Stability, Digestion, and Cellular Transport of Soy Isoflavones Nanoparticles Stabilized by Polymerized Goat Milk Whey Protein.

Soy isoflavones (SIF) are bioactive compounds with low bioavailability due to their poor water solubility. In this study, we utilized polymerized goat milk whey protein (PGWP) as a carrier to encapsulate SIF with encapsulation efficiency of 89%, particle size of 135.53 nm, and zeta potential of -35.16 mV. The PGWP-SIF nanoparticles were evaluated for their stability and in vitro digestion properties, and their ability to transport SIF was assessed using a Caco-2 cell monolayer model. The nanoparticles were resistant to aggregation when subjected to pH changes (pH 2.0 to 8.0), sodium chloride addition (0-200 mM), temperature fluctuations (4 °C, 25 °C, and 37 °C), and long-term storage (4 °C, 25 °C, and 37 °C for 30 days), which was mainly attributed to the repulsion generated by steric hindrance effects. During gastric digestion, only 5.93% of encapsulated SIF was released, highlighting the nanoparticles' resistance to enzymatic digestion in the stomach. However, a significant increase in SIF release to 56.61% was observed during intestinal digestion, indicating the efficient transport of SIF into the small intestine for absorption. Cytotoxicity assessments via the MTT assay showed no adverse effects on Caco-2 cell lines after encapsulation. The PGWP-stabilized SIF nanoparticles improved the apparent permeability coefficient (Papp) of Caco-2 cells for SIF by 11.8-fold. The results indicated that using PGWP to encapsulate SIF was an effective approach for delivering SIF, while enhancing its bioavailability and transcellular transport.

Comparison of lipidome profiles in human milk from Chinese Han and Korean ethnic groups based on UPLC-QTRAP-MS/MS.

The composition of milk lipids varies across different ethnic sources. The lipidome profiles of Chinese Han human milk (HHM) and Chinese Korean human milk (KHM) were investigated in this study. A total of 741 lipids were identified in HHM and KHM. Twenty-eight differentially expressed lipids (DELs) were screened between the 2 milk groups; among these, 6 triacylglycerols (TGs), 13 diacylglycerols (DGs), 7 free fatty acids (FFAs), and 1 monoglyceride (MG) were upregulated in KHM. Carnitine (CAR) was upregulated in HHM. Most DELs showed a single peak distribution in both groups. The correlations, related pathways and diseases of these DELs were further analyzed. The results demonstrated that DG, MG, and FFAs showed highly positive correlations with each other (r >0.8). The most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database (HMDB) pathways were inositol phosphate metabolism, and α-linolenic acid and linolenic acid metabolism, respectively. Major depressive disorder-related FFA (20:5) and FFA (22:6) were more abundant in KHM, while HHM showed more obesity-related CAR. These data potentially provide lipidome information regarding human milk from different ethnicities in China.

Effects of Panax notoginseng Saponins Encapsulated by Polymerized Whey Protein on the Rheological, Textural and Bitterness Characteristics of Yogurt.

Panax notoginseng saponins (PNSs) have been used as a nutritional supplement for many years, but their bitter taste limits their application in food formulations. The effects of PNS (groups B, C, and D contained 0.8, 1.0 and 1.2 mg/mL of free PNS, respectively) or Panax notoginseng saponin-polymerized whey protein (PNS-PWP) nanoparticles (groups E, F, and G contained 26.68, 33.35 and 40.03 mg/mL of PNS-PWP nanoparticles, respectively) on the rheological, textural properties and bitterness of yogurt were investigated. Group G yogurt showed a shorter gelation time (23.53 min), the highest elastic modulus (7135 Pa), higher hardness (506 g), higher apparent viscosity, and the lowest syneresis (6.93%) than other groups, which indicated that the yogurt formed a stronger gel structure. The results of the electronic tongue indicated that the bitterness values of group E (-6.12), F (-6.56), and G (-6.27) yogurts were lower than those of group B (-5.12), C (-4.31), and D (-3.79), respectively, which might be attributed to PNS being encapsulated by PWP. The results indicated that PWP-encapsulated PNS could cover the bitterness of PNS and improve the quality of yogurt containing PNS.

Physicochemical and Functional Properties of Thermal-Induced Polymerized Goat Milk Whey Protein.

