Annals of Microbiology (2019)69:279-289 htps/doi.org/10.1007513213-018-1418-z ORIGINAL ARTICLE CrossMark Monitoring the microbial community succession and diversity of Liangzhou fumigated vinegar during solid-state fermentation with next-generation sequencing Jianmin Yun'.Fengqin Zhao1.Wenwei Zhang'.Haijiao Yan'.Fengyun Zhao1.Duiyuan Ai1 Received:22 May 2018/Accepted:3 December 2018/Published online:7 January 2019 degli studi di Milano2019 Abstract This study reveals the microbial community succession and diversity during the whole solid-fermentation processes of naturally fermented Liangzhou fumigated vinegar(LZFV).Dynamics and diversity of microbial community succession in"Daqu"starter and other fermentation stages(starch saccharification,alcoholic fermentation,and acetic acid fermentation)were monitored using a metagenomic approach involving high-throughput sequencing.Meanwhile,dynamic changes of characteristic flavor com- pounds of vinegar were determined by gas chromatog raph(GC)analysis.The result showed that the microbiota composition exhibited rich diversity.Twenty-five bacterial and 18 fungal genera were found in the whole fermentation process where Lactobacillus,Acetobacter,Aspergillus,Saccharomyces,and Alternaria were the predominant microorganisms.Alpha diversity metrics showed that bacterial diversity in Daqu was greater than that in AF and AAF.By contrast,fungal diversity increased from Daqu to AF and decreased in the initial stage(5-8 days)of AAF then remained relatively steady.Hence,these results could help understand dynamics of microbial community succession in continuous fermentation of traditional Chinese vinegars.The LZFV fermentation is a continuous process with spontaneous growth that affects the dynamics of microbial communities.Continuous changes of micro-environment conditions in substrate affect the diversity and structure of microbiota.Microbial growth and metabolism were closely related to the changes in the physicochemical characteristics of the cultures.The microbial flora composition showed rich diversity.and with the increase in brewing time and the change in micro-ecological environmental conditions;the microbial community showed a complex dynamic changes. Keywords Microbial community succession.Diversity.Solid-state fermentation.Liangzhou fumigated vinegar High-throughput sequencing Introduction in Western countries that use only one or a few microor- ganisms to ferment(Zhao and Li 2005).It generally in- As a flavoring agent and an important feedstock,vinega volves preparation of the starter "Daqu"(made from bar- has a long history of over 3000 years in China (Xu et al. ley and pea by spontaneous growth of the microorganisms 2011).Liangzhou fumigated vinegar (LZFV)is one of on/in it),starch saccharification (SS),alcoholic fermenta- Chinese traditional vinegar with local characteristic, tion(AF).and acetic acid fermentation(AAF)(Chen et al. which is naturally fermented by a mixture of various mi- 2009:Wu et al.2013).The special fermentation technol- croorganisms(i.e.,yeasts,molds,and bacteria)in the sol- ogy is also reflected in simultaneous SS and AF after id state (Fig.1).This process differs from similar products inoculating Daqu starter and yeast-in general,this pro- ess lasts 3 days in a closed tank at room temperature- the fermented product is called "Jiupei."This is followed ☒Jianmin Yun by an open-style process of AAF after Jiupei is mixed Yunjianmin@gsau.cdu.cn with some aged vinegar mother"Cupei"-the acetic acid fermentation product)in a fermentation vat,and this con- College of Food Science and Engineering.Gansu Agricultural tinues for about 10 days(Zhao and Yun 2016).This fer- ty.No.I Yin men Village,Anning District, mentation,with its coexistence of various microorgan- isms,provides numerous enzymes for synthesis of flavor ②Springer
ORIGINAL ARTICLE Monitoring the microbial community succession and diversity of Liangzhou fumigated vinegar during solid-state fermentation with next-generation sequencing Jianmin Yun1 & Fengqin Zhao1 & Wenwei Zhang1 & Haijiao Yan1 & Fengyun Zhao1 & Duiyuan Ai1 Received: 22 May 2018 / Accepted: 3 December 2018 /Published online: 7 January 2019 # Università degli studi di Milano 2019 Abstract This study reveals the microbial community succession and diversity during the whole solid-fermentation processes of naturally fermented Liangzhou fumigated vinegar (LZFV). Dynamics and diversity of microbial community succession in BDaqu^ starter and other fermentation stages (starch saccharification, alcoholic fermentation, and acetic acid fermentation) were monitored using a metagenomic approach involving high-throughput sequencing. Meanwhile, dynamic changes of characteristic flavor compounds of vinegar were determined by gas chromatograph (GC) analysis. The result showed that the microbiota composition exhibited rich diversity. Twenty-five bacterial and 18 fungal genera were found in the whole fermentation process where Lactobacillus, Acetobacter, Aspergillus, Saccharomyces, and Alternaria were the predominant microorganisms. Alpha diversity metrics showed that bacterial diversity in Daqu was greater than that in AF and AAF. By contrast, fungal diversity increased from Daqu to AF and decreased in the initial stage (5–8 days) of AAF then remained relatively steady. Hence, these results could help understand dynamics of microbial community succession in continuous fermentation of traditional Chinese vinegars. The LZFV fermentation is a continuous process with spontaneous growth that affects the dynamics of microbial communities. Continuous changes of micro-environment conditions in substrate affect the diversity and structure of microbiota. Microbial growth and metabolism were closely related to the changes in the physicochemical characteristics of the cultures. The microbial flora composition showed rich diversity, and with the increase in brewing time and the change in micro-ecological environmental conditions; the microbial community showed a complex dynamic changes. Keywords Microbial community succession . Diversity . Solid-state fermentation . Liangzhou fumigated vinegar . High-throughput sequencing Introduction As a flavoring agent and an important feedstock, vinegar has a long history of over 3000 years in China (Xu et al. 2011). Liangzhou fumigated vinegar (LZFV) is one of Chinese traditional vinegar with local characteristic, which is naturally fermented by a mixture of various microorganisms (i.e., yeasts, molds, and bacteria) in the solid state (Fig. 1). This process differs from similar products in Western countries that use only one or a few microorganisms to ferment (Zhao and Li 2005). It generally involves preparation of the starter BDaqu^ (made from barley and pea by spontaneous growth of the microorganisms on/in it), starch saccharification (SS), alcoholic fermentation (AF), and acetic acid fermentation (AAF) (Chen et al. 2009; Wu et al. 2013). The special fermentation technology is also reflected in simultaneous SS and AF after inoculating Daqu starter and yeast—in general, this process lasts 3 days in a closed tank at room temperature— the fermented product is called BJiupei.