| Yak milk is one of the main sources of high quality protein for Tibetan residents and the most advantageous and potential specialty industry of Tibetan animal husbandry.Commercialization of yak milk is one of the important channels for western herders to expand their income sources in recent years,and also one of the major needs of the country to accelerate the improvement of living standards in minority areas.The commercialization of yak milk needs to go through many processing procedures,and heat treatment is a necessary part.Since milk is rich in unsaturated lipids,endogenous oxidative enzymes,metal catalysts and other oxidative stress environments,yak milk proteins will inevitably suffer from oxidative damage during heat treatment,which may cause functional changes in the protein;the protein oxidation products that accumulate during processing,storage and subsequent digestion may change the intestinal microecology and thus affect human intestinal health.Therefore,exploring the change patterns of proteins closely related to nutritional quality during commercial processing of yak milk and elucidating the effects of the corresponding derivatives on intestinal health are essential for the exploration of an accurate understanding of the nutritional and biological properties of yak milk proteins.In this study,we firstly investigated the degree of oxidation of yak milk proteins by sterilization at different temperatures;secondly,we used proteomics combined with bioinformatics to identify the protein functions and the corresponding functional pathways that changed significantly during the heat treatment;finally,we performed animal experiments on yak milk proteins at different temperatures by macrogenomic sequencing and non-targeted metabolomics.Finally,we analyzed the effects of oxidized proteins on intestinal flora structure and intestinal health,and the changes of metabolic processes,and established the relationship between thermal oxidative modification of yak milk proteins and in vivo organism functions and intestinal health.The specific research results are as follows:(1)The carbonyl group,dimeric tyrosine content and surface hydrophobicity of yak milk protein increased with increasing temperature after sterilization at different temperatures of 65℃,90℃ and 120℃;the endogenous fluorescence intensity,disulfide bond and total sulfhydryl content decreased with increasing temperature;heat treatment increased the particle size of yak milk protein and decreased the absolute value of zeta potential,among which120℃ was the most significant.The results of Fourier transform infrared spectroscopy showed that the protein secondary structure changed to different degrees,the relative content of α-helix increased and then decreased with increasing temperature,the relative content of random curl did not change significantly,the relative content of β-folding decreased and then increased,and the relative content of β-turning angle decreased gradually;the electron microscopic observation showed that the protein microstructure was fractured and folded and stacked.These results indicated that the higher the temperature the greater the degree of oxidation on yak milk proteins;causing changes in the structure of yak milk proteins;65℃ and 90℃ treatments had the least oxidation as well as structural changes on the proteins,and 120℃ had the most significant changes.(2)The function of yak milk proteins changed differently after different temperature treatments.62 protein functions were reduced and 49 protein functions were improved at 65°C,31 protein functions were reduced and 37 protein functions were improved at 90°C,and the greatest protein differences were observed at 120°C,where 327 protein functions were reduced and 308 protein functions were improved.These protein functions are focused on biochemical processes,cellular components and molecular functions.The corresponding functional changes on the pathways were: 4 pathways were upregulated and 7 pathways were downregulated at 65°C,5 pathways were upregulated and 4 pathways were downregulated at 90°C,and 11 pathways were upregulated and 33 pathways were downregulated at 120°C.These results indicate that lower temperature treatment of yak milk can better preserve the function of yak milk proteins and make the nutritional value of yak milk better preserved,which can have beneficial effects on the body after use,but too high temperature treatment of yak milk will aggravate the protein damage of yak milk and make the nutritional value of yak milk greatly reduced,and increase the probability of body diseases and affect the health level after consumption.(3)Macrogenomics analysis of mouse intestinal microorganisms revealed that a total of 66 taxonomic genera were changed,and the main difference species in the control group were Phoenicibacter and Longibaculum;in the 65℃group,the main difference species were E.faecalis,Chaetomium,Trichoderma,Beniciaceae,Eggerthella,and Sordariales and so on 33 species;90 ℃ group mainly for Lactobacillus,Bacillus spp.and Romboutsia three kinds;120 ℃group main difference genera are warty bacillus,Ackermannia,Frigoribacter,galactobacillus group,giant cocci,anode bacillus and so on 28 species.Functionally control group and 65℃ group will up-regulate the metabolism,translocation,cell replication and repair function of other amino acids,down-regulate the metabolism function of amino acid metabolism,cofactor and vitamin;90℃ group significantly increase the metabolism,translocation,cell replication and repair function of other amino acids,significantly down-regulate the carbohydrate metabolism,amino acid metabolism,biosynthesis of secondary metabolites,peptidoglycan biosynthesis and metabolism function The 120℃group significantly up-regulated the functions of translocation and energy metabolism and down-regulated the functions of amino acid metabolism.(4)Analysis using metabolomics techniques revealed 25 metabolic pathways in mice,including steroid hormone biosynthesis,vitamin B6 metabolism,phenylalanine metabolism,pantothenate and coenzyme A biosynthesis,and primary bile acid biosynthesis,among which the most significant metabolic pathways affected by differential metabolites were:phenylalanine metabolism,vitamin B6 metabolism,steroid hormone biosynthesis,and pantothenate and coenzyme A biosynthesis. |
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