fmicb-11-01878 August 3, 2020 Time: 12:3 # 1 ORIGINAL RESEARCH published: 05 August 2020 doi: 10.3389/fmicb.2020.01878 Microbial Community Dynamics and Metabolome Changes During Spontaneous Fermentation of Northeast Sauerkraut From Different Households Xiaozhe Yang1,2,3, Wenzhong Hu2,3*, Zhilong Xiu1, Aili Jiang2,3, Xiangyan Yang2,3, Gaowa Saren1,2,3, Yaru Ji1,2,3, Yuge Guan1,2,3 and Ke Feng2,3 1 School of Bioengineering, Dalian University of Technology, Dalian, China, 2 College of Life Science, Dalian Minzu University, Dalian, China, 3 Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian, China Sauerkraut, one of the most popular traditional fermented vegetable foods in northern China, has been widely consumed for thousands of years. In this study, the physicochemical characteristics, microbial composition and succession, and Edited by: Lin Lin, metabolome profile were elucidated during the fermentation of traditional northeast Jiangsu University, China sauerkraut sampled from different households. The microbial community structure Reviewed by: as determined by high-throughput sequencing (HTS) technology demonstrated that Zaixiang Lou, Jiangnan University, China Firmicutes and Proteobacteria were the predominant phyla and Weissella was the Beizhong Han, most abundant genus in all samples. Except for Weissella, higher relative abundance China Agricultural University, China of Clostridium was observed in #1 sauerkraut, Clostridium and Enterobacter in #2 *Correspondence: sauerkraut, and Lactobacillus in #3 sauerkraut, respectively. Meanwhile, Principal Wenzhong Hu [email protected] component analysis (PCA) revealed significant variances in the volatilome profile among different homemade sauerkraut. Acids and lactones were dominant in the #1 sauerkraut. Specialty section: The #2 sauerkraut had significantly higher contents of alcohols, aldehydes, esters, This article was submitted to Food Microbiology, sulfides, and free amino acids (FAAs). In comparison, higher contents of terpenes a section of the journal and nitriles were found in the #3 sauerkraut. Furthermore, the potential correlations Frontiers in Microbiology between the microbiota and volatilome profile were explored based on Spearman’s Received: 30 June 2020 Accepted: 16 July 2020 correlation analysis. Positive correlations were found between Clostridium, Enterobacter, Published: 05 August 2020 Lactobacillus, Leuconostoc, Weissella and most volatile compounds. Pseudomonas, Citation: Chloroplast, Rhizobium, Aureimonas, and Sphingomonas were negatively correlated Yang X, Hu W, Xiu Z, Jiang A, with volatile compounds in sauerkraut. This study provided a comprehensive picture of Yang X, Saren G, Ji Y, Guan Y and Feng K (2020) Microbial Community the dynamics of microbiota and metabolites profile during the fermentation of different Dynamics and Metabolome Changes homemade northeast sauerkraut. The elucidation of correlation between microbiota and During Spontaneous Fermentation of Northeast Sauerkraut From volatile compounds is helpful for guiding future improvement of the fermentation process Different Households. and manufacturing high-quality sauerkraut. Front. Microbiol. 11:1878. doi: 10.3389/fmicb.2020.01878 Keywords: homemade northeast sauerkraut, microbiota, metabolome, flavor, correlation Frontiers in Microbiology| www.frontiersin.org 1 August 2020| Volume 11| Article 1878 fmicb-11-01878 August 3, 2020 Time: 12:3 # 2 Yang et al. Microbiota and Metabolome in Traditional Northeast Sauerkraut INTRODUCTION MATERIALS AND METHODS Since ancient times, fermented foods are characterized by Sample Collection and Preparation extended shelf lives and improved organoleptic properties. All samples were collected from three different households Through fermentation by microorganisms, products exhibit located in Dalian, Liaoning Province, China. Sampling was special microbiota, flavor, and nutritional properties. Chinese carried out according to the process described by Yang et al. fermented vegetables are popular foods that date back to (2020). The samples were collected using sterile transfer pipes the 6000 bc (Azam et al., 2017). Fermented vegetables are from the pickle jar at three different sites: the top, middle, and usually prepared under non-sterile fermentation conditions, and bottom. The five obtained samples from each site (150 mL) were a number of microorganisms participate in the fermentation mixed evenly and packed in sterile self-sealing bags. Seventy-two process. Sauerkraut is one of the well-known traditional samples from three different households were collected at days 0, fermented vegetable in northern China and has been widely 3, 5, 7, 12, 17, 23, 30 and then transported to the laboratory for consumed for hundreds of years (Zhou et al., 2018). Generally, further analysis. cabbages are pretreated and immersed in a 0.5–3.5% salt solution, and then left at an ambient temperature (18–20◦C) for 1 month. Traditional northeast sauerkraut was fermented Physicochemical Analysis based on naturally occurring microorganisms present on the raw The pH value was measured by a pH meter (PHS-3G, INESA material, such as lactic acid bacteria (LAB), Enterobacteriaceae, Scientific Instrument Co., Ltd., Shanghai, China). Titratable Pseudomonadaceae, and yeasts (Wouters et al., 2013). This acidity (TA) was measured and expressed as lactic acid complex fermentation process can improve the nutrition and according to the method described by Yang et al.