Metagenomic analysis of kimchi, a traditional Korean fermented food

Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.     

酱香型白酒酿造酒醅中酵母菌多样性研究

为解析酱香型白酒酿造酒醅中酵母菌的菌群结构,获取酒醅中的主要酵母菌,采用高通量测序法分析酱香型白酒酒醅中酵母菌多样性及主要功能菌群,同时采用可培养分离方法获取酒醅中酵母菌活性菌株。从酱香型白酒下沙至五轮次酒醅中共检出59个属、129个种的酵母菌,分离得到酵母菌活性菌株41种,检测到的酵母菌种类与获得的酵母菌活菌在各香型白酒中最多。不同时期酒醅中的酵母菌种类和数量差异明显,其中下沙、造沙轮次以Pichia kudriavzevii为绝对优势酵母菌;一至五轮次随着轮次的递增,酒醅中优势酵母菌的种类增多,其中主要的优势酵母菌有Pichia kudriavzevii、Pichia manshurica、Zygosaccharomyces bailii、Saccharomyces cerevisiae、Candida apicola。酱香型白酒酒醅中蕴藏着极其丰富的酵母菌资源,对酵母菌菌群结构的解析有助于科学地认识酱香型白酒酿造过程中产酒与风味代谢机理,为发酵过程的调控提供一定依据。     

Traditional fermented foods

For centuries, diverse plant and animal materials have been fermented by various bacteria, yeasts, and fungi to make excellent foods. The kinds of micro organisms used in traditional fermentation are restricted to a relatively few genera, including Aspergillus, Rhizopus, Mucor, Actinomucor, Monascus, Saccharomyces, Neurospora, Acetobacler, Bacillus, and Lactobacillus. The two principal advantages of food fermentations over other processes are to add flavor and to prevent spoilage. Fermented fish is a common food in the Orient and may have been the first product made by fermentation. Flavor is especially important in vegetable diets based on bland foods such as rice. Shoyu is the best, known oriental food fermentation, and it is very widely used as a flavoring agent. Be sides this fermentation, there are a large number of additional ones not so well known outside the Orient, whose products serve as seasoning or flavoring agents. Miso and natto are prepared from soybeans in Japan. Sufu is a cheese like product made from soybean milk in China. Tempeh and ontjom are Indonesian foods prepared from soybeans and peanuts, respectively. These food fermentations are discussed with emphasis on how they are produced and the flavor formed.     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.     

Dynamics and diversity of a microbial community during the fermentation of industrialized <Emphasis Type="Italic">Qingcai</Emphasis> paocai,a traditional Chinese fermented vegetable food,as assessed by Illumina MiSeq sequencing,DGGE and qPCR assay

Paocai is a traditional Chinese fermented food and typically produced via spontaneous fermentation. We have investigated the microbial community utilized for the fermentation of industrialized Qingcai paocai using the combination of Illumina MiSeq sequencing, PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) assay. Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were identified by both MiSeq sequencing and PCR-DGGE. The dominant genera observed in the fermentation were Lactobacillus, Pseudomonas, Vibrio and Halomonas. Most genera affiliated with Proteobacteria or Bacteroidetes were detected more often during the earlier part of the fermentation, while Lactobacillus (affiliated with Firmicutes) was dominant during the later fermentation stages. Fungal community analysis revealed that Debaryomyces, Pichia and Kazachstania were the main fungal genera present in industrialized Qingcai paocai, with Debaryomyces being the most dominant during the fermentation process. The quantities of dominant genera Lactobacillus and Debaryomyces were monitored using qPCR and shown to be 10⁹–10¹² and 10⁶–10¹⁰ copies/mL, respectively. During the later fermentation process of industrialized Qingcai paocai, Lactobacillus and Debaryomyces were present at 10¹¹ and 10⁸ copies/mL, respectively. These results facilitate further understanding of the unique microbial ecosystem during the fermentation of industrialized Qingcai paocai and guide future improvement of the fermentation process.     

蜡状芽孢杆菌发酵南极磷虾生成活性物质的研究

南极磷虾具有分布量广、营养价值高、贮藏量大等特点,是一种重要的海洋蛋白新资源,有待开发利用。从深海泥样中分离筛选出一株能够以南极磷虾为唯一碳氮源生长的蜡状芽孢杆菌,利用该菌发酵南极磷虾。对发酵条件进行了初步优化,研究了发酵液的抗氧化能力以及发酵液中风味物质的种类和含量的变化情况。结果显示,30℃恒温培养48 h,DPPH清除率为44.05%,总酚含量为0.059 2 mg/m L,亚铁离子螯合能力为92.23%,还原力为200.13μmol/L,总蛋白含量为2.952 g/100 g,有机酸组成丰富,游离氨基酸含量(227.540 2 mg/m L)比发酵前(106.5796 mg/m L)增加一倍。本研究使南极磷虾这一丰富的资源得到最大限度的利用,同时也使南极磷虾的生物活性物质能够在生产中发挥潜在的作用。     