Goat milk whey protein products are a hard-to-source commodity. Whey protein concentrate was directly prepared from fresh goat milk. The effects of the heating temperature (69-78 °C), time (15-30 min), and pH (7.5-7.9) on the physicochemical and functional properties of the goat milk whey protein were investigated. The results showed that the particle size of the samples significantly increased (p < 0.05) after heat treatment. The zeta potential of polymerized goat milk whey protein (PGWP) was lower than that of native goat milk whey protein. The content of the free sulfhydryl groups of PGWP decreased with increasing heating temperature and time, while an increase in surface hydrophobicity and apparent viscosity of PGWP were observed after heat treatment. Fourier Transform Infrared Spectroscopy analysis indicated that heat treatment and pH had considerable impacts on the secondary structure of goat milk whey protein. Transmission electron microscope images revealed that heat induced the formation of a large and uniform protein network. Additionally, the changes in the physicochemical and structural properties contributed to the improvement of the emulsifying and foaming properties of goat milk whey protein after heat treatment. The results may provide a theoretical basis for the applications of polymerized goat milk whey protein in related products.

Insights from 4D Label-Free Proteomic Analysis into Variation of Milk Fat Globule Membrane Proteins of Human Milk Associated with Infant's Gender.

Milk fat globule membrane (MFGM) protein profiles of breast milk collected from women in northeast China with male or female babies were investigated using a four-dimensional (4D) label-free proteomic technique. Altogether, 2538 proteins were detected and quantified and 249 were differentially expressed, with 198 decreased proteins compared to the samples of mothers with female babies. Different proteins associated with infant's gender were principally located in nuclear. The differentially expressed proteins were mainly involved in gene ontology (GO) functions of the cellular process, binding, and cell and found to be distributed in lipid-related biological processes and molecular functions to a large extent. The pathway of neurodegeneration-multiple disease ranked top for the altered proteins. The screened proteins were observed to contain some proteins related to typical functions of immunity, lipid metabolism, digestion, and growth and development. 114 proteins formed a relatively compact network (269 interactions) and dolichyl-diphospho-oligosaccharide-protein glycosyltransferase subunit 2 interacted the most with other proteins as the hub protein. MFGM proteins of breast milk were affected by the sex of offspring, and these findings may provide useful information for reasonable adjustments of infant formula powder specifically for boys or girls in the market.

Deciphering the difference of casein fraction in human milk associated with infant gender using quantitative proteomics.

Human milk is an ideal natural food for infants, and the infant's gender may have impact on protein composition of breast milk. In this study, we used 4D label-free quantitative proteomics techniques to identify and quantitatively analyze casein fraction in breast milk secreted for male and female infants. The results showed that a total of 2064 proteins were identified in human milk, and 95 of them were differentially abundant proteins. Compared to breast milk secreted by mothers of female infants, 21 proteins were up-regulated, and 59 proteins were down-regulated in breast milk secreted by mothers of male infants. The most abundant domain among the differentially abundant proteins was the immunoglobulin V-set domain, which may be involved in immune regulation. Gene Ontology functional analysis revealed that, the main biological processes, molecular functions, and cellular components corresponded to cellular process, binding, and cell part, respectively. The Kyoto Encyclopedia of Genes and Genomes pathways were mainly associated with human diseases and metabolism, with biosynthesis of cofactors being the most involved pathway. The results contribute to our understanding of the composition of casein in breast milk, and may provide information about the nutritional differences in breast milk from mothers of newborns of different genders.

Proteomic characterization and comparison of milk fat globule membrane proteins of Saanen goat milk from 3 habitats in China using SWATH-MS technique.

