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Structure and Diversity of Bacterial Communities in the Fermentation of Da-Jiang

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Structure and Diversity of Bacterial Communities in the Fermentation of Da-Jiang Annals of Microbiology (2018) 68:505–512 https://doi.org/10.1007/s13213-018-1355-x ORIGINAL ARTICLE Structure and diversity of bacterial communities in the fermentation of da-jiang Pengfei Zhang 1 & Rina Wu1 & Ping Zhang1 & Yiming Liu2 & Dongbing Tao1 & Xiqing Yue1 & Ying Zhang1 & Jing Jiang1 & Junrui Wu1 Received: 15 December 2017 /Accepted: 28 June 2018 /Published online: 8 July 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract Da-jiang is the traditional soybean fermented food which is popular in the world for a long time. In order to improve the quality and nutritional value of da-jiang, structure and diversity of bacterial communities in the fermentation of da-jiang were analyzed. Illumina MiSeq platforms coupled with bioinformatics approach were used in this study. In the first 28 days, the trends of bacterial abundance were similar in different regions which are increasing firstly, decreasing secondly, and rising again. The quantity of bacteria in post-fermentation is lower than pre-fermentation. In the fermentation of da-jiang, Firmicutes and Proteobacteria are the dominant phyla. The dominant genera in da-jiang from different regions are different: Tetragenococcus (58.1–73.0%) is the dominant genus in da-jiang from Xinmin; Leuconostoc (9.2–25.7%) is the dominant genus in da-jiang from Tieling; Acinetobacter (8.7–25.1%) and Leuconostoc (12.4–22.0%) are the dominant genera in da-jiang from Shenyang. Additionally, Weissella, Lactobacillus, Staphylococcus, Erwinia,andPseudomonas also were found in da-jiang. It is identified that Leuconostoc steadily existed in all da-jiang samples. These results demonstrate the diversity of microbes in traditional fermented da-jiang, which will probably provide a data basis for choosing starter culture for da-jiang industrial fermentation. Keywords Da-jiang . Bacteria diversity . Leuconostoc . Illumina Miseq Introduction Da-jiang, a kind of important soybean paste in the northeast of China, has 2000 years of history (Xu 2013;Geetal.2012). Fermentation is one of the oldest and most economical ways It is one of four major traditional fermented soybean products of producing and preserving foods (Hugenholtz 2013). Due to in China. As we all know, the flavor of da-jiang has a strong the improvement of nutritional value and sensory attributes, relationship with microbial metabolism. The usual methods of fermentation became popular in many cultures. At present, making da-jiang are as follows: soybeans are soaked and fermented foods are part of the daily intake (Chaves-López steamed to make 6 cm × 5 cm × 5 cm bricks, then naturally et al. 2014). China has an ancient civilization with a long fermented for 1 to 2 months; next, we mix the fermented history. Chinese has founded many fermented foods, which bricks with brine in a certain ratio, stir daily, and ferment for has unique flavor and nutrition. The typical fermented foods 1 to 2 months; finally, we can get delicious da-jiang. The are da-jiang, sufu, Puer tea, cheese, and vinegar. The produc- whole fermentation of da-jiang usually lasts for 6 months. tion of fermented food is becoming easier and easier along There are approximately three different stages in da-jiang fer- with the development of technology. However, people always mentation. The first period is degradation of components in prefer foods, which are made by traditional methods. raw materials by a series of complex enzymes secreted by microorganisms. In this stage, the most significant thing is the degradation of protein and starch, which can generate * Junrui Wu kinds of small molecule materials that will be helpful to the [email protected] growth of microorganisms in later fermentation. The second 1 College of Food Science, Shenyang Agricultural University, stage is the flavor formation phase. Various biochemical reac- Shenyang 110866, People’s Republic of China tions occur during the formation of flavor, such as Maillard 2 Department of Foreign Language, Shenyang Agricultural University, reaction which react between the reducing sugar and amino Shenyang 110866, People’s Republic of China compounds and generate melanin and flavor substances. The 506 Ann Microbiol (2018) 68:505–512 third stage is flavor re-balance period. Volatile flavoring sub- Materials and methods stances are mainly formed by microorganisms in the environ- ment that secrete enzymes. Ester is one of the typical flavor Sample collection substances, which are generated from alcohols and organic acids under the action of enzymes secreted by salt-tolerant Seventeen da-jiang samples were collected from three differ- yeast and bacteria (Sun 2007;Liu2010). ent regions (Xinmin (T), Tieling (Z), Shenyang (S)) in Natural fermentation is a spontaneous mixed-culture Liaoning Province of China. All samples were prepared by fermented process under appropriate temperature and humid- traditional fermented methods in the same year and collected ity conditions. This traditional food processing method has