Sub-culturing Of Kefir Grains In Goat Milk And Its Impact On The Structure Of Kefir Grains And Fermentation Properties Of Goat Milk Kefir

Kefir grains,as a unique symbiotic association of different microbiota,can be used for the production of traditional kefir.Different functional properties of goat milk have been studied.However,the microbiota in kefir grains is strongly influenced by the geographical origin and sub-culturing environment.It is difficult to maintain the stability of goat milk kefir's quality.High-throughput sequencing technique was applied for the changes in bacterial and fungal diversity of three different kefir grains which were originated from China(CN),Germany(DE)and United States of America(USA)when sub-cultured in goat milk for 2 to 4 months.And the changes in kefiran(exopolysaccharide in kefir grains),protein and peptides of different kefir grains were also investigated.Based on the symbiotic association and the interactions among microbiota in kefir grains,a recombination system(combined with kefir grains CN and DE at different ratios)has been developed.Effects of bacteria and fungi on the physicochemical properties,volatiles and peptides(digested by microbiota in kefir grains)of goat milk kefir were studied in the system.Results were used for targeted screening and constructing kefir starter culture for goat milk fermentation.Finally,effects of polymerized whey protein and pectin(PP)system on physical properties and volatile compounds of goat milk kefir and kefir mild were investigated.Results were used for selecting a suitable thickening system for goat milk kefir manufacture.The main conclusions were drawn as follows:(1)After sub-culturing in goat milk for 2 to 4 months,amplicon sequencing was applied for the changes in bacteria and fungi of three kefir grains(CN,DE and USA).Results of?diversity shown an autogenic succession phenomenon among microbiota in kefir grains.Taxonomic analysis displayed that Lactobacillus and Acetobacter was the pre-and sub-dominant genus in different kefir grains,respectively.At species level,for the beginning of sub-culturing(?2 months),Lactobacillus helveticus and Lactobacillus kefiranofaciens were the dominant species in kefir grains CN and DE;while for the sub-culturing time increased(3?4 months),Lactobacillus kefiranofaciens has been the predominant species in these two kefir grains samples.For the analysis of fungi,at genus level,for the beginning of sub-culturing(?2 months),Kazachstania was the predominant genus in kefir grains CN and DE;while for the sub-culturing time increased(3?4 months),contents of Kluyveromyces and Saccharomyces were increased.It is found that the microbiota are stable for the sample from USA during the whole sub-culturing process.These results may be contributed to the quorum sensing signal which was metabolized by microbiota and interactions among bacteria(especially Lactobacillus sp.)and fungi(mainly yeasts).At the beginning of sub-culturing,due to the changes of fermentation substrate,contents of bacteria and fungi in kefir grains were unstable.The Acetobacter start to proliferate.Then,the metabolite of Acetobacter and some yeasts could stimulate Lactobacillus kefiranofaciens to proliferate and regained the dominant position.Finally,a restabilized symbiotic association in kefir grains was rebuild.The results of?diversity analysis supported this finding.(2)After sub-culturing in goat milk for 4 months,proteomic platform was applied for the analysis of protein and peptides in three kefir grains.Except S-layer protein from special Lactobacillus sp.,varies of?_s1-casein,?_s2-casein and?-casein from goat milk were found from kefir grains.These proteins could form aggregated through covalent interaction with kefiran to maintain the morphological stability of grains.Additionally,the number of hydrophilic/hydrophobic peptides which were hydrolyzed by extracellular proteases were found from three different kefir grains.There were a large number of bioactive peptides,including DKIHPF,LGPVRGPFP,QEPVLGPVRGPFP.(3)After sub-culturing in goat milk for 4 months,spectroscopy,chromatography and microscopic imaging techniques were applied for the analysis of structure of kefiran of these three different kefir grains.Fourier transform infrared spectroscopy(FTIR)spectrum shown that kefiran extracted from kefir grains belonging to?-D-glucopyranose,and the sugar configuration type is?