Saanen goats are among the major dairy goats in China. In present study, variation of milk fat globule membrane proteins profile of Saanen goat milk caused by geographic location was investigated using sequential window acquisition of all theoretical fragment ions data-independent acquisition mass spectrometry based proteomic approach. A total of 1,001 proteins were quantified in goat milk collected from 3 habitats of China [Guangdong (GD); Inner Mongolia (IM); Shannxi (SX)]. Most of the proteins were found to act cellular process of biological process, cell of cellular component, binding of molecular function after Gene Ontology annotation and metabolic of pathway indicated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Differentially expressed proteins (DEP) for GD versus IM, GD versus SX, IM versus SX were identified to be 81, 91, and 44, respectively. Gene Ontology enrichment analysis showed that the greatest DEP for 3 groups (GD vs. IM, GD vs. SX, IM vs. SX) were cellular process, cellular process and organonitrogen compound biosynthetic process/immune system process for biological process. For cellular component, the largest number of DEP for 3 comparison groups were organelle, organelle and organelle/intracellular. For molecular function, DEP of the 3 comparison groups were expressed most in structural molecule activity, binding and anion binding, respectively. Pathways with the majority of DEP were ribosome, systemic lupus erythematosus and primary immunodeficiency/systemic lupus erythematosus/amoebiasis/PI3K-Akt signaling pathway for GD versus IM, GD versus SX and IM versus SX, severally. Protein-protein interaction network analysis showed that DEP interacted most were 40S ribosomal protein S5, fibronectin and Cytochrome b-c1 complex subunit 2, mitochondrial for GD versus IM, GD versus SX and IM versus SX, separately. Data may give useful information for goat milk selection and milk authenticity in China.

Comparative proteomics analyses of whey proteins from breastmilk collected from two ethnic groups in northeast China.

The current study aims to investigate differences in whey protein of breastmilk of volunteered mother collected from two ethnic groups (Korean and Han) in China using data-independent acquisition (DIA) based proteomics technique. The total detected 624 proteins were principally allocated to cellular process of biological process (BP), cell and cell part of cell component (CC) and binding of molecular function (MF) according to Gene Ontology (GO) annotation; and carbohydrate metabolism of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Among the 54 differently expressed proteins, 8 were related with immunity. Enrichment data showed that intracellular of GO functions and viral myocarditis of KEGG pathways were most significantly enriched (p < 0.05). Protein-protein interaction (PPI) network suggested that 40S ribosomal protein S27a and 60S ribosomal protein L10a which interacted most with other proteins ranked the top two hub proteins by MCC (Maximal Clique Centrality) method. This study may have guiding role for development of infant formula powder for specific infants of Han or Korean groups according to responding breastmilk composition.

Investigation of the differences in the effect of (-)-epigallocatechin gallate and proanthocyanidins on the functionality and allergenicity of soybean protein isolate.

In this study, the differences in effects of (-)-epigallocatechin gallate (EGCG) and proanthocyanidins (PC) on the functionality and allergenicity of soybean protein isolate (SPI) were studied. SDS-PAGE demonstrated that SPI-PC conjugates exhibited more high-molecular-weight polymers (>180 kDa) than SPI-EGCG conjugates. Structural analysis showed that SPI-PC conjugates exhibited more disordered structures and protein-unfolding, improving the accessibility of PC to modify SPI, compared to SPI-EGCG conjugates. LC/MS-MS demonstrated that PC caused more modification of SPI and major soybean allergens than EGCG, resulting in a lower abundance of epitopes. The successful attachment of EGCG and PC to SPI significantly increased antioxidant capacity in conjugates. Furthermore, SPI-PC conjugates exhibited greater emulsifying activity and lower immunoglobulin E (IgE) binding capacity than SPI-EGCG conjugates, which was attributed to more disordered structure and protein-unfolding in SPI-PC conjugates. It is implied that proanthocyanidins may be promising compounds to interact with soybean proteins to produce functional and hypoallergenic foods.

Comparative proteomics of human milk casein fraction collected from women of Korean and Han ethnic groups in China.

Introduction: Human breast milk provides neonates with indispensable nutrition and function. Milk protein is one of the main constituents of breast milk. Human milk profiles can be influenced by many factors. Methods: The present study aimed to investigate the difference in casein isolated from mature milk of healthy mothers of Korean and Han ethnic groups in China using data-independent acquisition (DIA) proteomics. Results: A total of 535 proteins were identified and quantified in casein fraction samples from both groups. A total of 528 proteins were annotated to 52 Gene Ontology (GO) terms, the majority (94.13%) of which were distributed in the cell and cell parts of the cellular component. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 106 proteins were involved in 23 pathways, the greatest (36.79%) in carbohydrate metabolism. There were 39 differentially expressed proteins (DEPs)-10 upregulated and 29 downregulated-between Korean and Han milk. The GO function of blood microparticles and KEGG pathway of Staphylococcus aureus infection for DEPs were the most significantly enriched (p < 0.05). Protein-protein interaction analysis revealed a network with 23 DEPs in 47 interactions, and the fibrinogen alpha chain ranked first as the hub protein. Discussion: These data may provide useful technical guidance for the development of specific infant foods for certain populations.