^ This is followed by an open-style process of AAF after Jiupei is mixed with some aged vinegar mother BCupei^—the acetic acid fermentation product) in a fermentation vat, and this continues for about 10 days (Zhao and Yun 2016). This fermentation, with its coexistence of various microorganisms, provides numerous enzymes for synthesis of flavor * Jianmin Yun Yunjianmin@gsau.edu.cn 1 College of Food Science and Engineering, Gansu Agricultural University, No.1 Yingmen Village, Anning District, Lanzhou 730070, Gansu, China Annals of Microbiology (2019) 69:279–289 https://doi.org/10.1007/s13213-018-1418-z
Sta Alcohol Inoculation mash Seed culture 7 (7th days) Microbe Fumigating Corn Fresh Ripening 50% 70℃for sorghum culture amplify (30 days) Saccharification Acetic acid and alcohol Wheat fermentation Water fermentation Bran Rice hull Mature culture Canned -Storing Raw Decoction vinega Fig.1 The traditional brewing processing of the Liangzhou fumigated vinegar and functional substances,such as organic acids,amino acids. Materials and methods (AC and the e et a Sampling sition and Dagu and the original cultures (the solid fermentative substrate of i ZEV)used in this study werecollected from yimin food industry cess.However.the microbial diversity and its dynamic change Company.located in Wuwei,Gansu,China.To monitor the mi- are significant for the quality and characterstics of the fermenta crobial succession and diversity during solid-state fermentation tion products(Chen et al.2013).In order to control the fermen ally collected six cultur nples(1.3,5,8,10,an ring LZFV ays)from in the for eredTI.T3.T5.T8.T10 The culture independent techniaue hased on a next and T13 res ectively.Approximately 200gper sa amole was col generation sequencing system,such as pyrosequencing lected and placed inosterile bluecap bottles and immediately nas been described a a more complete alterative to captur stored inan ice-box.The samples were storedat-80Cuntil used the complexity of th for genomic DNA extraction and physicochemical analysis 2012 ne 20 201 Su oduced usi Physicochemical analysis ntly applied to some famous vin solid-state fermentation technology (Nie et al.2013:Lu An an oximately 10-g sample was homogenized with 30 ml et al.2016;Nie etal.2017).However,it has never been used of de-ionized water,and the pH was measured using a pH to study the microbiota present in LZFV fermentation.Due meter(Mettler Toledo,USA).The total acidity was analyzed to th lack of unders ing of th ction m crobial com on sig sta im h munity success fermentation process 2013) phenolphth n as the nthe work wo sitio dinitr odas described by Miller (199).and function.and succession of microorganisms in different the amino nitrogen was analyzed according to thomas and stages of LZFV fermentation process in order to provide Ingledew (1990).The ethanol concentration in samples was references for related enterprises to improve process control determined by high-performance liquid chromatography ac and vinegar quality. cording to Wu et al.(2013).Gas chromatograph-flame Springer
and functional substances, such as organic acids, amino acids, acetoin (ACT), and the volatile component ligustrazine (He et al. 2004; Xu et al. 2007). Because of the uniqueness and complexity of raw materials, technology, and environmental conditions of LZFV fermentation, it is difficult to study the composition and succession of the microbial community during fermentation process. However, the microbial diversity and its dynamic change are significant for the quality and characteristics of the fermentation products (Chen et al. 2013). In order to control the fermentation process well, and ensure vinegar quality, it is necessary to explore the changes in the microbial community during LZFV fermentation. The culture-independent technique based on a nextgeneration sequencing system, such as pyrosequencing, has been described as a more complete alternative to capture the complexity of the communities present in different fermented products (Roh et al. 2010; Jung et al. 2011; Illeghems et al. 2012; Ercolini 2013). Such a system was recently applied to some famous vinegars produced using solid-state fermentation technology (Nie et al. 2013; Lu et al. 2016; Nie et al. 2017). However, it has never been used to study the microbiota present in LZFV fermentation. Due to the lack of understanding of the function microbial community succession in the traditional fermentation process, vinegar flavor control is still mainly by virtue of experience. So, in the present work, we researched the composition, function, and succession of microorganisms in different stages of LZFV fermentation process in order to provide references for related enterprises to improve process control and vinegar quality. Materials and methods Sampling Daqu and the original cultures (the solid fermentative substrate of LZFV)usedinthisstudywerecollected fromYiminFood Industry Company, located in Wuwei, Gansu, China. To monitor the microbial succession and diversity during solid-state fermentation, we periodically collected six culture samples (1, 3, 5, 8, 10, and 13 days) from the same location. All these culture samples were collected at a depth of approximately 25 cm fromthe upper surface in the fermentation vat, and were numbered T1, T3, T5, T8, T10, and T13, respectively. Approximately 200 g per sample was collected and placed into sterile blue-cap bottles and immediately stored in an ice-box. The samples were stored at−80 °C until used for genomic DNA extraction and physicochemical analysis. Physicochemical analysis An approximately 10-g sample was homogenized with 30 ml of de-ionized water, and the pH was measured using a pH meter (Mettler Toledo, USA). The total acidity was analyzed by titration using standardized solution (0.1 M sodium hydroxide) with phenolphthalein as the indicator (Nie et al. 2013). Residual sugar amount was estimated using the 3,5- dinitrosalicylate method as described by Miller (1959), and the amino nitrogen was analyzed according to Thomas and Ingledew (1990). The ethanol concentration in samples was determined by high-performance liquid chromatography according to Wu et al. (2013). Gas chromatograph-flame Fig. 1 The traditional brewing processing of the Liangzhou fumigated vinegar 280 Ann Microbiol (2019) 69:279–289