every 7 days during fermentation. Before collection, da-jiang been reserved in China without disappearance for many cen- in the bowl needs to be mixed. The collected da-jiang was turies. The microbial diversity in fermentation system is influ- loaded in sterile zipper lock bag and immediately stored at enced by the fermented method. As we all know, different − 80 °C waiting for further analysis. This work was approved microorganisms are responsible for different fermented stages by the Shenyang Agricultural University. All work was ap- and play different essential roles (Kim and Chun 2005; Zhang proved by relevant Chinese laws and institutional guidelines. et al. 2011). In recent years, studies on microbial diversity in traditional fermentation can often be seen. Everyone knows Determining pH, total acidity, and amino nitrogen that the traditional fermentation is very complicated. at different fermented stages Therefore, research on microbial diversity can not only help to analyze the relationship between metabolites and microor- SH-2100 pH meter (OHAUS, Jiangsu, China) was used to ganisms but also help to propose a potential approach to mon- measure pH of da-jiang. Total acidity (TA) of da-jiang was itor the traditional fermentation. determined by alkali titration as follows: to determine total The traditional methods for detecting food-related micro- acidity as lactic acid, 25 ml of properly diluted sample was organisms are mainly culture-dependent method and colony titrated with 0.01-N NaOH to pH 8.1. The determination of counts. However, these methods are limited to exploring the AN was as follows: firstly, formol nitrogen (FN) and ammo- variation and structure of microbial communities. Firstly, nium nitrogen were determined according to Chinese National culture-dependent method may be only suitable for part of Standard (CNS, 2002). To determine FN, diluted sample and microbes. Secondly, some microbes cannot be accurately dis- formalin solution were adjusted to pH 8.1, then 20 ml of criminated based on their appearance. Currently, only a few formalin solution was mixed with 25 ml of diluted samples microbes were identified in soybean paste, such as for 3 min, and titrated to pH 8.1 with 0.01-N NaOH. Then, Lactobacillus plantarum, Leuconostoc, Bacillus subtilis,and amino nitrogen (AN) was determined by subtraction of am- Enterococcus faecium. Previous limitations of culture- monium nitrogen from FN according to CNS (Chinese dependent method on research on microbial diversity have National Standard (CNS) 2002). For each analysis, three rep- been largely overcome by advances in molecular biology licates tests were performed. technology, which provide a rapid and high-resolution de- scription of microbial communities. In fact, molecular biology Determination of LAB changes methods have generally supported the traditional results, but theadvancedtechniqueshaveidentifiedmuchhighermicro- The 10-fold-concentrated cells were serially diluted (100 to bial diversity in microecology than previously expected 106) in 0.9% sterile saline solution, and 100 μlofproperly (Navarro et al. 2013). High-throughput sequencing technolo- diluted samples was spread on de Man, Rogosa, and Sharpe gy is a promising method, because it can generate hundreds of (MRS, Merck, pH 5.7) agar plates, then incubated for 1–2days thousands of sequences within a short time which can cover at 37 °C for counting of lactic acid bacteria (LAB). The col- the complex microbial communities as well as low-abundance onies formed (25–250) were calculated as log CFU per milli- microorganisms (Ercolini et al. 2012; Quigley et al. 2012). liter of da-jiang. Thus far, this technology has been used to explore the bacterial composition in fermented food samples, such as botrytized DNA extraction and PCR amplification wines (Bokulich et al. 2012), Chinese traditional sourdough (Liu et al. 2016), and grapes from a Californian region Firstly, 20-mL PBS buffer was added into 8-g sample and (Bokulich et al. 2014). Therefore, in order to provide a theo- vortexed for 5 min. The mixture was centrifuged at 1000 g retical basis for the understanding of the traditional fermenta- for 10 min. The supernatant was collected. Next, 20-ml PBS tion for da-jiang, we performed high-throughput sequencing buffer was added into the precipitate and vortexed for 5 min. technology (Illumina MiSeq platform) to analyze bacterial We centrifuged the mixture at 3000 g for 10 min and collected diversity in 17 da-jiang samples, which are from different the supernatant again. The above supernatant is mixed and fermented stages and different areas. centrifuged at 12,000 g for 10 min. The resulted precipitate Ann Microbiol (2018) 68:505–512 507 was washed with sterilized water and mixed with 700-μlTE using UCLUST. The highest abundance sequence in each buffer. Total DNA of da-jiang is rapidly extracted using the OTU is selected as the representative sequence. All the anal- CTAB method. Thirty microliter SDS and 3-μl protease K yses were performed using the QIIME program. The phylo- were added into the mixture, which then was incubated at genetic affiliation of each OTU representative sequence was 37 °C for 60 min.
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