Digestion behavior, in vitro and in vivo bioavailability of cannabidiol in emulsions stabilized by whey protein-maltodextrin conjugate: Impact of carrier oil.

An emulsion delivery system may be affected significantly by oil phase composition in terms of digestion behavior and bioavailability of the delivered substance. In this study, emulsions loaded with cannabidiol (CBD) were prepared with medium chain triglyceride (MCT), long chain triglyceride (LCT) or MCT/LCT(1:1) as carrier oil and whey protein-maltodextrin conjugate as emulsifier, and the digestion behavior of emulsion and bioavailability of CBD were assessed in vitro and in vivo. The particle size of emulsions throughout the in vitro digestion process was in the order of MCT < MCT/LCT < LCT, and three emulsions showed consistent particle size changes: stable in oral phase, sharply increased in gastric phase, and decreased in small intestine. After intestinal digestion, about 90% of free fatty acids (FFA) was released in MCT emulsion, followed by MCT/LCT (76%) and then LCT (45%). CBD was degraded during gastrointestinal digestion and the transformation stability of CBD in oil phase was in the order of LCT > MCT/LCT > MCT. Although CBD had higher bioaccessibility in MCT and MCT/LCT emulsions, the bioavailability of CBD in LCT was the highest (43%), followed by MCT/LCT (39%), MCT (33%). In vivo pharmacokinetic study showed that MCT/LCT and LCT were more favorable for CBD transport and absorption. The results may provide useful information for the construction of delivery systems, protecting CBD molecules, and improving their bioavailability.

A review on polyphenols and their potential application to reduce food allergenicity.

This review summarized recent studies about the effects of polyphenols on the allergenicity of allergenic proteins, involving epigallocatechin gallate (EGCG), caffeic acid, chlorogenic acid, proanthocyanidins, quercetin, ferulic acid and rosmarinic acid, etc. Besides, the mechanism of polyphenols for reducing allergenicity was discussed and concluded. It was found that polyphenols could noncovalently (mainly hydrophobic interactions and hydrogen bonding) and covalently (mainly alkaline, free-radical grafting, and enzymatic method) react with allergens to induce the structural changes, resulting in the masking or/and destruction of epitopes and the reduction of allergenicity. Oral administration in murine models showed that the allergic reaction might be suppressed by regulating immune cell function, changing the levels of cytokines, suppressing of MAPK, NF-κb and allergens-presentation pathway and improving intestine function, etc. The outcome of reduced allergenicity and suppressed allergic reaction was affected by many factors such as polyphenol types, polyphenol concentration, allergen types, pH, oral timing and dosage. Moreover, the physicochemical and functional properties of allergenic proteins were improved after treatment with polyphenols. Therefore, polyphenols have the potential to produce hypoallergenic food. Further studies should focus on active concentrations and bioavailability of polyphenols, confirming optimal intake and hypoallergenic of polyphenols based on clinical trials.

Effect of proanthocyanidins on protein composition, conformational structure, IgE binding capacities and functional properties in soybean protein.

This study was performed to determine the crosslinking formed by proanthocyanidins (PC) with respect to IgE binding capacities, functionality, structure and composition of soybean protein (SPI) following the alkali treatment at 60-100 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the formation of >180 kDa polymers, resulting from the formation of SPI-PC conjugates and protein cross-links. Structural analyses demonstrated that SPI-PC conjugates exhibited structural changes to unfold proteins and increase molecular flexibility. Liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) showed a decrease in unique protein and peptide numbers as well as major allergen and dominant epitopes abundance. When SPI was treated with PC under the alkali treatment at 80 °C, it exhibited a maximum reduction (68.8 %) in the immunoglobulin E (IgE) binding capacity and a maximum increase in DPPH radical scavenging activities (6.11-fold), ABTS + radical scavenging activities (4.80-fold), foaming stability (6.1 %) and emulsifying activity (27.3 %), compared to the control SPI. Overall, this study demonstrates that alkali treatment at 60-100 °C to form SPI-PC conjugates has potential applications for producing hypoallergenic soybean products with the desired functionality, most especially from alkali treatment at 80 °C. Moreover, the addition of PC pronouncedly alleviates the undesirable functional properties in heated SPI.