Am Micobiol(201969279-289 281 detector(Agilent Techologies,USA)analysisafte sample were pooled and purified p to py cing azine (TTMP),and was applied to a Genome Sequencer FLX454 Titanium ethylacetate (EA)according to Richter et al.2013 and Zhao System(454 Life Sciences.Branford.Connecticut,USA) and Yun 2016. (Liu et al.2015:Polka et al.2015). Microbial viable cell count of the main functiona Multivariate statistical analyses The co mmunity-s rted software package v1.110 (Schlos The five functional microbial communities (including acetic acid bacteria,lactic acid bacteria,heat-resistant bacteria. munities and systematic layout planning methodology was yeasts,and molds)viable cell count were carried out accord- used to analyze the number of sequences assigned to eacl ing to the method of Zhao (2016) cluster(regarded as Operational Taxonomic Units,OTUs) DNA extraction and PCR amplification To des ce. Samples were homogenized using liquid nitrogen (flash-fro 1949)and Shannon-Weaver index (Haruta et al.2006) zen in liquid nitrogen and then rapidly thawed in a water-bath Principal component analysis was then performed to group at 65C for 2 min,and repeated three times).and approxi- microorganisms using Canoco for Windows v4.5 software was ther (Wageningen U Neth Venn diagrams were drawr 0 m d hierarch atmaps using 0-catmap s wa polypyrrolidone (PVPP.).pH 8.0].CTAB was use remove polysaccharides from vinegar samples,and PVPP was used for polyphenolic component adsorption. Tota Results DNA was de amlouk Nie et al.2013). Dynamic cha on DE.USA)at the OD260/OD280 ratios of 1.7-2.0.DNA integrity was assessed Dynamic chan s in physicochemical properties during the using agarose gel electrophoresis. whole fermentation are shown in Fig.2.During LZFV fer wo pairs of universal primers wer resp ctively used mentation,the temperature changed significantly.In the first to ampl lify th ba 4 regio 16S rDNA and the 28( AF), ed slowly from 22.6 c.t 43 ed to 329 C Con entration of residual s dually in amplify bacteria 16S rDNA.and ITSIF (5'-CTTG in the first 3 days:ethanol content in samples also constantly GTCATTTAGAGGAAGTAA-3)and 2043R(5'-GCTG rose and reached a maximum (6.9%.)at 3 days.The con- CGIICIICATCGTGC-3)to amplity the fungal ITS re tent of total acidity increas I rapidly from 0.45%(whw)in The P (25l)c reactio 04.58%at13da s.I he free amino nitroge te mM dN dually d .66to3.18.AAF of PCR were assessed by elec ophoresis on 1%(w/v)agarose gel. unds ofvinegar Temnerature nH ethanol total acidity and residual sugar were the key physicochemical metrics to mon Pyrosequencing itor AAF (Fig.2).The characteristic flavor compound 0 ACT,TTMP,an EA)ha significant The were a TTMP manufacturer's ol The purified at 3 davs of for tation.and then radually decreased fied using a spectrophotometer (NanoDrop Technolo) However.TTMP and EA continuously accumulated during Next,equal amounts (100 ng)of amplicons from different the whole fermentation process. 鱼Springe
ionization detector (Agilent Technologies, USA) analysis after headspace solid-phase micro extraction (Supelco, USA) was used to determine ACT, tetramethylpyrazine (TTMP), and ethylacetate (EA) according to Richter et al. 2013 and Zhao and Yun 2016. Microbial viable cell count of the main functional communities The five functional microbial communities (including acetic acid bacteria, lactic acid bacteria, heat-resistant bacteria, yeasts, and molds) viable cell count were carried out according to the method of Zhao (2016). DNA extraction and PCR amplification Samples were homogenized using liquid nitrogen (flash-frozen in liquid nitrogen and then rapidly thawed in a water-bath at 65 °C for 2 min, and repeated three times), and approximately 500 mg of sample was then added to the extraction buffer [100 mM Na3PO4, 100 mM Trise HCl, 100 mM ethylene diaminetetraacetic acid, 1.5 M NaCl, 1% cetyltrimethyl ammoniumbromide (CTAB, w/v), 2% polyvinylpolypyrrolidone (PVPP, w/v), pH 8.0]. CTAB was used to remove polysaccharides from vinegar samples, and PVPP was used for polyphenolic component adsorption. Total DNA was extracted according to previously described methods (Mamlouk et al. 2011; Nie et al. 2013). All DNA concentrations were determined using a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at OD260/OD280 ratios of 1.7–2.0. DNA integrity was assessed using agarose gel electrophoresis. Two pairs of universal primers were respectively used to amplify the bacterial V4 region of 16S rDNA and the fungal internal transcribed spacer (ITS) region: primers 520F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) were used to amplify bacteria 16S rDNA, and ITS1F (5′-CTTG GTCATTTAGAGGAAGTAA-3′) and 2043R (5′-GCTG CGTTCTTCATCGTGC-3′) to amplify the fungal ITS region. The PCR mixture (25 μl) contained 5 × Q5 reaction buffer, 5 × Q5 GC high enhancer, 2.5 mM dNTP, 5 ng of extracted total DNA, 10 μM of each primer and 1.25 U of Q5 polymerase. The sizes of PCR products were assessed by electrophoresis on 1% (w/v) agarose gel. Pyrosequencing The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The purified products were quantified using a spectrophotometer (NanoDrop Technologies). Next, equal amounts (100 ng) of amplicons from different samples were pooled and purified prior to pyrosequencing using the ethanol precipitation method. The amplicon mixture was applied to a Genome Sequencer FLX454 Titanium System (454 Life Sciences, Branford, Connecticut, USA) (Liu et al. 2015; Polka et al. 2015). Multivariate statistical analyses The community-supported software package v1.11.0 (Schloss et al. 2009) was used to describe and compare microbial communities and systematic layout planning methodology was used to analyze the number of sequences assigned to each cluster (regarded as Operational Taxonomic Units, OTUs). To describe microbial diversity (including dominance, richness, and equitability), based on OTU richness, we calculated biodiversity indices: Chao1, ACE, Simpson index (Simpson 1949) and Shannon–Weaver index (Haruta et al. 2006). Principal component analysis was then performed to group microorganisms using Canoco for Windows v4.5 software (Wageningen UR, Netherlands). Venn diagrams were drawn using Venny 2.0.2, and hierarchical clustering analysis was performed to