At present, China has approved 100 kinds of foods containing proteins for special medical purposes, including nutritionally complete formulas, specific nutritionally complete formulas, protein modules, and liquid formulas. Eighty-four percent of them are produced using whey protein as the protein source. This review examines these food formulations, focusing on the application of whey protein powders in them. Based on current regulatory requirements and the characteristics of whey protein, we discuss future prospects for the application of whey protein powders in foods for special medical purposes. This review provides support for the research and development of foods for special medical purposes (FSMP).

Infant formula generally contains 25%–30% fat content. Unsaturated fatty acids, such as linoleic acid, linolenic acid, docosahexaenoic acid, arachidonic acid, and eicosapentaenoic acid, are often added to infant formula in order to ensure its nutritional adequacy. There is a membrane structure composed of whey protein-casein complexes on the surface of fat globules in infant formula, which leads to the accumulation of a fat layer on the surface of milk powder particles. This structure makes the oil in milk powder concentrate on the surface, which increases the chance of contact with oxygen and water, making the oil easier to oxidize and hydrolyze. During processing and storage, infant formula is susceptible to oxygen, metal ions and other fat oxidation reaction conditions, leading to the deterioration of product quality. In this paper, the fatty acid composition, the structural characteristics of fat globules, and the mechanism of fat redox reaction in infant formula are reviewed, and the different fat extraction methods and analytical methods used to determine the peroxide value of infant formula are summarized and evaluated. This review hopes to provide a theoretical basis for maintaining the quality and safety of infant formula milk powder during processing, storage, and marketing.

The formulation and nutritional characteristics of nutritionally complete foods for special medical purposes for children aged 1 to 10 years approved and registered by the State Administration for Market Regulation were examined and analyzed based on their label instructions. The results showed that there were no significant differences in the distribution of label values of protein, fat, or carbohydrates between different nutritionally complete food products. The average energy supply ratios of the three nutrients were 13.5%, 33.0%, and 53.5%, respectively, which were all within the appropriate range. The average label value of protein was 0.79 g/100 kJ, and the proportion of high-quality protein was 100%. The average energy supply ratios of linoleic acid and α-linolenic acid were 4.5% and 0.9%, respectively, which were significantly higher than the levels defined in the General rules of food for special medical purposes (GB 29922-2013). The vitamin and mineral label values of most products were equal to or up to 200% higher than the lower limit specified in GB 29922-2013. Optional ingredients such as dietary fiber, choline, and taurine were frequently used without the addition of fluoride or nucleotides. The most frequently used sources of protein, fat, and carbohydrate were whey protein, medium chain triglycerides, and maltodextrin, respectively.

Next generation sequencing (NGS) is a new sequencing method invented at the beginning of the 21st century on the basis of first-generation sequencing technology. With its high throughput, high accuracy and abundant genetic data, NGS has been gradually applied to the dairy industry and has played an important role in ensuring the quality and safety of dairy products. On the one hand, this paper introduces the application of whole genome sequencing (WGS) technology to identify contaminated microorganisms, pathogen traceability, strain polymorphism, antibiotic resistance and virulence factor carrying status analysis. On the other hand, this paper focuses on the application of metagenomic sequencing technology in research on the application of microbial diversity, including microbial diversity investigation in the processing environment of dairy products, environmental microorganism monitoring of dairy factories, shelf-life assessment of dairy products, microbial risk monitoring of raw materials, and authenticity assessment of dairy products. Finally, this paper comprehensively analyzes and discusses the challenges and future prospects for the application of NGS technology in the field of dairy products.

Folic acid is a very important water-soluble vitamin in human milk, which is essential for embryonic development and growth. The folic acid in human milk often exists in the form of polyglutamic acid salts (≥ 4 glutamyl residues), such as 5-methyltetrahydrofolate. Folic acid deficiency may lead to birth defects such as neural tube defect and congenital heart disease. This article introduces the folic acid content in breast milk from different countries/regions, the factors influencing it and its health effects in order to provide a reference for the development of infant formula milk powder.

To evaluate the measurement uncertainty in the microbiological determination of the vitamin B6 content in milk powder, we estimated various uncertainty sources according to the National food safety standard for the determination of vitamin B6 in foods (GB 5009.154-2023) and the Evaluation and expression of uncertainty in measurement (JJF 1059.1-2012), and calculated combined standard and extended uncertainties. The results showed that the content of vitamin B6 in milk powder was (9.34 ± 1.23) mg/kg (k = 2, p = 95%). Through uncertainty component analysis, it was found that measurement repeatability and the preparation of standard solution had a significant impact on the determination of the vitamin B6 content in milk powder.