Heat-induced changes in epitopes and IgE binding capacity of soybean protein isolate.

The effects of heating temperature on epitopes, IgE-binding capacity, and conformation of soybean protein isolate (SPI) were investigated in this study. Indirect ELISA demonstrated that the IgE binding capacity of SPI was increased by 13.1 %-31.6 % after being heated at 60-100 °C for 20 min. SDS-PAGE demonstrated no changes in protein profiles, and native PAGE revealed the formation of aggregates. Structural analyses demonstrated the protein unfolding, appearing temperature-dependent, thus exposing conformational epitopes. Peptide mapping analysis revealed the changes in peptide profiles of major allergens (Gly m 4, Gly m 5, Gly m 6, P28, and Kunitz trypsin inhibitor). LC/MS-MS demonstrated that heating caused the masking or exposure of linear epitopes in Gly m 4 - Gly m 6 and P28. Therefore, heating caused structural changes to expose epitopes to increase IgE binding capacity in SPI. Patients with soybean allergy should avoid the heated SPI until the results of clinical trials are confirmed.

Protein modification, IgE binding capacity, and functional properties of soybean protein upon conjugation with polyphenols.

The modification, structure, functionality and IgE binding capacity of soybean protein (SPI) upon covalent conjugation with gallic acid (GA), caffeic acid (CA), and tannic acid (TA) under alkali treatment were assessed. SDS-PAGE showed the formation of SPI-polyphenol conjugates and the cross-linking of SPI. Protein unfolding in the conjugates was observed, characterized by a reduction in α-helix and an increase in UV ultraviolet absorption, surface hydrophobicity and free sulfhydryl groups. LC/MS-MS demonstrated that the modification of protein and major allergens varied with the types of polyphenols. Western-blot and ELISA demonstrated that SPI-polyphenol conjugates exhibited a significant reduced IgE binding capacity due to the masking or destruction of epitopes among Gly m 4, Gly m 5, Gly m 6 and P28, resulting from structural changes. Additionally, antioxidant capacity and emulsifying properties were increased in SPI-polyphenol conjugates. Therefore, polyphenol treatment may be a promising method to prepare hypoallergenic soybean products with desired functionality.

Promotion effect of the blend containing 2'-FL, OPN and DHA on oligodendrocyte progenitor cells myelination in vitro.

During early neurodevelopment of infant, myelination plays an essential role in brain connectivity and emergence of behavioral and cognitive function. Early life nutrition is an important factor to shape myelination and consequently cognitive appearance. To analyze the effects of additive nutrients, including 2'-fucosyllactose (2'-FL), osteopontin (OPN), docosahexaenoic acid (DHA), on neurocognitive function and brain structure, the current study evaluated the effects of different composition of breast milk nutrients on oligodendrocyte progenitor cells (OPCs) myelination with a neural primary cell model in vitro. The study showed that the three nutrients promoted the proliferation, maturation and differentiation of OPCs into mature oligodendrocytes (OLs) in each phage of the cell growth, and the effect of the nutrients blend is obviously stronger than that of the nutrient treatment alone, showing a synergistic effect in promotion of OPCs. The results of this experiment clarified the effects of 2'-FL OPN and DHA to promote myelination development of neural cells, and laid an experimental basis for further optimization of infant formula.

Effects of Radiation on Cross-Linking Reaction, Microstructure, and Microbiological Properties of Whey Protein-Based Tissue Adhesive Development.