draw heatmaps using Hemi 1.0.3.7-Heatmap Illustrator. Results Dynamic changes in physicochemical characteristics during the whole fermentation Dynamic changes in physicochemical properties during the whole fermentation are shown in Fig. 2. During LZFV fermentation, the temperature changed significantly. In the first 3 days (the AF), temperature increased slowly from 22.6 °C to 28.3 °C, followed by the AAF when temperature rose rapidly from 28.3 °C to 45.2 °C; after 8 days, it gradually fell to 32.9 °C. Concentration of residual sugar gradually increased in the first 3 days; ethanol content in samples also constantly rose and reached a maximum (6.9%, v/v) at 3 days. The content of total acidity increased rapidly from 0.45% (w/w) in original culture to 4.58% at 13 days. The free amino nitrogen also increased throughout the whole fermentation but pH gradually decreased from 4.66 to 3.18. AAF is a critical stage for production of organic acids, which are the dominant compounds of vinegar. Temperature, pH, ethanol, total acidity, and residual sugar were the key physicochemical metrics to monitor AAF (Fig. 2). The characteristic flavor compounds of LZFV (such as ACT, TTMP, and EA) had significant differences at the different stages of fermentation—among them, ACT, as a TTMP precursor, reached a peak of 1.46 g/100 g at 3 days of fermentation, and then gradually decreased. However, TTMP and EA continuously accumulated during the whole fermentation process. Ann Microbiol (2019) 69:279–289 281
282 301 1 r5.0 Total acidity(TA) -Residual sugart(RS】 4.5 nH tota 4.0 suga 35 and 3 25 15 10 .05 15 T3 6 (FAN) 20- EA) (TTMP 1.4 08- 04 02 0.0 , nctional microbia howed that the numb rof molds brewing process sed Du the first 5 da of for ntation the yeast had a small increase with the prolongation of fermenta In order to understand the influence of major functional mi- tion time.It reached the maximum on the fifth day,and then crobial communities on quality of vinegar well,the five fune- decreased.The presence of yeast was almost undetectable robial communities (including acetic acid bacte fter the tenth day.Howe ver,the number of bactena reache ae actena,heat-res (in Fig 3). 0n eigntn day. ong th m.acetic ≌Springer
Dynamic changes of main functional microbial communities cell counts in Liangzhou vinegar brewing process In order to understand the influence of major functional microbial communities on quality of vinegar well, the five functional microbial communities (including acetic acid bacteria, lactic acid bacteria, heat-resistant bacteria, yeasts, and molds) viable cell count were carried out (in Fig. 3). The results showed that the number of molds was the highest on the first day of fermentation, and gradually decreased as the fermentation progressed. During the first 5 days of fermentation, the yeast had a small increase with the prolongation of fermentation time. It reached the maximum on the fifth day, and then decreased. The presence of yeast was almost undetectable after the tenth day. However, the number of bacteria reached the maximum on the eighth day. Among them, acetic acid bacteria, lactic acid bacteria, and heat-resistant bacteria T1 T3 T5 T8 T10 T13 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 O O O O O O Time( day ) Temperature O Ethanol Total acidity( TA ) Residual sugar( RS ) pH The concentration of Ethanol( %,v/v )/TA( g/100g ) pH/The concentration of RS( g/100g ) T1 T3 T5 T8 T10 T13 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time( day ) Free amino nitrogen ( FAN ) Acetoin ( ACT ) Tetramethylpyrazine ( TTMP ) Ethyl acetate ( EA ) a bTemperature ć The concentration of /FAN/ACT/TTMP/EA( g/100g ) Fig. 2 Dynamic changes in physicochemical characteristics during LZFV fermentation. a described the dynamic changes in the key physicochemical metrics, including temperature, pH, total acidity, residual sugar, and ethanol concentration during LZFV fermentation. b described the characteristic flavor compounds (free amino nitrogen, acetoin, tetramethylpyrazine, and ethylacetate) of vinegar during LZFV fermentation 282 Ann Microbiol (2019) 69:279–289
Amn Microbiol (2019)67 283 8二Ac entation.Durin tolerate the high ethanol concentration died.In the AAF process,the acidogenic bacteria and extreme microorgan- isms grew rapidly and organic acids quickly accumulated and so bacteria with a low tolerance of acetic acid disap 4 er.fungal dive sity showed a different trend the sample with the most phylotypes was the transition period(5 days).but the initial (I day)and final fermenta- Tie Ti3 same trend.Thu d s tabod this gradually increased in the late stage of fermentation.and they were becoming the dominant communities.These were relat- Dynamics and succession of microbial community during whole fermentation ed to the accumulation of metabolites and changes in pH. Changes of microbial diversity in the whole fermentation ted (Figs.4 and 5). The dynamic changes in the microbial community dur The diversities of bacterial and fungal communities dur- ing the whole fermentation of LZFV were investigated ing the whole LZFV fermentation were systematically in- periodically.It showed that the microbial community (a composition dynamically changed-every sample hac owed th also ha some common OTU: d mples. and that all rich(413 and 388 OTUs)in matched the true diversity of LZFV.The Chaol analysis indicated that the sample in the early fermentation period (T1);however,for samples during the AAF process (T5 (1 day)consisted of more phylotypes than other pha T8,and T10).OTUs gradually declined(111-143 OTUs) nd the sample the transition period (3 days) nd then remained rel .Ioward OTU he end o the least mcrbial 0n, e trend in a terial div ty.This sho ed that most undanc (>50%)in Daqu and the TI sar le and bacteria oud adapt to the cuure nironment on the mon genera represented <10%.In contrast.common Sample ID 0.97 ACE Chaol Shannon Simpson Coverag 2423 43 0976 0s717 18153 16 2 0.6214 0998 T8 15809 111 222 13 1.1 0.4499 0.9973 19991 143 214 1.24 0.4162 0.9976 T13 17615 239 298 226 138 0.3733 0.9962 Values are the mes of three replicates Springer