To investigate the correlation between the beany odor of raw cow’s milk and feed types, this study selected two samples of raw cow’s milk with beany odor and five common feeds: soybean meal, alfalfa, flaked corn, oat grass, and corn silage, and measured their volatile flavor compounds by headspace solid phase microextraction-gas chromatography-mass spectrometry (GC-MS). Key flavor compounds were identified using relative odor activity value (ROAV). The results indicated that acids and aldehydes contributed primarily to the beany odor of raw milk. A total of 49 volatile substances were identified in the two milk samples, among which hexanoic acid, octanoic acid, decanoic acid, decanal, nonanal, (E)-2-octenal, and (E)-2-decenal were the main beany odor substances. A total of 257 volatile substances were detected in all feed samples, including decanal, nonanal, and (E)-2-octenal. According to ROAV analysis, the beany odor compounds were more abundant in the feed samples than in the milk samples. The relative contents of beany odor compounds in the feed samples followed the following decreasing order of alfalfa > oat grass > soybean meal > flaked corn > corn silage, and flaked corn exclusively contained vanillin, with a strong milky aroma.

The iodine content in liquid milk was determined via gas chromatography (GC). Through systematic screening and optimization of derivatizing reagents and derivatization time, higher measured values of the iodine content in liquid milk were obtained while solving the problem of poor repeatability of the traditional method. The optimized method was more efficient than the national standard method National food safety standard-determination of iodine in food (GB 5009.267-2020). The optimal pretreatment conditions were determined to be 1.0 mL of sulfuric acid, 2.0 mL of butanone, 0.5 mL of 3.5% (V/V) hydrogen peroxide, and derivatization reaction for 30 min. This study provides a reliable basis for this optimized scheme to achieve efficient and accurate detection of iodine in liquid milk and holds significant application value in the control of the iodine content in liquid milk.

In this study, to understand the differences in quality between high-fat and regular yogurts, we compared the changes in viable bacterial counts, acidity, and textural characteristics between homemade high-fat and regular yogurts as well as commercial yogurt (two samples for each type of yogurt) during the shelf life. The results showed that the fat content (16.7% and 17.0%) and energy (731 and 721 kJ/100 g) of high-fat yogurt were significantly higher than those of regular yogurt, and the carbohydrate content (3.6% and 2.3%) was significantly lower than that of regular yogurt. High-fat yogurt possessed a strong fatty flavor and a thick texture. The acidity (90.62 and 91.15 °T) and viable bacterial count (9.15 and 8.95 (lg (CFU/mL))) of high-fat yogurt were lower than those of regular yogurt, whereas the hardness (22.45 and 26.15 g), adhesiveness (0.59 and 0.72 mJ) and chewiness (0.86 and 1.03 mJ) were higher than those of regular yogurt. Compared to commercial yogurt, the acidity of homemade yogurts is more stable, but basically the same viable bacterial count and textural characteristics. In conclusion, high-fat yogurt has lower carbohydrate content, acidity and viable bacterial count, and the high fat content not only imparts a fatty flavor to this yogurt but also improves its textural properties and stability.

In this study, four prebiotics including inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and yeast β-glucan were added singly (inulin) or in different combinations (inulin + FOS + GOS and inulin + FOS + GOS + yeast β-glucan) to fermented milk containing Bifidobacterium animalis subsp. lactis PB200. The titratable acidity, pH, whey syneresis and total viable count of the fermented milk were measured during storage periods. Moreover, the rheological and textural properties were measured. The aim was to clarify the effect of different prebiotic combinations on the yogurt system in order to obtain the optimal probiotic combination. Compared with the control group with no prebiotic added, the addition of prebiotics significantly delayed the post-acidification, and promoted the growth of B. animalis subsp. lactis PB200. Besides, the titratable acidity of fermented milk added with inulin, FOS, GOS and β-glucan was the lowest , and the titratable acidity increased slowly and whey syneresis was less with the extension of storage time. The addition of all four prebiotics could maintain a higher viable count of B. animalis subsp. lactis PB200. The results of rheological and textural analysis showed that yogurt added with all four prebiotics had higher gel strength, hardness, cohesiveness, gumminess, adhesiveness and springiness, showing better texture. In summary, the addition of inulin, FOS, GOS and β-glucan resulted in better quality of probiotic fermented milk with B. animalis subsp. lactis PB200.

