| NiKeng Baijiu is a typical local Chinese strong-flavor Baijiu in Hebei Province.It is characterized by the use of traditional hand-made Jiuqu and mud pond fermentation.It is a traditional and extensive natural production mode.The product quality is easily affected by environmental conditions.Therefore,the study on exploring the composition and diversity of Jiuqu fungi community and screening of superior functional microorganisms,will provide a theoretical support for the standardized production and technological improvement of NiKeng liquor in the future.In this study,taking NiKeng Jiuqu as the research object,using the method of high-throughput sequencing,Biolog microplate analysis,traditional separation culturing to study the composition and diversity of the microbial community of Jiuqu.The results will provide scientific basis for improving the quality of Jiuqu and Baijiu.The main findings are as follows:1.Using a temperature sensor and oven drying method to measure the temperature and moisture content of Jiuqu,the results showed that in the five stages P1-P5 of the Jiuqu making process,the temperature of the Jiuqu rose rapidly and reached the highest temperature of 60? in the P3 stage and then decreased gradually,.Finally,the temperature of mature Jiuqu dropped to 35?.The moisture content of Jiuqu had been on a downward trend,and the moisture content of different parts had always been the inner layer>middle layer>surface layer.The temperature of the P1 Jiuqu during the whole fermentation stage was higher than other stages,and the moisture content of different parts of the Jiuqu displayed great difference,but the temperature and moisture of the mature Jiuqu in the P5 stage was not very different.2.Using Biolog technology to analyze the AWCD value,diversity index of the microbial community of Jiuqu and the utilization of 6 types of carbon sources.The results showed that the microbial AWCD value of the yeast in different periods of the first 8 hours did not change significantly,and then it began to increase exponentially.The AWCD value in the P1 stage was the largest and the microbial activity was the highest,while the microbial AWCD value in the P4 period was the lowest.The McIntosh index,Shannon index and Simpson index showed that the diversity index of P1 and P3 had been higher than that of the other stages in the whole cultivation stage,and the P4 stage had been significantly lower than the other four stages;According to the specific cultivation of 6 kinds of carbon sources,it was found that the functional diversity of Jiuqu microorganisms at different stages is relatively significant,and the utilization of different carbon sources was also different.The microorganisms in the whole stage had obvious utilization rates of amino acids,phenolic acids and carboxylic acids,but less utilization of amines,polymers and sugars.Various metabolic characteristics basically presented the tread of P1>P3>P2>P5>P4.3.High throughput sequencing was used to analyze ITS region of fungi in different fermentation stages to study the composition and diversity of fungal microbial community:(1)The total number of OTUs in the whole fermentation period was 231,of which 20 common OTUs were accounting for 8.66%,indicating that the similarity of OTUs in different fermentation stages was not high.The number of unique OTUs in P1 stage accounted for 30.3%of the total,indicating that the species diversity in this period was significantly higher than that in other periods.(2)According to alpha diversity analysis,the richness,evenness and diversity of Jiuqu fungal community had been decreasing first and then increasing.The diversity and richness of Jiuqu fungi were higher in P1 and P5.and P2 was higher The results of PC A analysis showed that except for P3 and P4,the samples of the other three periods were scattered,showing obvious differences,and the aggregation degree of Jiuqu species in P1 period was the worst;NMDS analysis showed that the distance between P2 and P5,P3 and P4 were relatively small,however,the distance between P1 and other stages was the farthest,which means the community structure of P1 was not very similar to other stages.(3)In this study,5 different phyla and 49 different genera of fungi were identified.At the phylum level,Ascomycota accounted for 75.78%of the total fermentation period.At the genus level,with the progress of fermentation,the dominant genera changed from Pichia?Candida and Aspergillus in P1 period to P2-P5 The results showed that the dominant genera of Thermoascus?Pichia and Rhizomucor were successional,the abundance of fungi in five different stages was different,and the abundance of fungi in P1 stage was different.4.Using the traditional microbial isolation and molecular identification methods,the cultivable strains were isolated and purified,and the changes of the number and species of fungal colonies in the fermentation process were analyzed,and their status in species and genus was preliminarily identified.The results showed that the number of fungal colonies increased during the heating period,and the number of mold reached the maximum of 15.6 lg CFU/g and the number of yeasts reached 11.6 lg CFU/g at 60?,and then began to decline,and the number of mold decreased to the minimum at the mature stage of Jiuqu,which was 8.1 lg CFU/g,but the yeast was not detected.With the fermentation process,the community succession of different parts of Jiuqu were changed,suggesting the phenomenon of new species formation and the disappearance of original species,and some strains showed gradually growth from the surface to the inside of Jiuqu.According to the morphological characteristics and molecular biological results,the species and genus status of these 20 isolates were identified.5.The strains with high esterifying power,high liquefying power,high fermenting power and high saccharifying power were screened from 12 representative fungal strains.The results showed that Pichia kudriavzevii,kazachstania turiciensis,Rhizopus oryzae and Aspergillus niger were better than other strains,which could be used for further experimental optimization. |
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