gradually increased in the late stage of fermentation, and they were becoming the dominant communities. These were related to the accumulation of metabolites and changes in pH. Changes of microbial diversity in the whole fermentation The diversities of bacterial and fungal communities during the whole LZFV fermentation were systematically investigated (Tables 1 and 2). The alpha diversity metrics (Tables 1 and 2) showed the richness and evenness of microbiota in vinegar samples. The four alpha diversity metrics were computed to ensure that all metrics closely matched the true diversity of LZFV. The Chao1 analysis indicated that the sample in the early fermentation period (1 day) consisted of more phylotypes than other phases, and the samples in the transition period (3–5 days) had the least microbial population among all samples. Shannon, Simpson and ACE metrics also indicated the same trend in bacterial diversity. This showed that most bacteria could adapt to the culture environment on the first day of fermentation. During the transition period, as ethanol gradually accumulated, bacteria that could not tolerate the high ethanol concentration died. In the AAF process, the acidogenic bacteria and extreme microorganisms grew rapidly and organic acids quickly accumulated, and so bacteria with a low tolerance of acetic acid disappeared with longer times of fermentation—causing a decline in microbial diversity. However, fungal diversity showed a different trend— the sample with the most phylotypes was the transition period (5 days), but the initial (1 day) and final fermentation period (13 days) samples had small populations. Shannon, Simpson, and ACE metrics also indicated the same trend. Thus, the transition period showed this environment was suitable for growth and metabolism of most fungi participating in fermentation. Dynamics and succession of microbial community during whole fermentation To evaluate the contribution of OTUs among the different cultures of LZFV fermentation, Venn diagrams were constructed (Figs. 4 and 5). The dynamic changes in the microbial community during the whole fermentation of LZFV were investigated periodically. It showed that the microbial community composition dynamically changed—every sample had some specific OTUs and also had some common OTUs to others. Bacterial components were rich (413 and 388 OTUs) in Daqu and the sample for the initial period of fermentation (T1); however, for samples during the AAF process (T5, T8, and T10), OTUs gradually declined (111–143 OTUs) and then remained relatively steady. Toward the end of fermentation, the number of OTUs also gradually increased. Specific bacterial genera had higher relative abundance (> 50%) in Daqu and the T1 sample, and common genera represented < 10%. In contrast, common T1 T3 T5 T8 T10 T13 0 1 2 3 4 5 6 7 8 9 Acetic acid bacteria Lactic acid bacteria Heat-resistant bacteria Yeasts Molds Microbial viable cell count (lgCFU/g) Time (day) Fig. 3 Succession of major functional microbial communities during the fermentation process Table 1 Bacterial community diversity metrics (distance < 0.03) during LZFV fermentation Sample ID Reads 0.97 OTU ACE Chao1 Shannon Simpson Coverage DQ 24128 413 297 275 2.44 0.1942 0.9976 T1 25516 388 323 308 3.04 0.1093 0.9977 T3 12812 141 147 137 0.46 0.8717 0.9969 T5 18153 116 133 112 0.83 0.6214 0.9981 T8 15809 111 222 137 1.11 0.4499 0.9973 T10 19991 143 214 164 1.24 0.4162 0.9976 T13 17615 239 298 226 1.38 0.3733 0.9962 Values are the means of three replicates Ann Microbiol (2019) 69:279–289 283
284 Table 2 Fungal c Sample ID Reads 0.97 OTU ACE Chaol Shannon Simpson Coverage DO 28380 07 96 107 0.2288 0.9993 25350 0 126 1.65 0.4196 0.9991 T3 3048 124 122 2.08 0.295 0.9993 T5 3477 195 19s 1.49 0.561 0.999 30 T13 2182 103 2.4 02215 0.9999 cates fungal genera had relative abundance >45%and specific Analysis of microbial composition and community genera%in Daqu,and the relative abundance of spe- structure in the whole fermentation cific genera in each sample was15%.This phenomenon that co an fo of the ba s. genera of >0.1%are shown in. ndance of al fermentation.Bacteria in Daqu also participated in the The microbial community structure analysis using high fermentation.but were not the dominant throughput sequencing showed a more diverse bacterial com- microorganisms- -the do ninant bacteria in the fermenta- munity compared with the fungal community in the LFV tion mainly came from the "vinegar mother"and the ex- ernal environment )The Daqu wa Fig.4 Venn diag T1 a t LZFV fe es (a- 17 252 206 fied at the 2 14 ates超 c 53 35 137 hd个 Springer
fungal genera had relative abundance > 45% and specific genera 0.1% are shown in Fig. 4. The microbial community structure analysis using highthroughput sequencing showed a more diverse bacterial community compared with the fungal community in the LZFV process. The bacteria changed more quickly than fungi (Table 3 and Fig. 6). The microbial community of Daqu was a b c Fig. 4 Venn diagrams showing specific and common OTUs of bacterial communities (a–c) during the LZFV fermentation. Samples were classified at the genus level. a Represents the contribution of OTUs (bacterial genus level) in Daqu starter (DQ) and the initial fermentation stage of LZFV fermentation process (T1, the 1st day culture sample; T3, the 3rd day culture sample). b Represents the contribution of OTUs (bacterial genus level) in the intermediate stage of fermentation during LZFV fermentation process (T3, the third day culture sample; T5, the fifth day culture sample; T8, the eighth day culture sample). c Represents the contribution of OTUs (bacterial genus level) in the last stage of fermentation during LZFV fermentation process (T8, the eighth day culture sample; T10, the tenth day culture sample; T13, the 13th day culture sample) Table 2 Fungal community diversity metrics (distance < 0.03) during LZFV fermentation Sample ID Reads 0.97 OTU ACE Chao1 Shannon Simpson Coverage DQ 28380 80 97 96 1.92 0.2288 0.9993 T1 25350 107 124 126 1.65 0.4196 0.9991 T3 30482 121 124 122 2.08 0.2951 0.9998 T5 34777 164 195 195 1.45 0.5614 0.9990 T8 29358 154 154 153 1.97 0.3590 0.9997 T10 30447 136 136 136 2.50 0.2415 0.9998 T13 21182 102 103 102 2.49 0.2215 0.9999 Values are the means of three replicates 284 Ann Microbiol (2019) 69:279–289
Ann Microbiol(2019169279-289 285 TUs of T3 a b 19 at the 30 (fung re s of OTUs (funga T10 o:T8 30 29 71 hth dav T13 Bacillu. the relative abundance of Acetobacter increased as AAF dominant bacterial genera and had relative abundance>5% progressed,indicating that they were the predominant fune The TI sample also had multitudinous bacteria-it contained tional microorganisms for AAF of vinegar fermentation. 5 genera,except bacterial composition was The 18S-26S rDNA ITS region analysis showed that the of 。h ie in T3.indicating that 286)The p was when ss Lactobacillus were the major functional mic and AF were carried out simultaneously.so Asperillus and the AF process.With increasing time.the relative abundance Saccharomyces were the dominant fungi and the total relative of Lactobacillus was gradually reduced and that of abundance of the two genera exceeded 50%:TI was 52.89 Acetobacter gradually increased.During the AAF.total and T3 was 70.78%.In addition.Alternaria.Monascus. Table3 The microbial Sample Bacteria Fungi diacttaoooamcge Phylum Class Order Family Genus Phylum Class Order Family Genu DO 6 12 20 42 55 4 11 2 3 TI 12 20 31 57 84 4 9 T3 13 22 27 4 4 22 27 4 24 3010 666 88I 361 23 444 456 292 446 66 Springer