(2019). enrich the flavor of sauerkraut. The nitrite concentration was measured using hydrochloride During fermentation, microbes play a vital role in the naphthodiamide by national standard of China GB 5009.33-2016 formation of flavor and enhancement of nutritive value. In (Mao et al., 2018). particular, LAB, the major bacterial community, possess enzymes capable of hydrolyzing many food constituents, including DNA Extraction and PCR Amplification carbohydrates, proteins, and lipids, into aroma precursors, and Microbial community genomic DNA from different samples to further convert them into a variety of aroma compounds was extracted using an E.Z.N.A. R soil DNA kit (Omega Bio-tek, (Thierry et al., 2015). It has been proven that sauerkraut has Norcross, GA, United States) according to manufacturer’s many benefits for human health, such as promoting digestion, instructions. The integrity of DNA was analyzed on 1% agarose lowering the cholesterol level, and improving the intestinal gel electrophoresis, and DNA concentration and purity were function (Rabie et al., 2011). determined with NanoDrop 2000 UV-vis spectrophotometer However, up to now, sauerkraut is usually homemade in (Thermo Fisher Scientific, Wilmington, DE, United States). northeast China and relies on spontaneous fermentation, making Primers pairs 515F (50-GTGYCAGCMGCCGCGGTAA-30) it difficult to the production of uniform and high-quality and 806R (50-GGACTACNVGGGTWTCTAA-30) were used to products. During spontaneous fermentation, the microbial amplify the hypervariable region V3–V4 of the bacterial 16S community will be altered by various factors, such as the rRNA gene by an ABI GeneAmp R 9700 PCR thermocycler kinds of raw material, the parameters of fermentation process, (ABI, CA, United States; Walters et al., 2016). PCR amplification and the conditions of geographical and climatic (Cao et al., conditions were as follows: pre-PCR for 3 min at 95◦C, 27 cycles 2017; Li et al., 2017). Therefore, it is essential to investigate of denaturation at 95◦C for 30 s, annealing at 55◦C for 30 s, microbial community characteristics to identify the primary elongation at 72◦C for 10 min, and final extension at 72◦C for and core functional microorganisms for improving fermentation 10 min. PCR amplification was performed in a mixtures, which management and exploring microbial resources as starter contained 4 mL buffer (5 × TransStart FastPfu), 0.4 mL DNA cultures to improve the safety and quality of sauerkraut. Polymerase (TransStart FastPfu), 2 mL dNTPs (2.5 mM), 0.8 mL Additionally, the correlation between the microbiota and volatile primer (5 mM), 10 ng genome DNA, and finally made up to 20 mL metabolites, and which genus boosts the accumulation of the with ddH2O. The amplified DNA was detected by 2% agarose gel major flavor metabolites during the fermentation of northeast followed by purification with the AxyPrep DNA Gel Extraction sauerkraut, is still unclear. Kit (Axygen Biosciences, Inc., Union City, CA, United States) In this study, the microbial composition and succession was according to the manufacturer’s instructions and quantification characterized by high-throughput sequencing (HTS) technology, using the QuantusTM Fluorometer (Promega, United States). and volatilome profile was analyzed via headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS- SPME/GC-MS). Meanwhile, the physicochemical properties Illumina Miseq Sequencing and and non-volatile metabolites were detected. Furthermore, the Processing of Sequencing Data correlation between the microbiota and volatile metabolites Purified amplicons were pooled in equimolar and paired-end was explored. This study could provide scientific basis for sequenced (2 × 300) using Illumina MiSeq sequencing (Illumina, optimizing and standardizing safety and flavor characteristics of Inc., San Diego, CA, United States) at Majorbio Bio-Pharm northeast sauerkraut. Technology, Co., Ltd (Shanghai, China). Frontiers in Microbiology| www.frontiersin.org 2 August 2020| Volume 11| Article 1878 fmicb-11-01878 August