Whey proteins, as high-quality proteins in milk, have high nutritional value and diverse functional properties. Genetic variations under different environments and post-translational modifications cause differences in the expression and structure of whey proteins, resulting in an increased diversity and complexity, which makes it more difficult to gain insights into whey proteins. With the continuous development of omics techniques, people have gained a deeper understanding of the composition and functional activities of whey proteins. With a view to providing a scientific theoretical basis for in-depth studies of bovine whey proteins and the development of personalized dairy products, this paper reviews the application of proteomics in the study of bovine whey proteins, and analyzes the similarities and differences in the types, molecular structures, metabolic pathways, and biological functions of whey proteins from different breeds of cow, lactation periods, feeding conditions, and processing methods.

Dairy products are an indispensable food in our lives. Protein is a major component of dairy products, and changes in the protein content greatly affect the quality and taste of dairy products. Proteomics is at the core of current post-genomics and systems biology approaches. In this paper, the concept, detection methods and data analysis methods of proteomics are reviewed, with emphasis on proteomic studies of differential proteomes in casein, milk fat globule membrane protein and whey protein, which are useful to distinguish dairy products from different sources. This review provide an effective working idea for authenticity identification and quality and safety assurance of dairy products.

The fat in breast milk exists in the form of fat globules, which are enveloped by a unique biophysical membrane called the milk fat globule membrane. The absence of fat globules will affect the structure and stability of fat droplets, which will in turn affect lipid digestion and absorption. In an effort to make the nutritional composition of milk powder approximate as closely as possible to that of breast milk, an extensive literature has developed concerning the fat globule membrane of cow’s milk, goat’s milk and other milks both domestically and internationally. With the aim of providing a reference for the resource development and application of milk fat globule membrane resources, this article reviews recent progress in understanding the composition of the milk fat globule membrane as well as the factors affecting the structure and functional characteristics of the milk fat globule membrane such as lactation stages, feeding regimes and milk sources as well as separation, thermal processing, homogenization and non-thermal processing, and it summarizes recent advances in breast milk fat globule membrane simulation and its application in infant formula and milk products for middle-aged and elderly people.

Human milk contains a large amount of vitamin C, which can meet the needs of newborns’ early growth and development. Vitamin C has been proven to have multiple functions such as preventing scurvy, treating hypoxic-ischemic encephalopathy and preventing placental abruption. This article provides an overview of the content of vitamin C in human milk in different countries/regions, the factors influencing it and its health effects on infants, hoping to provide a reference for the development of infant formula milk powder.

Protein is an important nutrient in the food system, and active ingredients (such as polyphenols, flavonoids, and terpenes) have a variety of physiological effects because of their unique chemical structures, such as antioxidant, antitumor, and chronic disease-preventive effects. They form complexes through covalent or noncovalent interactions, thus changing their functional structure, nutritional properties, and bioavailability. In this review, we summarize the application of multispectral methods, including ultraviolet-visible (UV-vis) absorption, fluorescence, Fourier transform infrared (FTIR), circular dichroism (CD) spectra, and molecular simulation techniques (molecular docking and molecular dynamics simulation) in the study of protein-active ingredient interaction. The principles, features, and examples of these techniques in the study of the interaction between protein and active ingredients are briefly described. This review also focuses on the application of molecular simulation in delivering active ingredients, regulating food flavor, and monitoring food quality based on protein-active ingredient systems, hoping to provide scientific reference for further study of the interaction between protein and active ingredients.

The milk fat globule membrane (MFGM) is a three-layered membrane that surrounds milk fat globules. Its main components include proteins, carbohydrates, and lipids, which are mainly derived from the mammary gland cell membrane. Different milk sources vary in the composition of MFGM proteins, which have good emulsifying properties, gastrointestinal stability and safety. Some MFGM proteins have diverse bioactivities including anti-inflammatory, antiviral and intestine regulatory effects and enhancing the exercise performance of muscles. This paper reviews the composition, physicochemical properties and bioactivities of MFGM proteins, which will provide a reference for the research and application of MFGM proteins in the dairy industry and related food additives.

A method was developed for simultaneous measurement of the levels of vitamins A, D3, K2, E, and β-carotene in infant formula milk powder using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The calibration curves for the five fat-soluble vitamins showed good linearity within the concentration range of 1–800 ng/mL. The average recoveries at three spiked levels were between 92.0% and 108.0% with relative standard deviations (RSDs) ranging from 0.4% to 2.6%. The limits of quantification (LOQs) of this method were 12.5–125 μg/100 g. The proposed method was characterized by simple sample pre-treatment, high quantitative sensitivity, and good accuracy and precision.