composed of 24 genera—Streptomyces, Bacillus, Thermoactinomyces, Lactobacillus, and Erwinia were the dominant bacterial genera and had relative abundance > 5%. The T1 sample also had multitudinous bacteria—it contained 25 genera, except Geobacillus, its bacterial composition was the same as Daqu, and there was no clearly dominant bacterial genus among them. Lactobacillus was the dominant bacterial genus with relative abundance highest in T3, indicating that Lactobacillus were the major functional microorganisms in the AF process. With increasing time, the relative abundance of Lactobacillus was gradually reduced and that of Acetobacter gradually increased. During the AAF, total abundance of Lactobacillus and Acetobacter was > 90%— the relative abundance of Acetobacter increased as AAF progressed, indicating that they were the predominant functional microorganisms for AAF of vinegar fermentation. The 18S-26S rDNA ITS region analysis showed that the dominant members of the fungal community in Daqu were Aspergillus (35.67%), Thermoascus (40.96%), and Monascus (12.86%). The period of 1–3 days was when SS and AF were carried out simultaneously, so Aspergillus and Saccharomyces were the dominant fungi and the total relative abundance of the two genera exceeded 50%: T1 was 52.89% and T3 was 70.78%. In addition, Alternaria, Monascus, a b c Fig. 5 Venn diagrams showing specific and common OTUs of fungal communities (a–c) during LZFV fermentation. Samples were classified at the genus level. a Represents the contribution of OTUs (fungal genus level) in Daqu starter (DQ) and the initial fermentation stage of LZFV fermentation process (T1, the first day culture sample; T3, the third day culture sample). b Represents the contribution of OTUs (fungal genus level) in the intermediate stage of fermentation during LZFV fermentation process (T3, the third day culture sample; T5, the fifth day culture sample; T8, the eighth day culture sample). c Represents the contribution of OTUs (fungal genus level) in the last stage of fermentation during LZFV fermentation process (T8, the eighth day culture sample; T10, the tenth day culture sample; T13, the 13th day culture sample) Table 3 The microbial composition of samples at different taxonomic levels Sample Bacteria Fungi Phylum Class Order Family Genus Phylum Class Order Family Genus DQ 6 12 20 42 55 4 11 21 28 43 T1 12 20 31 57 84 4 15 29 39 56 T3 5 7 13 22 27 4 18 32 46 64 T5 5 8 14 22 27 4 18 34 45 69 T8 6 8 13 21 28 4 14 28 43 66 T10 6 8 16 28 37 4 15 29 41 64 T13 6 11 21 43 56 4 16 27 36 54 Ann Microbiol (2019) 69:279–289 285
286 DO 8 T10 DO TI T3 T5 T8 T10 T13 Themoascus and wickerlamonnes also had higher contents grew ranidl-so samnles of this neriod contained not only eth and their relative abundances were>1%.The relative abundance anol but also acetic acid (Fig.2).The last stage was during 8 of Aspergillus,Saccharomyces,and Wickerhamonyces sharply 13 days,when the microbial community was almost stable,al- decreased after 3 days of fermentation indicating that these three though the acetic acid content consistently increased (Fig.2). was basically stable（e roximately 556)and Epicocc Mortere Cladospori Coc Discussion Monascus,and Wallemia appeared or their relative abundance ncreased- thus,we conjectured that they were the predominant functional microorganisms in the AAF process nyc Hierarchical cluster analysis of microorganisms etal.2013.2017).Xu monitored the microbial community during in the whole fermentation solid-state AAF of Zhengjiang aromatic vinegar using PCR. DGGE,and showed that there were 12 genera of bacteria and four ing the whole termentation, gehera were prod an Taniin Duliu adiesinytrmgCR DGGEanddcotfiedi8 tion of Dagu and TI had ome similarity those for and Ts fungi (Nie t al 2013).Nie detected 22 bacterial were identical,and those among T8.T10.and T13 were very genera in a study of the microbial community succession during similar.The samples of the AF process(T1 and T3)had similar the fermentation ofShanxiaged vinegar (Nie etal 2017). d the samples of AAF(T5 Our r lore the L during solid first stage was 3 days.with the t and high thm pout ofdata (2014) isms being aspergillus and The second stag Results showed that every sample contained more than 27 was3-8 days,and this was a preparatory phase of AAF.In this bacterial and 43 fungal genera,indicating that more microor stage,microbial composition changed quickly,many functiona ganisms were detected by high-throughput sequencing than microorganisms of AF weakened and those of AAF appeared or by PCR-DGGE.Therefore,obtaining microbial diversity ≌Springe
Thermoascus, and Wickerhamomyces also had higher contents and their relative abundances were > 1%. The relative abundance of Aspergillus, Saccharomyces, and Wickerhamomyces sharply decreased after 3 days of fermentation indicating that these three genera were the predominant functional microorganisms for the SS and AF processes. During the AAF, the relative abundance of Alternaria was basically stable (approximately 55%) and Epicoccum, Mortierella, Cladosporium, Cochliobolus, Monascus, and Wallemia appeared or their relative abundance increased—thus, we conjectured that they were the predominant functional microorganisms in the AAF process. Hierarchical cluster analysis of microorganisms in the whole fermentation Hierarchical cluster analysis was used to explore the changes in different microorganisms during the whole fermentation, and heatmaps of bacterial and fungal genera were produced (Fig. 7a and b, respectively). The bacterial community composition of Daqu and T1 had some similarity, those for T3 and T5 were identical, and those among T8, T10, and T13 were very similar. The samples of the AF process (T1 and T3) had similar fungal community composition, and the samples of AAF (T5, T8, T10, and T13) had identical fungal community composition. There were three stages during the LZFV fermentation. The first stage was 1–3 days, with the major functional microorganisms being Aspergillus and Saccharomyces. The second stage was 3–8 days, and this was a preparatory