| The preparation and structure-activity relationship of milk-derived antioxidant and ACE-inhibiting peptides is a research hotspots at home and abroad.Lactic acid bacteria are often used as fermentation strains,but these studies mainly focuse on the production of single peptides,and the milk source is mainly cow milk.Cow,goat and sheep milk are nutritious,and their composition is different,which will affect the growth of lactic acid bacteria and the release of peptides.However,there are few studies on the use of lactic acid bacteria to ferment goat milk and sheep milk to produce a variety of peptides.In this study,cow,goat and sheep milk were used as raw materials,and the ACE inhibition rate and antioxidant activity were used as indicators.First,the target strains MF55 and MF48 were screened from 74 strains of lactic acid bacteria,and the strains were identified.After two target strains cultured in cow,goat and sheep milk,ultrafiltration and LC-MS/MS were used to separate and identify ACE inhibitory peptides and antioxidant peptides.Then,the process conditions for fermenting cow,goat and sheep milk with strain MF55 were optimized.Research on cold storage of fermented milk was carried out.Finally,the metabolomics analysis of the two target strains was carried out.The following conclusions are drawn:1.Among the 74 strains of lactic acid bacteria that fermented cow,goat and sheep milk,67,67 and 65 strains had ACE inhibitory activity,respectively,and their ACE inhibition rates were 2.41%-70.47%,3.63%-70.73%and 1.40%70.05%,respectively.All strains of fermented cow,goat and sheep milk have antioxidant activity,DPPH free radical scavenging rate is 10.77%-64.58%.The primary screening screened out strains MF48,L221,B43,S1 and MF55.MF55 with strong ACE inhibitory activity and antioxidant activity and MF48 with only strong antioxidant activity of fermented cow,goat and sheep milk were obtained by rescreening.The 16S rDNA was identified as Lactobacillus bulgaricus and Lactobacillus paracasei,respectively.2.Ultrafiltration and LC-MS/MS were used to isolate and identify the peptides in cow,goat and sheep milk fermented by L.bulgaricus MF55 and L.paracasei MF48.It was found that TKLTEEEK peptides were found in all the fermented milk.Peptides EMPFPK,LNFLK,LTEEEK and other one peptide were found in all the milk fermented by MF55.Peptides ITVDDK、ITVDDKHYQK、LDQWLCEK and other two peptides were found in all the milk fermented by MF48,EGNPAHQK、FFDDK、GPFPILV and other six peptides were found in all the goat and sheep milk fermented by two strains,indicating that the proteases produced by the two strains have the same points,but the amino acid sequences of cow,goat and sheep milk proteins are different,resulting in different peptides,which were derived from casein and whey protein.3.The fermentation process of cow,goat and sheep milk by Lactobacillus bulgaricus MF55 was optimized by single factor experiment and customized experimental design.The optimal technological parameters of milk fermentation were 1.5%inoculum,37℃ for 10h,the ACE inhibition rate,DPPH free radical scavenging rate,ferrous ion chelation rate and sensory evaluation were 70.11%,69.20%,60.02%and 89.50,respectively.The optimal process parameters of goat milk fermentation were 2.5%inoculum,38℃ for 12h.The ACE inhibition rate,DPPH free radical scavenging rate,ferrous ion chelation rate and sensory evaluation were 70.12%,65.20%,78.02%and 64.39,respectively.The optimal process parameters of sheep milk fermentation were 1.2%inoculum,37℃ for 8.5h.The ACE inhibition rate,DPPH free radical scavenging rate,ferrous ion chelation rate and sensory evaluation were 69.13%,74.33%,72.11%and 78.86,respectively.4.During the cold storage process,the pH of the cow,goat and sheep milk fermented by L.bulgaricus MF55 gradually decreased,the acidity gradually increased,the water holding capacity increased gradually,the hardness,consistency and cohesion were higher than the fermentation end point,and the ACE inhibition rate of the milk decreased significantly,but the scavenging rate of DPPH free radicals remained basically unchanged,the ACE inhibition rate of goat and sheep milk decreased slightly,but the scavenging rate of DPPH free radicals decreased significantly.The number of viable bacteria first increased and then decreased,and was still higher than 108 CFU/ml after 4 weeks of cold storage,indicating that the strain MF55 was relatively stable during cold storage.5.The metabolomic analysis of L.bulgaricus MF55 and L.paracasei MF48 cultured in cow,goat and sheep milk was carried out by GC-MS.A total of 127 metabolites were identified from 6 groups of samples,and the metabolite groups were significantly different.The differential metabolites of strain MF55 after fermenting cow and goat milk were obviously more than those after fermenting goat and sheep milk,but the number of differential metabolites was similar between strains MF48 after fermentation of three kinds of milk.Different metabolites among the samples have multiple metabolic pathways and have similarities,such as glycerolipid metabolism,arginine biosynthesis and Dglutamate metabolism.The study found that different types of milk or different strains had significant effects on the types and contents of metabolites.In this study,L.bulgaricus MF55 and L.paracasei MF48 with strong peptideproducing ability of fermented cow,goat and sheep milk were obtained by screening.The amino acid sequences of ACE inhibitory peptides and antioxidant peptides were identified,and functional lactic acid bacteria fermented cow,goat and sheep milk was developed by using them,the metabolic differences between the two strains were compared,which provided theoretical and technical support for the development of bioactive peptide fermented milk. |
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