双孢蘑菇堆肥中真菌群落多样性分析

以农作物玉米秸和稻草、牛粪为原料,分别设计双孢蘑菇Agaricus bisporus堆肥配方并进行堆肥发酵,研究二者堆肥过程中真菌多样性。在建堆、一次发酵结束和二次发酵结束3个时期分别采集堆肥样品,提取总DNA,以真菌18S rDNA基因通用引物,进行PCR-DGGE扩增和序列分析。累计获得39条特异条带18S rDNA基因信息,分属于真菌14个属、藻类7个属和原生动物3个属。子囊菌是两种配方堆肥过程中的优势菌群,建堆时期的优势类群为Pichia和Wickerhamomyces,一次和二次发酵时期的优势类群为Chaetomium和Lecythophora。多样性指数分析显示,稻草配方微生物多样性大于玉米秸配方;主成分分析（PCA）显示,玉米秸配方一次发酵结束时期与稻草和玉米二次发酵结束时期聚为一类,说明玉米秸配方堆肥提前腐熟。     

Metabolite profiling and bioactivity of rice koji fermented by Aspergillus strains

In this study, the metabolite profiles of three Aspergillus strains during rice koji fermentation were compared. In the partial least squares discriminant analysis-based gas chromatography-mass spectrometry data sets, the metabolite patterns of A. oryzae (KCCM 60345) were clearly distinguished from A. kawachii (KCCM 60552) and only marginal differences were observed for A. oryzae (KCCM 60551) fermentation. In the 2 days fermentation samples, the overall metabolite levels of A. oryzae (KCCM 60345) were similar to the A. oryzae (KCCM 60551) levels and lower than the A. kawachii (KCCM 60552) levels. In addition, we identified discriminators that were mainly contributing tyrosinase inhibition (kojic acid) and antioxidant activities (pyranonigrin A) in A. oryzae (KCCM 60345) and A. kawachii (KCCM 60552) inoculated rice koji, respectively. In this study, we demonstrated that the optimal inoculant Aspergillus strains and fermentation time for functional rice koji could be determined through a metabolomics approach with bioactivity correlations.     

纤维素降解菌的分离筛选及对中药废弃物的固相发酵研究

中药在提取炮制后残留大量生物质被简单堆弃,而其中的木质纤维素等生物质能被微生物有效利用降解,筛选到16株对木质纤维素有降解效果的菌株,对其中8株降解效果明显功能菌株进行了深入研究,经16(18)S rDNA鉴定发现5株细菌主要为Bacillus属、Streptomyces属和Enterobacter属,3株真菌为Ascomycete属、Aspergillus属和Trichosporon属;利用8株菌固相发酵中药废弃物,通过监测堆体温度、pH等参数,发现细菌类在发酵初期能迅速提高堆体温度,而真菌类发酵腐熟过程较缓慢,提示利用细菌-真菌联合组成降解菌系是提高中药废弃物固相发酵的重要手段。     

洋河浓香型白酒发酵过程酒醅微生物群落结构解析及其与有机酸合成的相关性

【目的】解析江苏洋河酒厂浓香型白酒窖内发酵过程酒醅微生物群落结构,建立酒醅微生物与主要有机酸合成的关联性。【方法】通过宏基因组测序获得白酒发酵过程中微生物群落结构变化规律,利用主成分分析和偏最小二乘回归分析寻找酒醅中影响主要有机酸合成的关键微生物。【结果】根据微生物组成结构变化和有机酸合成变化规律,可将白酒窖内发酵分为两个时期(0–14 d和15–60 d)。其中窖内发酵0–15 d与主要有机酸合成相关的微生物数量显著高于15–60 d的。窖内发酵过程与主要有机酸合成相关的微生物包括7个菌属,分别为乳杆菌属(Lactobacillus)、葡萄球菌属(Staphylococcus)、酵母属(Saccharomyces)、Naumovozyma、伊萨酵母属(Issatchenkia)、嗜冷芽孢杆菌属(Psychrobacillus)和根霉属(Rhizopus)。【结论】本研究识别了白酒窖内发酵过程中与主要有机酸合成相关的核心和关键微生物,可为阐明白酒窖内发酵产酸机理和保障白酒品质的稳定性奠定研究基础和理论依据。     

Pectinolytic activity secreted by yeasts isolated from fermented citrus molasses

AIMS: The aim of this study was to obtain improved strains of pectinolytic yeasts adapted to the conditions of an industrial fermentation process, which was continuously operated to convert citrus molasses into ethanol. METHODS AND RESULTS: The starter yeast of the industrial fermentation process was a commercial baker's yeast, which was capable of growing without forming any secretion halo of pectinase activity on solid medium. Nevertheless, isolates showing secretion of pectinolytic activity on plates were obtained from the fermentation process. The secretion of pectin-degrading activity by isolates on plates was repressed by galactose and improved as the result of colony aging on polygalacturonic acid plates at 30 degrees C. Liquefaction of polygalacturonate gels as well as the splitting of the pectin-degrading activity into a wall-linked and a supernatant fraction were also observed when the starter yeast was propagated under agitation in liquid medium containing pectin. CONCLUSIONS: Isolates capable of secreting pectinolytic activity on plates were predominant at the end of the citrus molasses fermentation. Nevertheless, the sizes of the secretion haloes on plates were not necessarily an indication of the levels of pectinolytic activity secreted in the liquid medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Improved pectinolytic strains of Saccharomyces can be used as a source of pectinases for a variety of applications. This organism also participates in plant deterioration processes.     