phase of AAF. In this stage, microbial composition changed quickly, many functional microorganisms of AF weakened and those of AAF appeared or grew rapidly—so samples of this period contained not only ethanol but also acetic acid (Fig. 2). The last stage was during 8– 13 days, when the microbial community was almost stable, although the acetic acid content consistently increased (Fig. 2). Discussion Many microbiological studies have been performed to examine the process oftraditionalChinese vinegar fermentation by cultureindependent techniques, especially PCR-denaturing gradient gel electrophoresis (DGGE) (Haruta et al. 2006; Xu et al. 2011; Nie et al. 2013, 2017). Xu monitored the microbial community during solid-state AAF of Zhengjiang aromatic vinegar using PCRDGGE, and showed that there were 12 genera of bacteria and four genera of fungi during the fermentation process (Xu et al. 2011). Nie researchedthemicrobialsuccessionanddiversityduringsolidstate fermentation of Tanjin Duliu mature vinegar using PCRDGGE, and identified 18 genera of bacteria and eight genera of fungi (Nie et al. 2013). Nie detected 22 bacterial and six fungal genera in a study of the microbial community succession during the fermentation of Shanxi aged vinegar (Nie et al. 2017). Our research explored the microbial succession and diversity during solid-state fermentation of LZFV by highthroughput sequencing, a revolutionary technology characterized by low cost and high-throughput of data (Zhu et al. 2014). Results showed that every sample contained more than 27 bacterial and 43 fungal genera, indicating that more microorganisms were detected by high-throughput sequencing than by PCR-DGGE. Therefore, obtaining microbial diversity Fig. 6 Phylogenetic taxonomy for bacterial (a) and fungal (b) dynamic changes at the genus level during LZFV fermentation. All genera are shown in Fig. 6, which the relative taxonomic abundance are more than 0.1% 286 Ann Microbiol (2019) 69:279–289
Ann Microbiol(2019169279-289 Other Alternaria Corvnehacterium Saccharopolyspora Streptomyces Cladosporium Chryseobacterium Cochliobolus Monascus Monographella Thermoactinomyces actobacillus haeosphaeriaceae Pleosporaceae accharomyces hermoascus luconobacter Klebsiella Wallemia Acinetobacter Wickerhamomyces seudomonas Ignatzschineria 5.052140.773.68 412-1620893.38 ms in whole fer Red represents otive and black information in Daqu will help us to understand its impact on continuously present in Daqu,and they contribute to the evo the quality of vinegar products.Like many other Daqu types. lution offlavor and activities ofenzymes such as amylases and heng ct a most species in these genera are resistant to high temperature. was enhanced significantly with increasing of TTMP(Han Many Streptomyces strains can be used for isolating enzymes 2013).Bacillus is the main genus involved in TTMP produc such as a-galactosidases which can hydrolyze raffinose family tion(Larroche et al.1999 Xiao et al.2009 Zhu et al.2010) ainly ramlinose and stachyose,ant Ihe Tungi neede for LZF tation 20107 ly one genus, nging detected in high ter ature dagu samples (Zhu et al od and showeda rend.Asr rgillus (35.67 2014-Yaoet al 2014 2015)that exhibit high enzyme produc Thermoascus (40 96)and Monascus (12 86)were tion,such as of cutinolytic esterase and alkaline protease(Fett regnant in the LZFV Daqu fungal community.This was et al.2000:Divakar et al.2006).Bacillus was reported to be very different for Shanxi-Daqu (Wu et al.2004:Chen 鱼Springe
information in Daqu will help us to understand its impact on the quality of vinegar products. Like many other Daqu types, Liangzhou-Daqu is made in a hot environment (35–60 °C). We found that the dominant bacterial genera in Daqu were Streptomyces, Bacillus, Thermoactinomyces, and Erwinia— most species in these genera are resistant to high temperature. Many Streptomyces strains can be used for isolating enzymes such as ɑ-galactosidases which can hydrolyze raffinose family oligosaccharides—mainly raffinose and stachyose, antinutritional factors in legumes of soybean—to increase nutritional value (Cao et al. 2010). Thermoactinomyces have been detected in many high-temperature Daqu samples (Zhu et al. 2014; Yao et al. 2014, 2015) that exhibit high enzyme production, such as of cutinolytic esterase and alkaline protease (Fett et al. 2000; Divakar et al. 2006). Bacillus was reported to be continuously present in Daqu, and they contribute to the evolution of flavor and activities of enzymes such as amylases and proteinases (Zheng et al. 2011) in vinegars and liquors. Our previous study showed that TTMP was the characteristic flavor compound of LZFV, and that flavor of the vinegar product was enhanced significantly with increasing of TTMP (Han 2013). Bacillus is the main genus involved in TTMP production (Larroche et al. 1999; Xiao et al. 2009; Zhu et al. 2010). The fungi needed for LZFV fermentation almost all came from Daqu—only one unclassified genus, belonging to Phaeosphaeriaceae, appeared in the fermentation period and showed a rising trend. Aspergillus (35.67%), Thermoascus (40.96%), and Monascus (12.86%) were regnant in the LZFV Daqu fungal community. This was very different for Shanxi-Daqu (Wu et al. 2004; Chen Fig. 7 Hierarchical cluster analysis of microorganisms in whole fermentation. Red represents positive and black represents negative; the gradient from blue to red represents the microbial relative abundance gradually rising Ann Microbiol (2019) 69:279–289 287
28 et al.2009)and Duliu-Dagu (Nie et al.2013)with diversity and structure of microbiota.These results will be Saccharomyces more abundant,but Thermoascus less quite useful for understanding the relationshin between mi abundant.The different microbial flora might be the main croorganisms and the characters or quality of vinegar.Thus reason why the flavor characteristics of the vinegars dif- we can conclude that the traditional solid-state fermentation fer.For LZFV fermentation,we found that Aspergillus process of LZFV is extremely complex.Its essence is the the SS bacillu. microbial community in in the AAF process,and Lactobacillus had the highest relative abundance in the early fermentation period and declined in the AAF stage casodas5acaand ew Many mic ganisms that Compliance with ethical standards could not tolerate high alcohol died and competition abated. so that Lactobacillus could grow quickly.However,after al- cohol accumulated,Cupei was added to the vat witha abundan ited the would anisn other micr and manual edonce daily ygen lim rapidly.and acetic acid accumulated most