Quality Characteristics And Microbial Community Evolution Of Aspergillus-type Douchi During Fermentation

Aspergillus-type douchi is a kind of seasoned non-staple food with a long history in China,with functions such as anti-oxidation,lowering blood pressure,and thrombolysis.Nowadays,extensive production processes were still used in stages of Aspergillus-type douchi,which may caused instability and increased risks of venenation.Prior to develop scientific artificial fermentation,it need to own comprehensive insights on quality properties and microbial succession rules to douchi wth original process,while previous researches still not investigate its rules in-depth.Therefore,the paper compared the quality properties and vital enzyme activities of Aspergillus-type douchi at different stages.Then microbes succession were explored from absolute and relative abundance using molecular identification technology.Finally,preliminary evaluation of environmental tolerance and potential of dominant microbes was performed.The specific process and results were as follows:1.During six fermentation stages of douchi,the fermentation tank temperature rose and then fell,and around 45?C mostly,with pH values steadily dropping to 4.86 at the end.The salt content remained at around 6%,and the amino nitrogen and total acid showed a steady upward trend,while a steady downward trend for reducing and total sugar.The color and hardness of douchi turned black and smaller respectively,the main flavor with maotai began to form on day15 and strengthened on day21.The activities of neutral protease,alpha-amylase were higher,while those of cellulase,lipase and beta-glucosidase were lower by two to three orders of magnitude,the activities of all vital enzymes were continuous decline.2.By culturable method,it found that bacteria accounted for the vast majority during fermentation,and the abundance of bacteria,lactic acid bacteria and fungi all showed a downward trend.Twelve of the eighteen isolated strains were bacteria,the dominant species were Bacillus velezensis,Staphylococcus gallinarum,and Weissella jogaejeotgali.Absolute abundance of bacteria and fungi was significantly decreased using real-time quantitative PCR,the bacteria was two to three orders of magnitude ahead of fungi.Absolute abundance of Staphylococcus has fallen sharply since day10,while roughly stable for Bacillus.High-throughput sequencing of bacteria showed that Firmicutes were dominant phylum,and Staphylococcus was the only dominant genus wholly,its abundance increased first and then decreased,with a maximum of 93.6%.The proportion of Bacillus increased gradually in the latter stages,and Lactic acid bacteria,represented by Weissella and Lactobacillus,were mainly distributed in the initial and last stages.Further analysis found that the overall bacterial structure had undergone major changes,and the pathways to nucleotide metabolism,membrane transport,replication,and repair showed a continuous downward trend.The redundancy analysis indicated that pH and temperature were the main environmental factors for succession of the bacterial community in douchi.3.The stress environment test on the dominant bacteria found that Weissella and Lactobacillus were less tolerant to stress factors,four species were weak in high temperature tolerance,which were resistant to salinity,low pH and other factors.Among them,Staphylococcus was hardly affected by 6% salt growth,and colonies cultured on the plate was much less with smaller morphology.For fermentation test of each bacterium,the flavor produced by Weissella and Lactobacillus was frankincense mainly,while the characteristics of Bacillus and Staphylococcus were strong soy and maotai flavor respectively.The activity of neutral protease in the fermentation of various bacteria was significantly higher than that in the natural fermentation of Douchi,while the activity of lipase,beta-glucosidase and cellulase were all low.Overall,the paper taking the changes of quality properties and enzyme compositions in douchi as pointcuts,from the perspective of absolute and relative abundance with various tools,the change rules of microbes was clarified,which was the fundamental factor to quality properties alterations of douchi.By culture experiments,the environmental tolerance and fermentation potential of dominant microbes were evaluated preliminarily,so as to provide opinions to explore further rules for Aspergillus-type douchi,and establish the experimental basis for further steps of strain combination fermentation.