Microbial Community Flora In Kazak Artisanal Cheese And Analysis On The Formation Of Flavor Compounds

With a long history of Kazak cheese manufacture in Xinjiang,there forms a variety of flavor compounds in this natural fermented dairy products.At present,Kazak artisanal cheese is still adopted for the traditional production process,lead to the complex microbial community flora and unstable flavor.Therefore,it is a key role to investigate the correlation between microbial community structure and flavour metabolism,solve the problem of cheese flavor quality instability and standardized production based on characteristic flavor of Kazak cheese.The dissertation based on the Kazak traditional artisanal cheese from different regions in xinjiang as the research object,and analyzed the characteristic flavor components fingerprint and the variety of microbial community structure,then further investigated the correlation between succession of microbial community and flavor metabolism.The relationship between dominant yeasts and the formation of cheese characteristic flavor was clarified by enhancement inoculation of enzyme producing strains,and the results were provided theoretical basis for the high quality,standardized production of the Kazak cheese.The main results of this paper are as follows:(1)HS-SPME/ GC-MS was used to analyze the volatile compounds from 20 local ethnic minority farmhouses.A total of 52 major volatile compounds were detected in the cheese samples,and the major characteristic flavor,including acids(acetic acid,butyric acid,heptanoic acid,and octylic acid),alcohols(ethanol,phenyl ethanol and 3-methylbutanol),esters(ethyl acetate,ethyl hexanoate and ethyl octanoate),ketones(2-nonanone and 2-heptanone),aldehydes(hexanal,heptanal,benzaldehyde,2-decenal and nonanal),were determined by GCO flavor intensity and sensory evaluation,forming characteristic flavor components fingerprint of Kazak cheese.(2)Based on three criteria,high abundance ratios,flavor correlation and co-occurrence rate,Lactobacillus and Streptococcus were determined the dominate bacterial community in kazak cheese through high-throughput sequencing,accounted for 69.59 % and 21.31 %,respectively.For fungus,there were Issatchenkia,Debaryomyces,Kluyveromyces,Pichia,Torulaspora and Geotrichum,accounted for 36.04%,22.13%,17.32%,7.72%,6.12% and 4.05% respectively.Spearman's showed that the proportion of co-correlation above 5.0% were Torulaspora(6.55%),Kluyveromyces(5.95%),Pichia(5.95%),Issatchenkia(5.365%).Lactobacillus,Streptococcus and Kluyveromyces were the primary contributors to the cheese flavor according to the combination of microbial and flavor components.Based on the mentioned standards above,the core characteristic microbial in kazak cheese were Kluyveromyces,Pichia,Issatchenkia,Torulaspora,Debaryomyces,Lactobacillus and Streptococcus?(3)HS-SPME/GC-MS and HPLC were applied to detect the volatile compounds,fatty acid and amino acid concentration to define the flavor variation in Hazak cheese ripening through hydrolysis of protein,lipid degradation and decomposition of lactose and deflection.Cheese flavor metabolism in the inital ripening process was significant different,especially free amino acid.The concentration of amino acid increased significantly during cheese ripening,and reach the maximum in the 10 th day(P < 0.05),which corresponding with protease activity.In later period(10-30 days)of cheese ripening,amino acid metabolism was changed and caused the reduction of corresponding flavor,such as metabolism of phenylalanine and leucine form 2-phenyl ethanol and 3-methylbutanol.The activity of cheese lipase was the highest in the 15 d,the fat metabolism was shifted to butyric acid,caproic acid,caprylic acid and decanoic acid.The main alcohols(ethanol,2-phenyl ethanol and 3-methylbutanol)and carboxylic acids formed the corresponding esters,such as ethyl caproate,octanoic acid ethyl ester,ethyl butyrate and decanoic acid ethyl ester,and reached the higheast concentration 11.07 mg·kg-1 in 15 days.The metabolic pathway of deflection of different stages is main origin form cheese.flavor.(4)Illumina-Hiseq sequencing was carried out for the Kazak cheese in different ripening stages.The relative abundance of Lactobacillus and Streptococcus were decline,while Acetobacter and Lactococcus were significant increased.According to the prediction analysis of the PICRUSt,the metabolic function of bacterial flora was most active in 10 d in cheese ripening,with the amino acid metabolism up to 16%,which was consistent with the changes of amino acids and the predict function results.The core yeast Kluyveromyces,Torulaspora,Issatchenkia and Pichia were significantly changed at 20 d in cheese ripening.Fluorescence quantitative PCR was used to monitor microbial community succession in cheese ripening,and gene copy number of bacterial biomass was up to the higheast with 9.6 lg copies·g-1 in the 10 d and 7.1 lg copies·g-1 for fungus at 10 d,which were corresponding to high-throughput sequencing results.O2 PLS analysis found that bacterial populations were related to the variation of amino acid.Acetobacter,Lactococcus,bacillus exhibited positive correlation with Glu,His,Ile,Pro and(P < 0.05,R > 0.9).For fungus,Issatchenkia played the key role in forming ethyl acetate,hexanoic acid,3-methylbutanol,phenyl ethanol,n-decanoic acid,Leu and Phe(P < 0.05,R >0.90).The abundance of Kluyveromyces showed positive correlation with phenyl ethanol,ethyl butyrate and pentadecylic acid(P < 0.05,R >0.90).These results preliminarily revealed the relationship between microbial community succession and flavor substance formation in the process of cheese maturation.(5)Based on the above results,establishing various yeast cheese fermentation system,exploring the feasibility of Kazak cheese standardization production,screening key enzymes(lipase,protease,?-galactosidase,esterase)which influence the formation of flavor to determined the core enzyme production microbes,which were 5 strains of K.marxianus(5-7,5-11,N-9,24-5 and 27-2),2 strains of K.lactis(NTE and N-S),3 strains of P.kudriavzevii(NX,4-5 and 10-8).Cheese fermentation system was clarified by enhancement inoculation of enzyme producing strains,and P.kudriavzevii N-X play a role in keeping the characteristic flavor of acetic acid,ethyl acetate,and 3-methylbutanol.K.marxianus 24-5 had a strong ability to break down lactose,and produce benzene ethanol,3-methylbutanol.The characteristic of producing enzymes and the formation of cheese flavor exhibited obvious relevance.P.kudriavzevii N-X and K.marxianus 24-5 were cultured during cheese process,significantly promoting the formation of acetic acid,phenyl ethanol,ethyl acetate and 3-methylbutanol.This research provids a theoretical basis for the standardized production of Kazak cheese.