at the end of fer mentation.This explains why the relative abundance of Acetobacter was low at 3 days and increased after 5 days These results are consistent with the LZFV fermentation pro cess (An 201) References tion h casts and affect both the technological p microbial stability of the final products through production of Zhou ZG lactic acid (Yousifet al.2010).Our research also showed that a-gct abundanc 147 c2010.09.007 ntribute to in in the precursor of esters in fermentation (Li2014).and were the flavor-forming bacteria during the aaf process.in addi QS. (2013)Mon tion,A/ternaria was also found during the entire process and its relative abundance was stable during the AAF process,but er P.Ellaiah P(2006) ear.It was so det ss par der Ercolini)High-th cing and me Conclusions on Micmbiol 9:3143155. The present wor Fett WE,Wijey C.Mo was iny g hut alse of vinegar fermentation.The LZFV fermentation is a con- dvinegar.Master.Gnsu Agri Univ tinuous process with spontaneous growth that affects the dynamics of microbial communities.Continuous changes E.NaganoM.rh of micro-environment conditions in substrate affect the raditional pot fermentation of rice vinegar assessed by PCR Springer
et al. 2009) and Duliu-Daqu (Nie et al. 2013) with Saccharomyces more abundant, but Thermoascus less abundant. The different microbial flora might be the main reason why the flavor characteristics of the vinegars differ. For LZFV fermentation, we found that Aspergillus, Saccharomyces, Wickerhamomyces, and Lactobacillus played important roles in the SS and AF process, Acetobacter were predominant functional microorganisms in the AAF process, and Lactobacillus had the highest relative abundance in the early fermentation period and declined in the AAF stage. As used for Shanxi-aged vinegar, AF was carried out in a static and anoxic environment suitable for the survival of Saccharomyces and Lactobacillus. Alcohol gradually increased as Saccharomyces grew. Many microorganisms that could not tolerate high alcohol died and competition abated, so that Lactobacillus could grow quickly. However, after alcohol accumulated, Cupei was added to the vat with abundant other microorganisms and manually stirred once daily—this process would introduce oxygen. The increased oxygen limited the growth of Lactobacillus, so aerobic Acetobacter grew rapidly, and acetic acid accumulated most at the end of fermentation. This explains why the relative abundance of Acetobacter was low at 3 days and increased after 5 days. These results are consistent with the LZFV fermentation process (An 2011). Lactobacillus populations played a strong role in improving the taste of vinegar. They provide substrates for esterification by yeasts and affect both the technological properties and microbial stability of the final products through production of lactic acid (Yousif et al. 2010). Our research also showed that abundance of Bacillus, Solibacillus, and Lactococcus increased at the end of the fermentation stage, and these are related to flavor-forming. Bacillus is involved in the synthesis of ACT and TTMP. Solibacillus might contribute to increases in the precursor of esters in fermentation (Li 2014), and were the flavor-forming bacteria during the AAF process. In addition, Alternaria was also found during the entire process and its relative abundance was stable during the AAF process, but its function remains unclear. It was also detected during the whole fermentation of Shanxi-aged vinegar (Nie et al. 2017). Conclusions The present work revealed that a complex microbial community was involved in spontaneously fermented LZFV. Not only naturally selected species gradually grew and reproduced in the fermentation process, but also promoters of vinegar fermentation. The LZFV fermentation is a continuous process with spontaneous growth that affects the dynamics of microbial communities. Continuous changes of micro-environment conditions in substrate affect the diversity and structure of microbiota. These results will be quite useful for understanding the relationship between microorganisms and the characters or quality of vinegar. Thus, we can conclude that the traditional solid-state fermentation process of LZFV is extremely complex. Its essence is the microbial community succession and a series of biochemical reactions caused by them, to which vinegar flavor formation is closely related. Funding information This study was supported by the Ministry of Science and Technology, P. R. China, under the Nature Science Foundation of China (NSFC) (NO. 31360405), and the Gansu Province Key Laboratory of Food Science and Technology, Gansu Agricultural University, is gratefully acknowledged. Compliance with ethical standards Conflict of interest All authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References An ZG (2011) Microbial succession and metabolism during traditional brewing process of Liangzhou fumigated vinegar. Master degree dissertation. Gansu Agri Univ, Lanzhou (In Chinese) Cao YN, Yuan TZ, Shi PJ, Luo HY, Li N, Bai YG, Yang PL, Zhou ZG, Zhang ZF, Yao B (2010) Properties of a novel α-galactosidase from Streptomyces sp. S27 and its potential for soybean processing. Enzym Microb Technol 47:305–312. https://doi.org/10.1016/j. enzmictec.2010.09.007 Chen FS, Li L, Qu J, Chen CX (2009) Cereal vinegars made by solid-state fermentation in China. In: Solieri L, Giudici P (eds) Vinegars of the world. Springer, Milan, pp 243–259 Chen QS, Liu AP, Zhao JW, Qin OY (2013) Monitoring vinegar acetic fermentation using a colorimetric sensor array. Sensors Actuators B Chem 183:608–616. https://doi.org/10.1016/j.snb.2013.04.033 Divakar G, Sunitha M, Vasu P, Udayashanker P, Ellaiah P (2006) Optimization of process parameters for alkaline protease production under solid-state fermentation by thermocline happily PEE 14. Indian J Biotechnol 5:80–83 Ercolini D (2013) High-throughput sequencing and metagenomics: moving forward in the cultural-independent analysis of food microbial ecology. Appl Environ Microbiol 79:3148–3155. https://doi.org/10. 1128/AEM.00256-13 Fett WF, Wijey C, Moreau RA, Osman SF (2000) Production of cutinolytic esterase by filamentous bacteria. Europe PubMed Central Plus 31:25–29 Han QH (2013) Study on the relationship between microbial community and vinegar flavor in the traditional brewing process of Liangzhou fumigated vinegar. Master degree dissertation. Gansu Agri Univ, Lanzhou Haruta S, Ueno S, Egawa I, Hashiguchi K, Fujii A, Nagano M, Igarashi Y (2006) Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR- 288 Ann Microbiol (2019) 69:279–289