In this study, a prediction model based on Fourier transform mid-infrared spectroscopy was developed for the rapid detection of β-lactoglobulin in milk. The content of β-lactoglobulin in 260 different batches of milk samples was determined as reference by high performance liquid chromatography (HPLC), and the mid-infrared spectra of raw milk were collected using an Fourier transform mid-infrared spectrometer. The effective wavebands were selected, and the background noise was eliminated by Savitsky-Golay (SG) smoothing, first-order derivative or second-order derivative pretreatment before the establishment of the β-lactoglobulin prediction model by using partial least squares regression (PLSR). The results showed that SG five-point smoothing second-order derivative was the optimum preprocessing method. The PLSR model exhibited good prediction accuracy with correlation coefficient (R2) of 0.932 for the calibration set, root mean square error of calibration (RMSEC) of 0.049%, R2 of 0.923 for the calibration set, and root mean square error of prediction (RMSEP) of 0.057%.

Benzalkonium chloride and didecyl dimethyl ammonium chloride residues in infant formula milk powder sold from 2022 to 2024 were detected and statistically analyzed. Based on the European Union’s allowable daily intake for benzalkonium chloride and the German Federal Institute for Risk Assessment’s allowable daily intake for didecyl dimethyl ammonium chloride, a point estimation method was used to provide estimated values applicable to the percentiles of the available sample quantities. The detection rates of C12-benzalkonium chloride C14-benzalkonium chloride, C16-benzalkonium chloride, and didecyl dimethyl ammonium chloride in infant formula milk powder were 23.3%, 16.8%, 15.4%, and 16.0%, respectively; the average residue levels from lower bound (LB) to upper bound (UB) were 10.96–12.18 and 3.71–4.13 μg/kg, respectively. The average exposure levels (LB–UB) to benzalkonium chloride for infants and young children aged 0–6, 7–12, and 13–36 months were 0.281–0.297, 0.036–0.047, and 0.026–0.032 μg/(kg mb·d), respectively. The average exposure levels (LB-UB) to didecyl dimethyl ammonium chloride were 0.091–0.095, 0.007–0.011, and 0.013–0.016 μg/(kg mb·d), respectively. The hazard quotient (HQ) for both substances was less than 1. The contamination levels of benzalkonium chloride and didecyl dimethyl ammonium chloride in infant formula milk powder sold from 2022 to 2024 were found to be at an acceptable level.

To solve the difficulty of storing and transporting yak butter, yak butter microcapsule powders were prepared using a mixture of maltodextrin and sodium caseinate as the wall material, a mixture of molecular distilled monoglycerides and sucrose ester as the emulsifier and guar gum as the stabilizer by vacuum freeze-drying or spray drying. The basic physicochemical properties (moisture content, solubility, rheology, surface oil content, encapsulation efficiency, and yield), microscopic structure, and thermal stability of the two as-prepared samples were compared. The results showed that the microencapsulated powders prepared by the two methods were milky white in color and free of impurities, and had a fresh aroma. Both of them exhibited high encapsulation efficiency, (90.17 ± 1.07)% and (92.12 ± 0.32)%, respectively. The yield of freeze-dried microcapsule powders, (98.42 ± 0.38)%, was significantly higher than that of spray-dried microcapsule powders, (25.77 ± 1.34)%, and the average particle size (D[4,3] = 16.695 μm) was smaller than that of spray-dried powder (D[4,3] = 24.945 μm). The solubility and fluidity of the spray-dried powder were superior to those of the freeze-dried powder. Under a scanning electron microscope (SEM), the surface of spray-dried powder was smooth and spherical in shape, while the freeze-dried powder was irregular. The thermal stability of the spray-dried powder was better. In summary, when considering economic benefits, the spray-dried powder holds greater significance for industrial production.

Milk-derived extracellular vesicles (MEVs), originating from mammary epithelial cells, are nanoscale double-layered membrane vesicles transporting bioactive molecules like proteins, nucleic acids, and lipids, being crucial for intercellular communication and immune regulation. Although milk from minor animal species is not commonly found in the market due to its lower production, it possesses unique nutritional value and potential health benefits and is therefore worth exploring. This review covers the isolation and identification techniques, morphological characteristics, composition and biological characteristics of MEVs from goat, camel, yak, donkey, buffalo, porcine and horse milk. It points out the challenges and future directions in studying MEVs from minor dairy species, aiming to provide a theoretical foundation and scientific guidance for further research and application of MEVs so as to foster the development of the minor species milk industry.

