Biodiversity Of Chinese Traditional Sourdough Microbiota And Its Flavor Metabolism

Sourdough,also known as Laomian,Jiaozi et al.in China,has been used as a traditional starter in making the staple food Chinese steamed bread(CSB)for more than 1000 years.CSB fermented with sourdough,known as Laomian Mantou,has always been preferred by consumers.However,there is a lack of systematic research and understanding on its microbial composition and metabolites production during fermentation in China.In this work,we have collected sourdough samples from different regions in China to illuminate the microbial composition in Chinese traditional sourdough,and inoculated doughs with the isolated strains to investigate the production and changes of the volatiles during dough fermentation and studied the gene expressions of the main microbes by using RNA sequencing.The yeast and lactic acid bacteria(LAB)in 15 sourdough samples collected from different regions in China were investigated by culturing.In total,11 species of LAB were identified,namely Lactobacillus sanfranciscensis,Lactobacillus crustorum,Lactobacillus brevis,Lactobacillus plantarum,Pediococcus pentosaceus,Lactobacillus paralimentarius,Lactobacillus mindensis,Lactobacillus helveticus,Lactobacillus rossiae,Enterococcus faecium and Leuconostoc mesenteroides.L.sanfranciscensis was found dominant in Chinese traditional sourdough.As for yeast,6 species were found in the samples,including Saccharomyces cerevisiae,Candida humilis(Kazachstania humilis),Wickerhamomyces anomalus,Pichia kudriavzevii,Saccharomycopsis fibuligera and Pichia membranifacien.Among them,S.cerevisiae was the most abundant.Specifically,P.membranifacien had never been found in Chinese traditional sourdough before.High-throughput sequencing analysis of the bacteria in the 15 sourdough samples showed that most of the bacteria in the sourdough were LAB,mainly Lactobacillus,followed by Weissella and Leuconostoc,which three are more abundant and widely distributed among the samples.Other species that are relatively abundant in individual samples include the genera Hymenobacter,Ralstonia,Acetobacter,Methylobacterium,Acinetobacter,Pantoea,Enterobacter,Citrobacter,Aeromonas,Rhodococcus and Microbacterium.HTS also showed that L.sanfranciscensis was the dominant species in our Chinese traditional sourdough and was the only species found in all samples,with a content of more than 50%in most samples.Other LAB species of relatively high levels include Weissella confusa,Leuconostoc mesenteroides,Lactobacillus plantarum,Lactobacillus rossiae and Lactobacillus brevis.Our study showed that the composition of sourdough microbes is both uniformed and unique and has no obvious correlation with the geographical distribution.The results showed that leavening ability of the six yeasts was C.humilis(Kazachstania humilis)HB13>S.cerevisiae 9Y3>W.anomalus HN9>P.kudriavzevii 11Y11>S.fibuligeraHN13>P.membranifaciens 14Y6.Specially,P.membranifaciens 14Y6 showed no leavening ability.A total of 41 volatile compounds were found in the Chinese steamed bread fermented with different yeasts,including 11 alcohols,11 aldehydes,5 ketones,9 acids,3 esters and 2 other compounds.Among them,S.cerevisiae 9Y3 produced more compounds.Volatile compounds showed different changes during dough fermentation by S.cerevisiae:(a)most of them increased with the progress of fermentation,such as ethyl acetate,ethanol,2,3-pentanedione,2-methyl 1-propanol,3-methyl-1-butanol;(b)some of the volatiles slightly reduced or hardly changed,such as hexanal,butanol,pentanol,acetic acid,butyrolactone and pentanoic acid;(c)the content of 3-methylbutanal and acetoin first increased and then decreased.Compared with the dough which was fermented for 6 h,the expression of 85 genes in S.cerevisiae was significantly up-regulated in doughs fermented for 12 h.These genes are mainly involved in the synthesis and metabolism of carbohydrates(disaccharides,oligosaccharides),amino acids and organic acids,as well as the transmembrane transport of organic acids.When inoculated for sourdough fermentation,the LAB strains significantly accelerated the rate of acid production after 4 h and showed the highest acid-producing ability in 4-10 h.A total of 33 volatile compounds were found dough fermented for 12 h,including 5 alcohols,11 aldehydes,6 ketones,5 acids,3 esters and 3 other compounds.The dough fermented by heterofermentative LAB mainly feature ethanol,acetic acid,ethyl acetate and ethyl lactate,while dough with homofermentative LAB contained more aldehydes,ketones.In the sourdough fermented by L.sanfranciscensis,ethanol,hexanol,ethyl acetate,ethyl lactate,acetic acid,butyric acid,valeric acid and hexanoic acid gradually increased with the progress of fermentation.The results of gene expression analysis showed that after 12 h of fermentation,the growth and proliferation rate of L.sanfranciscensis decreased significantly,but the metabolic rate was not.Cofermentation of L.sanfranciscensis and S.cerevisiae did not affect the final acid amount produced by L.sanfranciscensis,though some genes related to the production of lactic acid and acetic acid were down-regulated in L.sanfranciscensis.Meanwhile,the expression of genes related to amino acid metabolism and peptidoglycan synthesis in L.sanfranciscensis in the mixed-fermented dough was significantly higher than in the individual fermentations,suggesting that cofermentation may have some effects on the growth and reproduction of L.sanfranciscensis,though plate counts didn't show that.However,plate counts showed that the presence of L.sanfranciscensis favored the growth and reproduction of S.cerevisiae in the dough.The volatiles of the dough fermented by the mix of the two microbes contains the compounds produced by each,but the yield was mostly less than the sum of the two alone fermentations,indicating that there was an interaction between them.