Whey proteins are mainly a group of small globular proteins. Their structures can be modified by physical, chemical, and other means to improve their functionality. The objectives of this study are to investigate the effect of radiation on protein−protein interaction, microstructure, and microbiological properties of whey protein−water solutions for a novel biomaterial tissue adhesive. Whey protein isolate solutions (10%, 27%, 30%, 33%, and 36% protein) were treated by different intensities (10−35 kGy) of gamma radiation. The protein solutions were analyzed for viscosity, turbidity, soluble nitrogen, total plate count, and yeast and mold counts. The interactions between whey proteins were also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and scanning electron microscopy. The viscosity of protein solution (27%, w/w) was increased by the treatment of gamma radiation and by the storage at 23 °C. The 35 kGy intensity irradiated soluble nitrogen (10%, w/w) was reduced to about half of the sample treated by 0 kGy gamma radiation. The effects of gamma radiation and storage time can significantly increase the viscosity of whey protein solutions (p < 0.05). Radiation treatment had significant impact on soluble nitrogen of whey protein solutions (p < 0.05). SDS-PAGE results show that the extent of oligomerization of whey protein isolate solutions are increased by the enhancement in gamma radiation intensity. Photographs of SEM also indicate that protein−protein interactions are induced by gamma radiation in the model system. Consistent with above results, the bonding strength increases by the addition of extent of gamma radiation and the concentration of glutaraldehyde. Our results revealed that the combination of gamma-irradiated whey protein isolate solutions and glutaraldehyde can be used as a novel biomaterial tissue adhesive.

Investigation of differences in allergenicity of protein from different soybean cultivars through LC/MS-MS.

Soybean allergy is a health-threatening issue and identifying raw soybeans with low allergenicity is important for producing hypoallergenic soybean products. Soybean allergy is mainly triggered by soybean proteins. In this study, the protein profiles, allergen compositions, and epitopes in protein from different soybean cultivars (R1, R2 and R3) were evaluated by SDS-PAGE and LC/MS-MS, and their allergenicity was assessed by indirect ELISA and Western blot analysis using the serum IgE of patients allergic to soybeans. The lowest allergenicity was observed in R3, probably resulting from the low concentration of Gly m 4-Gly m 6. The allergenicity of soybeans is affected by multiple allergens rather than a single allergen. Venn diagram, PCA, heatmap, and peptide map analyses have shown the differences in protein and peptide profiles among soybean proteins from different soybean cultivars. Epitope analysis further demonstrated that low contents of dominant epitopes in Gly m 4 and Gly m 5 contributed to low allergenicity in R3, although R3 contained high contents of no-dominant epitopes.

Peptide profiles and antioxidant capacity of extensive hydrolysates of milk protein concentrate.

Milk protein concentrate was hydrolyzed using one-step enzymatic hydrolysis. Both the peptide profiles and antioxidant activities of the resulting extensive hydrolysates of milk protein concentrate (EMPH) were analyzed using a peptidomics approach based on liquid chromatography-tandem mass spectrometry. The results demonstrated that the degrees of hydrolysis of the 4 EMPH by Alcalase-Protamex, Alcalase-Protease A 2SD, Alcalase-Flavorzyme, and Alcalase-ProteAXH were 12.02%, 16.85%, 15.87%, and 15.77%, respectively. Using size exclusion chromatography, 99.85% of the peptides in the Alcalase-Protease A 2SD hydrolysate were shown to have a molecular weight of <3 kDa. A total of 33 common peptides were identified in the EMPH by liquid chromatography-tandem mass spectrometry, 16 of which were identified as bioactive peptides using bioinformatics. The peptide profiles and the coverage of master proteins of the 4 EMPH were different. The EMPH also exhibited strong free radical scavenging capacity, as indicated by the results of the 1,1-diphenyl-2-picrylhydrazyl radical, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroxyl radical, and reducing power assays. The results of this study provided useful information on the peptide profiles and antioxidant activity of EMPH.

Differences in Protein Profiles of Kefir Grains from Different Origins When Subcultured in Goat Milk.

Proteins not only serve as a nitrogen source for microorganisms but are the main skeleton of kefir grains. After subculturing in goat milk for 4 months, proteins and peptides in three kefir grains from China, Germany, and the United States were analyzed. Except for the S-layer protein from special Lactobacillus sp., αs1-casein, αs2-casein, and ß-casein from goat milk were found in kefir grains. These proteins could form aggregates through a covalent interaction with polysaccharides to maintain the morphological stability of the grains. Furthermore, they were highly related to the microbiota in kefir grains. Additionally, a number of hydrophilic/hydrophobic peptides that were hydrolyzed by extracellular proteases were found from kefir grains. A correlation may exist between peptides and Lactobacillus sp. in kefir grains. Bioactive peptides, including DKIHPF, LGPVRGPFP, and QEPVLGPVRGPFP, were found from these kefir grains. The results indicated that goat milk as a substrate affects the protein and peptide composition of kefir grains.