, calcineurin B subunit type 1, serum alkaline protease inhibitor, DNA-dependent metalloproteinase SPRTN subtype X3, and serine hydroxymethyltransferase.

2. The camel milk protein as described in claim 1, characterized in that, The camel milk protein is obtained by the following method: using fresh camel milk as a substrate, adding pre-fermented milk as a natural starter, and carrying out solid-state fermentation in a constant temperature environment of 25±1℃, with timed stirring 3 times a day, fermenting for 48-72 hours to obtain naturally fermented camel milk; transferring the naturally fermented camel milk to a centrifuge tube, centrifuging at 4℃, scraping off the upper fat layer after centrifugation, and repeating the centrifugation operation until the fat layer is completely removed to obtain camel milk protein; preferably, the pre-fermented milk is made from fresh camel milk by conventional lactic acid fermentation under the action of Lactobacillus bulgaricus and Streptococcus thermophilus.

3. The camel milk protein as described in claim 2, characterized in that, The fresh camel milk is sourced from preferred varieties, originating from Altay, Tacheng, and Urumqi.

4. The camel milk protein as described in claim 2, characterized in that, The centrifugation conditions were 4000 g for 20 min.

5. The camel milk protein according to any one of claims 1-4, characterized in that, The camel milk protein has an average particle size of 614.80±61.45 nm and a zeta potential of -8.9±5.4 mV. And / or, the fluorescence intensity of the camel milk protein's surface hydrophobicity is 7323.44±1032.12, and the free thiol content is 17.23±2.81 μmol / g.

6. The camel milk protein according to any one of claims 1-4, characterized in that, The relative contents of β-sheet and β-turn of the camel milk protein are lower than those of fresh camel milk.

7. The camel milk protein according to any one of claims 1-4, characterized in that, The camel milk protein described herein exhibits upregulated protein expression compared to fresh camel milk. The upregulated proteins include one or more of the following: antimicrobial peptide-7-like protein, antimicrobial peptide-2-like isoform X2, CXC motif chemokine, CC motif chemokine, CD59 glycoprotein, α-lactalbumin, κ-casein, whey acid protein, aminopeptidase, superoxide dismutase (copper-zinc type), E3 ubiquitin protein ligase, mucin-4, mucin-15, clathrin light chain, β-2-glycoprotein 1, ferritin, properin, CD44 antigen, insulin-like growth factor II, and protein disulfide isomerase.

8. The camel milk protein according to any one of claims 1-4, characterized in that, The camel milk protein, compared to fresh camel milk, includes one or more of the following functional properties: a) Possesses antibacterial, anti-inflammatory, and metabolic regulatory potential; b) Functional characteristics shifted from basal metabolism to immune defense; c) Specific enrichment of ribosomes and muscle cytoskeleton pathways.

9. A method for preparing the camel milk protein according to claim 1, characterized in that, The method includes: using fresh camel milk as a substrate, adding pre-fermented milk as a natural starter, and carrying out solid-state fermentation in a constant temperature environment of 25±1℃, stirring three times a day for 48-72 hours to obtain naturally fermented camel milk; transferring the naturally fermented camel milk to a centrifuge tube, centrifuging at 4℃, scraping off the upper fat layer after centrifugation, and repeating the centrifugation operation until the fat layer is completely removed to obtain camel milk protein; preferably, the pre-fermented milk is made from fresh camel milk by conventional lactic acid fermentation under the action of Lactobacillus bulgaricus and Streptococcus thermophilus.

10. An application of a camel milk protein, wherein the camel milk protein is used as a food or dairy product in one or more of the following situations: a) Possesses antibacterial, anti-inflammatory, and metabolic regulatory potential; b) Functional characteristics shifted from basal metabolism to immune defense; c) Specific enrichment of ribosomes and muscle cytoskeleton pathways; The camel milk protein is as described in any one of claims 1-8.

Images