China is blessed with minor species milk resources. Minor species milks from different sources have their own distinctive characteristics in terms of nutrient composition and content as well as functional activity. Oligosaccharides, as important bioactive ingredients in milk, have many significant biological functions. With the aim of providing theoretical reference for the development and utilization of oligosaccharides in minor species milk, this paper reviews recent progress in the research on the types, structures and contents of oligosaccharides in goat milk, donkey milk, buffalo milk, camel milk and horse milk, and it also summarizes the bioactive functions of milk oligosaccharides such as probiotic, antibacterial and anti-inflammatory properties as well as promoting brain development.

Goat’s milk contains a rich variety of oligosaccharides with various physiological functions, and its content and structure are close to those of human milk oligosaccharides. Goat’s milk can be regarded as a natural alternative source of human milk oligosaccharides with potential applications in the food field. This paper briefly introduces the composition, content and structure of oligosaccharides in goat’s milk, analyzes the factors affecting the composition and content of oligosaccharides in goat’s milk, and focuses on the comparison of various preparation methods such as membrane separation, chromatography and biosynthesis. In addition, the pre-treatment methods for and the characteristics of different techniques for the detection of goat’s milk oligosaccharides are summarized, and finally, the probiotic functions of goat’s milk oligosaccharides as well as the prospects and challenges for their application in the future research are discussed. Finally, the probiotic functions of goat’s milk oligosaccharides and future prospects and challenges for their research and application are discussed.

A high-throughput method for the screening and quantitation of 54 pesticide residues in fresh milk was developed by quick, easy, cheap, effective, rugged, safe (QuEChERS) extraction followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The study optimized the composition of mobile phase, extraction solvent, extraction solvent volume, extraction salt packet formulation, and purification packet formulation. Furthermore, the matrix effect was evaluated and methodological evaluation were carried out. The results showed that the linear range was 5.0–100.0 μg/L for all 54 pesticides, with correlation coefficients (r) higher than 0.999. The limit of quantification (LOQ) was 10.0 μg/kg for all target analytes, and the average recoveries from spiked samples were 67.78%–119.38%, with relative standard deviations (RSDs) (n = 6) lower than 9%. The method is simple, rapid, sensitive, and accurate. It can meet the requirements of the simultaneous detection and analysis of 54 pesticide residues in fresh milk.

According to the opinions of the state administration for market supervision on standardizing the use of rapid food detection, the performance of commercial colloidal gold immunochromatographic test strips for rapid in situ identification of bovine-, ovine- and caprine-derived ingredients in dairy products were evaluated in terms of application scope, detection limit, cross-reaction rate, sensitivity, false negative rate, false positive rate and coincidence rate with the results of the reference method. The results showed that the application scope of the test strip for bovine-derived ingredients was raw milk, and the detection limit was 5% (V/V); the application scope of the test strip for ovine- and caprine-derived ingredients was raw ovine and caprine milk, and the detection limit was 0.3% (V/V). Both test strips had no cross-reaction with enrofloxacin, sulfamethazine, dexamethasone, tetracycline or aflatoxin M1. The sensitivity, false positive rate and false negative rate of the test strips were 100%, 0% and 0%, respectively. The coincidence rate with the results of the reference method was high. In conclusion, the colloidal gold immunochromatographic test strips have high accuracy, and can be used for rapid in situ screening of adulterated milk.

An analytical method for the determination of 3-monochloropropane-1,2-diol esters (3-MCPDEs) and glycidyl esters (GEs) in infant formula milk powder was established using gas chromatography-mass spectrometry (GC-MS). Based on optimized lipid extraction and derivatization conditions, the method was evaluated for spiked recovery, linear relationship, precision, limit of detection (LOD) and limit of quantification (LOQ). Quantification was carried out using the internal standard method. The proposed method showed good linearity in the range of 5–500 ng (r > 0.999), with an LOD of 0.006 mg/kg and an LOQ of 0.015 mg/kg. The recovery rates at three spiked concentration levels of 10, 100 and 500 ng ranged from 95.6% to 100.9%, with relative standard deviations (RSDs) less than 5.03%. The results of GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) for 3-MCPDEs and GEs in several batches of infant formula samples did not significantly differ. In addition, compared with GC-MS/MS, the GC-MS method was simpler and easier to operate with lower cost and could be promoted in different levels of laboratories. Therefore, this method is suitable for determining the content of 3-MCPDEs and GEs in infant formula milk powder.

