Analysis Of Microbial Community And Its Effect On Malt Quality In The Process Of Malting

Beer is one of the oldest beverages known to mankind.it is a low-alcohol beverage produced by using malt as the main raw material,adding hops and fermenting with purebred yeast.At present,China has become a superpower in beer production and consumption in the world.With the improvement of the quality of life,the demand for medium-and high-grade beer is increasing,and the quality of malt has been paid more and more attention.Malt manufacturing refers to the process of malting beer barley into malt by soaking,sprouting and drying under artificially controlled conditions.it is a complex biological process.In addition to the germination of barley itself to promote the synthesis of enzymes and the degradation of grain structure,it also includes the growth and reproduction of rich microbial flora.In the process of malting,the interaction between these microorganisms and a series of complex metabolic and biochemical reactions make the microbial community interact with grain metabolism in the process of malting,and finally affect the quality of malt products.However,the specific succession and functional mechanism of microflora in the process of wheat production are not clear.In addition,due to the relatively open malting environment and non-sterile barley raw materials,there is a risk of contamination by some unfavorable microorganisms in the malting process,which leads to the instability of malt quality between different batches under the same production process.In order to inhibit harmful bacteria and improve the characteristics of malt,some microorganisms with good characteristics have been widely used in malt production.However,at present,there is still a great shortage of suitable microbial resources for wheat production.In view of the above problems,this paper uses high-throughput sequencing technology combined with traditional separation and culture methods to scientifically and deeply analyze the complex microbial communities in the process of wheat production,and reveal the composition and diversity of microbial species.A better understanding of the potential mechanism of microbial community succession will contribute to the management and optimization of wheat microecology to ensure product quality.In addition,mining more functional microbial resources in the process of malting and exploring the potential harmfulness of different microorganisms and controlling them in actual production is one of the effective ways to improve the quality stability and safety of malt products.The main contents and results of this thesis are as follows:(1)Analysis of microbial community structure and diversity in wheat making process based on high-throughput sequencingTaking the microbial communities in the samples of different wheat production stages as the research object,based on the second generation sequencing data of Miseq,the community diversity,composition structure and dynamic succession were analyzed.In addition,multivariate statistical analysis was used to reveal the potential correlation and functional mechanism of microbial communities.A total of 57 fungal genera from2 mycetes and 65 bacterial genera from 4 dominant bacteria were detected in wheat samples.13 genera such as unclassified＿o＿Saccharomycetales,Filobasidium,Alternaria and Candida were identified as the dominant fungal genera in the process of wheat production.15 genera,such as Acinetobacter,Pantoea,Lactococcus,Weissella and so on,are the dominant bacteria in the process of wheat production.Diversity analysis showed that the microbial diversity and richness changed sharply in the wheat soaking stage,and then gradually tended to be stable.The results of community structure analysis showed that there was little difference in the colony structure of dominant microorganisms among the samples in the process of malting,but the relative abundance of different genera in the samples varied with the process of malting,which may be related to the interaction between microorganisms and environmental conditions in the process of malting.In addition,it is also related to the interaction between various microorganisms.Through correlation network analysis,a total of 44 nodes and 133 pairs of significant correlations were obtained.The connectivity of 12 nodes was ≥ 10 and classified as co-occurrence microbiota.It includes 8 bacterial genera and 4 fungal genera.To a certain extent,it reveals that the relationship between various complex microorganisms and other abiotic factors participate in driving the succession of microecology in the process of malting.Based on the results of extender sequencing,the phenotypic function of bacterial community during wheat production was predicted.The results showed that the relative abundance of facultative anaerobic bacteria was higher than that of aerobic bacteria,and the relative abundance of Gramnegative bacteria was higher than that of Gram-positive bacteria.In addition,the relative abundance of Gram-negative bacteria,biofilm formation,potential pathogenic bacteria and stress-tolerant bacteria have the same trend in the process of malting.These bacteria may affect the quality characteristics and safety of malt.The prediction of fungal function showed that the functional nutritional types of fungi in barley grains were mainly animal pathogens-endophytes-plant pathogens-woody saprophytes.However,in the process of wheat production after wheat soaking,saprophytic nutrition type is the dominant functional type.These saprophytic bacteria may also obtain nutrients by assisting in the degradation of barley cell walls.(2)Isolation and identification of culturable microorganisms in the wheat production processThrough the traditional plate separation method,using morphological observation and microbial DNA sequence analysis,all kinds of culturable microorganisms in the process of wheat production were isolated,purified and identified.A total of 108 strains of fungi were isolated from samples collected in different wheat production stages,which were preliminarily identified as 2 phyla,6 classes,11 orders and 24 fungal species.A total of 103 strains of culturable bacteria were isolated and identified as 19 bacteria belonging to 3 phyla,3 classes,7 orders and 11 families.A total of 37 strains of lactic acid bacteria positive for gram staining and negative for catalase test were isolated and purified by MRS medium plate,which were preliminarily identified as 7species of 4 genera and 2 families.The results show that there are abundant microbial resources in the whole malting ecosystem,and these microorganisms play various important roles in the malting ecosystem and affect the quality of malt products in varying degrees.(3)Preliminary study on the extracellular enzyme activities of fungi from the maltmaking ecosystemThe plate screening method was used to screen the activities of protease,amylase,xylanase and cellulase of cultured fungi in the process of wheat production.A total of16 enzyme-producing strains,6 fungal protease-producing strains,6 amylaseproducing strains,7 xylanase-producing strains and 12 cellulase-producing strains were screened.The results showed that the fungal community was the potential producer of malt hydrolase system.For example,Cladosporium halotolerans protease and cellulase activity were the strongest,Alternaria sorghi showed higher xylanase activity,and Rhodotorula mucilaginosa could decompose starch better.(4)effect of infection of four dominant bacteria on malt quality.Based on the results of high-throughput sequencing in chapter 2 and isolation and identification in chapter 3,four dominant bacteria with the highest relative abundance and the highest isolation frequency were screened.The suspensions of four dominant bacteria were inoculated into wheat soaking water to simulate the contamination of bacteria in the process of wheat production.The main quality indexes of malt,such as malt saccharification power,storage value and α-amino nitrogen,were analyzed after malting in order to clarify the effects and differences of dominant bacteria on the main quality characters of malt.The results showed that the infection of Pseudomonas oryzae,Acinetobacter johnsonii and Atlantibacter hermannii could reduce the malt pool value and the content of α-amino nitrogen in wort,and affect the protein degradation of malt.And significantly reduced the malt saccharification power and leaching rate,seriously affecting the subsequent wort fermentation.Among them,the influence of A.johnsonii is the most significant.The infection of glutamate bacillus Glutamicibacter mishrai had no significant effect on the quality of malt.The infection of four strains caused the increase of wort turbidity,among which A.johnsonii had the greatest effect.In addition,it was found that the effects of bacterial infection on malt quality of different barley genotypes were different.