宁夏老面的微生物群落解析、发酵菌株筛选与复配发酵特性研究

Metagenomic Analysis of Microbiota in Ningxia Traditional Sourdough, Screening of Fermentative Strains, and Evaluation of Combined Fermentation Properties

摘要:

为明确解析宁夏老面中的微生物群落结构并开发面团复合发酵菌剂，对从宁夏自北向南不同地区采集的12份家庭自然发酵老面，采用宏基因组学技术解析6份老面的微生物群落；通过菌株的分离纯化与初步鉴定，筛选出12份老面中的优势菌株，并对优势菌株进行分子生物学鉴定；对优势菌株的产酸速率、耐酸能力、产乙醇能力、面团发酵能力等发酵性能进行测定；将优势菌株进行复配，测定优势菌株对面团的发酵能力和发酵面团的游离氨基酸组成。结果表明，6份宁夏老面中的微生物群落以乳酸菌和酵母菌为核心微生物类群，细菌群落呈现明显组成差异，优势菌包括旧金山果糖乳杆菌（24.54%）、乳酸片球菌（15.99%）、戊糖片球菌（8.20%）和肠膜明串珠菌（7.69%）等；真菌群落结构相对集中，主要由酿酒酵母（90.99%）主导，仅个别样品以涎沫假丝酵母（73.73%）为主。通过分离培养，从12份老面中共获得26株乳酸菌与18株酵母菌；以产酸能力为指标对26株乳酸菌进行初筛，其中10株菌的产酸量≥15 g/L，表现出高产酸潜力；以产气能力为指标对18株酵母菌进行初筛，其中11株菌在12 h内产气量即超过杜氏管体积的3/4，表现出快速、高效的产气性能。对初筛获得的21株菌（11株酵母菌、10株乳酸菌）的PCR扩增产物进行测序，鉴定发现筛选得到的11株酵母菌中7株为酿酒酵母（Saccharomyces cerevisiae），1株为库德里阿兹威毕赤酵母（Pichia kudriavzevii），1株为葡萄牙棒孢酵母（Clavispora lusitaniae），2株为布拉迪酵母（Saccharomyces boulardii）；10株乳酸菌中，8株为乳酸片球菌（Pediococcus acidilactici），2株为植物乳植杆菌（Lactiplantibacillus plantarum）。对10株乳酸菌的产酸速率进行评价，最终筛选出2株乳酸菌用于复配实验；对11株酵母菌的耐酸能力、产乙醇能力与面团发酵力进行评价，最终筛选出3株酵母菌用于复配实验。将筛选获得的2株乳酸菌（植物乳植杆菌M6-1、乳酸片球菌M10-1）与3株酵母菌（库德里阿兹威毕赤酵母Y1-1、酿酒酵母Y6-2和布拉迪酵母Y10-2）进行复配，共构建25组菌种复配组合；复配实验结果表明，4株菌组合（M6-1、M10-1、Y6-2、Y10-2）的面团发酵性能最佳，同时其发酵面团中游离氨基酸总量最高，达到1253.24 mg/kg。该组合在发酵面制品生产中展现较好的应用潜力，印证了乳酸菌与酵母菌的协同作用及老面核心功能菌群的地位。希望研究可为传统面制品的复合发酵剂开发提供菌种资源与理论依据。

Abstract:

To elucidate the microbial community structure of Ningxia traditional sourdough and develop a composite starter culture for dough fermentation, 12 traditionally fermented sourdough samples were collected from north to south across Ningxia. Metagenomic sequencing was performed on six of these samples to analyze their microbial communities. Subsequently, dominant strains were isolated and purified from all 12 samples, preliminarily identified, and then subjected to molecular identification. Their fermentation properties, including acid production rate, acid tolerance, ethanol production capacity, and dough leavening ability, were evaluated. Finally, selected dominant strains were combined in different formulations, and the dough leavening ability as well as the free amino acid composition of the fermented dough were determined.Results showed that the microbial communities in the six sourdough samples were dominated by lactic acid bacteria (LAB) and yeasts as core microbiota. Bacterial communities exhibited significant compositional differences, with dominant bacteria including Fructilactobacillus sanfranciscensis (24.54%), Pediococcus acidilactici (15.99%), Pediococcus pentosaceus (8.20%), and Leuconostoc mesenteroides (7.69%). Fungal communities were relatively uniform, largely dominated by Saccharomyces cerevisiae (90.99%), except for one sample where Candida zeylanoides (73.73%) prevailed. Through isolation and culture, a total of 26 LAB and 18 yeast strains were obtained from the 12 sourdough samples. Preliminary screening based on acid production capacity identified 10 LAB strains with acid production ≥15 g/L, indicating high acid-producing potential. Screening based on gas production capacity identified 11 yeast strains that filled more than three quarters of a Durham tube within 12 h, demonstrating rapid and efficient gas production. PCR amplification and sequencing of the 21 preliminarily screened strains (11 yeasts, 10 LAB) revealed that the 11 yeast strains belonged to three genera: seven were Saccharomyces cerevisiae, one was Pichia kudriavzevii, one was Clavispora lusitaniae, and two were Saccharomyces boulardii.Among the 10 LAB strains, eight were Pediococcus acidilactici and two were Lactiplantibacillus plantarum. Evaluation of acid production rates led to the selection of two LAB strains for co fermentation experiments. Evaluation of acid tolerance, ethanol production capacity, and dough leavening ability led to the selection of three yeast strains. The selected strains – two LAB (Lactiplantibacillus plantarum M6 1, Pediococcus acidilactici M10 1) and three yeasts (Pichia kudriavzevii Y1 1, Saccharomyces cerevisiae Y6 2, and Saccharomyces boulardii Y10 2) – were used to construct 25 different consortia. Results of the co fermentation experiments showed that the four strain combination (M6 1, M10 1, Y6 2, Y10 2) exhibited the best dough leavening performance and also yielded the highest total free amino acid content in the fermented dough, reaching 1253.24 mg/kg. This consortium demonstrates good application potential in fermented flour based product manufacturing and confirms the synergistic effect of LAB and yeasts as the core functional microbiota in sourdough. This study provides valuable strain resources and a theoretical basis for the development of composite starter cultures for traditional flour based products.