-.The particle size(213.72±17.65 nm and 436.90±32.85 nm),molecular weight(1.17×10⁷ Da,1.07×10⁶ Da and 1.63×10⁷ Da,1.77×10⁶ Da)and heat stability of kefiran extracted from kefir grains CN and DE were higher than that of from USA.The surface structure of kefiran in kefir grains CN and DE was smoother and curled than that of in USA.Such a series of differences are related to the microbiota of kefir grains from different origin.Their symbiosis and metabolism differences lead to changes in the structure and properties of kefiran,which constitute the grain skeleton,and indirectly affect the interaction ability of these polysaccharides and proteins.(4)The results showed that the bacterial and fungal microflora in kefir grains from different origins are complexed and their impact on protein digestion and aromatic profile of goat milk kefir.Compared with?-casein,?_s1-casein,?_s2-casein,?-casein and?-lactalbumin,?-lactoglobulin was hardly to be hydrolyzed by extracellular protease from microbiota in kefir grains.Cluster analysis indicated that the different content of Lactobacillus genera in kefir grains was highly associated with the protein digestion ability in goat milk kefir.Contents of volatile compounds(such as alcohols,esters and ketones)in goat milk kefir were also correlated to the bacteria and fungi in kefir grains(especially for Saccharomyces cerevisiae,Lactobacillus helveticus and Lactobacillus kefiranofaciens).Hierarchical clustering analysis indicated that a high similarity of volatiles in goat milk kefir fermented by kefir grains CN and DE than that of by USA.(5)The physical and chemical properties of goat milk kefir samples which were fermented by reconstituted kefir grains(kefir grains CN and DE in different proportion:20DE80CN,40DE60CN,60DE40CN and 80DE20CN)and the correlation between the microbiota and volatile compounds were investigated.All the physicochemical indexes of goat milk kefir complied with relevant international standards.Compared with other kefir samples,goat milk kefir 80DE20CN showed significant improvement in particle size distribution,apparent viscosity and other physical properties.Results from high-throughput sequencing platform showed that abundance of bacteria and fungi were found in goat milk kefir.Bacteria such as Macrococcus caseolyticus,Enterococcus faecalis,Enterococcus faecium,Enterococcus hirae,Enterococcus xinjiangensis,Enterococcaceae bacterium,Lactobacillus helveticus,Lactobacillus kefiranofaciens,Lactobacillus kefiri and Lactobacillus sunkii,and fungi such as Kazachstania unispora and Kluyveromyces marxianus were dominant in goat milk kefir,respectively.Cluster results indicated that a strong symbiotic homeostasis system was found in kefir grains.Even if the microbial ratio of kefir grains was artificially changed,the dominant symbiotic system was able to maintain its initial composition.Results of volatiles confirmed that goat milk kefir 80DE20CN had the highest“Fruity”flavor(account for 33.44%).Partial least squares regression analysis(PLSR)model showed that the explanatory ability of bacterial and fungal species in goat milk kefir to volatiles was 0.744 and0.512,respectively.Positive correlations among Lactobacillus helveticus and Ethyl acetate,Amyl acetate and Ethanol,Kazachstania unispora and Octyl formate,1-Heptanol and Hexanoic acid were found from PLSR model.(6)The correlations between the microbiota and peptides in goat milk kefir fermented by reconstituted kefir grains were investigated.The peptides released by in-vitro digestion of goat milk kefir were explored.Except?-lactoglobulin,most of casein and whey proteins in goat milk could be hydrolyzed by the physiological activities of microbiota in kefir,and a total of 9 bioactive peptides were found from the kefir samples.The explanatory ability of Lactobacillus sp.to self-hydrolytic peptides in goat milk kefir was 0.212,and the Lactobacillus helveticus had a highly positive correlation with the bioactive peptide DKIHPF(>5.19%).After in-vitro digestion,highest contents of bioactive peptides(account for 4.38%)were found in goat milk kefir 80DE20CN,and the prediction result of bioactive peptides contents(account for 36.39%)also supported this conclusion.(7)Effects of polymerized whey protein and pectin(PP)system on particle size distribution,rheological properties,molecular weight distribution of polysaccharides,microstructures and volatile compounds of goat milk kefir mild and kefir were investigated.Cluster analysis indicated that the PP system addition could increase the particle size distributions and rheological properties of both kefir products.Molecular weight distribution of polysaccharides shown that an intermolecular aggregation occurred between pectin and exopolysaccharides(produced by microorganisms from starter cultures).Micrographs suggested that polymerized whey protein interacted with casein network to form a dense structure.However,PP system did not change the characteristics flavor of goat milk kefir mild and kefir.