| Inner Mongolia handmade cheese is the traditional national cheese in China.The widely distributed grasslands of Inner Mongolia makes the climate production areas and raw milk of cheese to be different,which affects the microbial fermentation quality of Inner Mongolia cheese.At the same time,adding sucrose to the original Inner Mongolian handmade cheese has become a popular way in order to cater to the tastes of non-Mongolian people.But whether adding sucrose changes the microbial diversity and safety,the nutritional content and flavor of Inner Mongolian handmade cheese or not is still unknown.In this study,a total of 11 handmade cheese samples with zero or sucrose addition from different grasslands in Inner Mongolia(Hulunbeier Grassland,Horqin Grassland,Ordos Grassland,Xilinguole Grassland and Ulanqab Grassland)were selected as the research object.High-throughput sequencing technology,basic physical and chemical properties analysis,and detection of volatile components and free amino acids were used to study the diversity of the microbial community structure of these cheese,as well as the influence and function of microorganisms on the nutritional composition and flavor formation of the handmade cheese.At the same time,the microbial community structure,the basic physical and chemical properties of cheese,the volatile components and the type of free amino acids of the handmade cheese with zero or sucrose addition were analyzed,to explore the effects of sucrose addition on the diversity and safety of cheese microbial community,as well as the nutritional content and flavor substances of cheese.Finally,based on whole-genome sequencing technology,we explored the wholegenome of a thermophilic bacterium(Thermus CEJ2-1)which was derived from sucrose adding handmade cheese,and analyzed its metabolic pathways and functions in the process of cheese acid fermentation.The main findings are as follows:1.We have constructed a high-throughput sequencing library with sufficient depth.The number of OTUs and Chao1 index of handmade cheese samples from Wulanchabu grassland and Xilinguole grassland in central Inner Mongolia were generally lower than handmade cheese samples from Hulunbeier,Horqin and Ordos grasslands in the east and west,and the OTU number and Chao1 index of all zero-added handmade cheese were significantly lower than the sucrose-added handmade cheese samples.The UPGMA and PCoA analysis based on the Weighted Unifrac distance showed that the microbial community structure of all handmade cheese samples from the Ulanqab grassland and the Xilinguole grassland in central Inner Mongolia was clearly distinguished from cheese samples from Hulunbeier,Korqin grassland in the east,and Ordos grassland in the west.At the same time,the microbial community structure of all zero-added handmade cheese samples were significantly different from other sucrose-added samples.2.Species annotation results indicated that the predominance of lactic acid bacteria in the handmade cheese samples from Wulanchabu and Xilinguole grasslands(relative abundance of 86.6%~98.6%)in central Inner Mongolia is generally higher than that from Hulunbeier,Horqin Ordos grassland cheese samples(relative abundance 37.9%~84.4%).Lactococcus were the absolute dominant bacterial group(relative abundance 73.8%~87.9%)among all starter microorganisms in zero added artisanal cheese,while Streptococcus were the most dominant bacterial group(relative abundance 25.8%~84.0%)among all starter microorganisms in the sucroseadded manual cheese sample.At the same time,the proportion of some special groups in the non-starter microbial flora has increased significantly.Such as the non-pathogenic bacteria Thermus,which accounted for 12.3% of the relative abundance in the EJ2 sample of Ordos,and the pathogenic bacteria Acinetobacter accounting for 8.7% in the KJ sample from Korqin,Enterococcus accounting for 1.9% in the HJ sample from Hulunbeier,and Bacteroides accounting for 2.5% in the EJ1 sample from Ordos.3.The contents of moisture,pH,NaCl,fat,protein,lactose and sucrose of the handmade cheese in Inner Mongolia did not show special regularity with the distribution of grassland.However,the content of moisture,pH,lactose and sucrose of Inner Mongolia handmade cheese with sucrose addition were generally higher than that of the sample with zero addition;while the content of fat and protein of Inner Mongolia handmade cheese with sucrose addition were generally lower than that of the sample with zero addition.At the same time,Spearman correlation analysis showed that the above physical and chemical factors were significantly correlated with the number of OTUs in the sequencing library and Chao 1 species richness index.4.A total of 13 acids,19 hydrocarbons,17 alcohols,10 aldehydes,11 ketones,22 esters,2 furans,5 olefins,and 2 pyrazines were detected in the cheese samples.These volatile components did not show a certain regularity with the distribution of the grasslands,but the principal component analysis results showed that the Inner Mongolian handmade cheese with zero addition and sucrose addition had a clearly differentiated composition of volatile components.Among them,the zero-added handmade cheese samples generally have higher relative contents of acids,furans,olefins and pyrazines,and lower relative contents of esters than sucrose-added samples.At the same time,we determined that there was a high Spearman positive correlation coefficient(ρ> 0.6)between 29 volatile substances and 24 microbial genera.Apart from 10 volatile components which were positively related to 5 lactic acid bacteria,all other volatile components were positively related to non-starter microorganisms.At the same time,the results of 14 kinds of free amino acids detected in EJ1 and EJ2 from Ordos,XS1 and XJ1 from Xilinguole showed that adding sucrose may form a unique type of free amino acid composition which differs from the zero-added cheese sample.5.The Thermus CEJ2-1S1 strain isolated from EJ2 samples from Ordos was whole genome sequenced.KEGG annotation results showed that the strain can catabolize lactose,produce intermediate product pyruvate through the EMP pathway,and then pyruvate entered the lactate metabolism pathway,acetate metabolism pathway,propionic acid metabolism pathway and tricarboxylic acid cycle metabolism pathway,producing lactic acid,acetic acid,propionic acid,succinic acid,malic acid and fumaric acid.The milk fermentation test verified that Thermus CEJ2-1S1 can produce the above-mentioned organic acids at 70℃.The highest concentration of citric acid it produced may be used as a carbon source substrate for Lactobacillus MXS11-1S4 and other starter lactic acid bacteria for fermentation metabolic processes.Based on the above results,we speculated that sucrose added in Inner Mongolia handmade cheese may replace lactose as the main carbon source used by cheese microbes,resulting in a decrease in the content of the starter lactic acid bacteria in the cheese microbial community,weakening the degree of acidification of cheese fermentation,and thus promoting the growth of non-starter microbial flora and leading to the increased predominance of conditional pathogenic bacteria,and the consumption of fat and protein in raw milk,producing more abundant and special volatile substances and free amino acids,which ultimately leads to Inner Mongolia handmade cheese nutrient composition and flavor changed significantly.The Thermus strain isolated from the EJ2 sample in Ordos may has a metabolic pathway which was similar to lactic acid bacteria to produce a variety of organic acids and lower the pH of the cheese.It also provided sufficient citric acid substrate for the fermentation of lactic acid bacteria and played a role in assisting acid-producing fermentation. |
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