Liquid chromatography-mass spectrometry was employed to determine the change of free amino acid contents in hard yak cheeses with different salt levels during 6 months of ripening. The results showed that a total of 23 free amino acids were detected, the major ones being aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, threonine and cysteine, together representing 94% to 95% of the total free amino acid content. The content of each free amino acid showed an increasing trend between months 1 and 6. In the late stage of maturation, the contents of threonine, asparaginate, aspartic acid, valine, proline and phenylalanine increased with increasing salt addition, the contents of alanine, histidine, arginine and tyrosine decreased, and the contents of leucine, isoleucine, serine, glycine and tryptophan first increased and then decreased. The contents of of glutamic acid, leucine, isoleucine, aromatic amino acid, cysteine, glycine and serine were significantly higher in long-ripened cheese with 1.3% salt addition than in those with higher salt contents. This study provides theoretical support for the development of low-salt hard yak cheese.

The optimal parameters for ultrasonic-assisted enzymatic extraction of polysaccharides from Potentilla anserina L. using a mixture of cellulase, pectinase and papain (2:1:0.5) were determined as follows: enzyme dosage 49 U/g, ultrasonic time 100 min and pH 4.4. Under these conditions, the maximum yield of polysaccharide extract from Potentilla anserina L. (PAPE) of (6.89 ± 0.08)% was obtained. Furthermore, the effect of PAPE on yak yoghurt fermentation was investigated. The results showed that PAPE could significantly enhance the viable count and acid-producing capacity of Streptococcus thermophilus CICC 6038, Lactobacillus bulgaricus CICC 20247 and Lactobacillus fermentum HY01 in a dose-dependent manner. Yoghurt added with PAPE at 2 g/100 mL concentration had a hardness of (163.0 ± 9.9) g, a viscosity of (?182.9 ± 1.6) g·s, a consistency of (69.7 ± 1.7) g·s, a cohesiveness of (410.0 ± 6.0) g and it had a good taste, with a sensory score of 7.78; its antioxidant capacity in terms of scavenging capacity against hydroxyl, superoxide anion and 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) cation radicals increased significantly compared with the control group. This study provides new ideas for improving the quality and added value of yak yogurt.

This study explored changes in the acidity of sheep milk across different lactation periods and the key factors affecting the acidity of sheep milk. The pH, titratable acidity, fat content, protein content and composition, total sugar content, mineral content, density, lactose content, fatty acid composition and content were analyzed, and the correlation between physicochemical indexes and acidity was investigated. The results indicated that the acidity of raw sheep milk was closely related to lactation stages. The highest acidity was observed in the early lactation period, which then gradually decreased as lactation progressed. Acidity changes were primarily associated with the protein content, fat content, fatty acid composition, total sugar content, lactose content, mineral content, and density of the milk. Among these, the protein content showed the strongest correlation with acidity changes, whereas fatty acid content had a relatively low correlation with acidity variations.

The enzymatic hydrolysate of Zhongdian yak milk casein was fractionated by ultrafiltration into two fractions: molecular mass > 3 kDa and < 3 kDa. The cell proliferation activity and antioxidant activity of the two fractions were determined. The bioactive peptides derived from yak milk casein were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics and molecular docking. The results showed that the proliferation rate of RAW264.7 cells was above 80% in the presence of either the molecular mass > 3 kDa or < 3 kDa fraction. Both fractions had the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) cation radical. The cell proliferation and free radical scavenging activity of the molecular mass < 3 kDa fraction were better than those of the > 3 kDa fraction. A total of 895 peptides were identified from the molecular mass < 3 kDa fraction, of which 96 were known bioactive peptides, mainly antioxidant peptides (39.6%) and angiotensin-converting enzyme inhibitory peptides (30.2%), indicating that Zhongdian yak casein is an important source of bioactive peptides. Based on their activity and hydrophobicity, antioxidant peptides GYF and RPW were selected from the predicted 20 potential antioxidant peptides. The results of molecular docking showed that the binding energies of GYF and RPW with ABTS cation and DPPH radicals were all negative, indicating that they had binding potential and exerted antioxidant activity mainly by forming hydrogen bonds and hydrophobic interactions.