Diversity and dynamics of lactic acid bacteria in <Emphasis Type="Italic">Atole agrio</Emphasis>, a traditional maize-based fermented beverage from South-Eastern Mexico,analysed by high throughput sequencing and culturing

The purpose of this work was to analyse the diversity and dynamics of lactic acid bacteria (LAB) throughout the fermentation process in Atole agrio, a traditional maize based food of Mexican origin. Samples of different fermentation times were analysed using culture-dependent and -independent approaches. Identification of LAB isolates revealed the presence of members of the genera Pediococcus, Weissella, Lactobacillus, Leuconostoc and Lactococcus, and the predominance of Pediococcus pentosaceus and Weissella confusa in liquid and solid batches, respectively. High-throughput sequencing (HTS) of the 16S rRNA gene confirmed the predominance of Lactobacillaceae and Leuconostocaceae at the beginning of the process. In liquid fermentation Acetobacteraceae dominate after 4 h as pH decreased. In contrast, Leuconostocaceae dominated the solid fermentation except at 12 h that were overgrown by Acetobacteraceae. Regarding LAB genera, Lactobacillus dominated the liquid fermentation except at 12 h when Weissella, Lactococcus and Streptococcus were the most abundant. In solid fermentation Weissella predominated all through the process. HTS determined that Lactobacillus plantarum and W. confusa dominated in the liquid and solid batches, respectively. Two oligotypes have been identified for L. plantarum and W. confusa populations, differing in a single nucleotide position each. Only one of the oligotypes was detected among the isolates obtained from each species, the biological significance of which remains unclear.     

乳杆菌在发酵乳饮料中的应用

为了制备更优质的乳酸菌饮料,本论文主要以鼠李糖乳杆菌、瑞士乳杆菌、副干酪乳杆菌、嗜酸乳杆菌四种典型的乳酸杆菌为研究菌种,详细分析它们在乳品发酵过程中pH、酸度变化,货架期内的活菌数变化以及代谢产生的有机酸种类和耐胃酸的能力,以确定乳杆菌在乳饮料中应用的最优条件,分析其应用于乳饮料中的可行性。本文为乳杆菌在乳饮料中的应用提供了参考依据。     

Functional compounds in fermented buckwheat sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Analyses of bacterial communities in meju,a Korean traditional fermented soybean bricks,by cultivation-based and pyrosequencing methods

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before.     

Variability of in vitro ruminal fermentation and nutritional value of cell‐disrupted and nondisrupted microalgae for ruminants

The objective of this study was to investigate ruminal fermentation and the nutritional value of different microalgae products (MAP) for ruminants, including inter‐ and intra‐genera variability. Furthermore, the effect of mechanical cell disruption was also evaluated. Cell‐disrupted and nondisrupted MAP of four genera were investigated using the Hohenheim Gas Test. The investigations included characterization of gas production (GP), production of volatile fatty acids (VFA) and methane, organic matter digestibility, and energetic value as well as utilizable crude protein at the duodenum and ruminally undegradable crude protein (RUP). Furthermore, a three‐step enzymatic in vitro system was used to estimate intestinal digestibility of RUP (IDP). Ruminal fermentation was low for all investigated microalgae genera, as indicated by overall low GP, low production of VFA, and low ruminal protein degradation. Nevertheless, all microalgae genera were characterized by high RUP concentrations (236–407 g/kg dry matter; passage rate = 8% hr^?1), indicating that microalgae might be a promising protein source for high‐performing ruminants. Low IDP (26%–49% of RUP) considerably contradicted this potential. Mechanical cell disruption in general enhanced the extent of ruminal fermentation of MAP but, as RUP was decreased and IDP was hardly affected, mechanical cell disruption appears not to be necessary when microalgae are intended for application as a protein source for ruminants. Because of the high variability in the characteristics of the nutritional value, general means are inappropriate to characterize the nutritional value of MAP. In conclusion, suitability of microalgae as a protein source for ruminants might be limited because of low IDP, although further studies are necessary to prove these findings in vivo.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.     

火龙果汁发酵饮品的研制

火龙果不但营养丰富而且具有一定的保健功能。本试验以新鲜的红皮白肉型火龙果果实的榨汁为原料,分别对发酵工艺条件、蔗糖添加量、柠檬酸用量、稳定剂用量等条件进行了研究,通过单因素试验和正交试验,得到火龙果汁发酵饮料的最佳工艺配方。采用该工艺配方制备的发酵饮料澄清透明,具有清新的果香和淡淡的醇香,酸甜可口,口感顺滑。