Diversity analysis of lactic acid bacteria in Korean rice wines by culture-independent method using PCR-denaturing gradient gel electrophoresis

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was used to determine the presence and diversity of lactic acid bacteria (LAB) in takju, a traditional Korean rice wine. Bacterial DNAs were extracted from 15 commercial rice wines and amplicons of partial 16S rRNA genes were separated by DGGE and intense bands were sequenced. Lactobacillus (Lb.) paracasei, Lb. plantarum, and Leuconostoc pseudomesenteroides were detected in all samples and Lb. harbinensis and Lb. parabuchneri were found with above 80% frequency of occurrence. Unknown species of Lactobacillus were also widely detected. This result revealed that, regardless of products and raw materials, the distribution profiles of LAB in takju products have a common pattern comprising of above predominant species and, furthermore, takju can be regarded as a LAB-rich fermented food providing various probiotics.     

Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce,using nested PCR-denaturing gradient gel electrophoresis and the plate count method

The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.     

Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis

Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method.     

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.     

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.     

传统分离培养结合PCR-DGGE技术分析传统乳制品中的乳酸菌

采用传统培养分离方法结合聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对传统乳制品中乳酸菌种群结构进行研究。结果表明:用传统分离与分子鉴定方法得到5种乳酸菌,其中,乳酸片球菌(Pediococcus acidilactici)为优势菌群,对通过PCR-DGGE方法得到的9条16S rDNA条带序列进行了比对,结果表明瑞士乳杆菌(Lactobacillus helveticus)的丰度最高,是酸奶样品中主要的优势菌群,乳酸片球菌(Pediococcus acidilactici)为次优势菌群。传统分离法与PCR-DGGE技术结合能够更有效、更全面地分析传统乳制品中微生物的群落结构及优势菌群。     

Isolation and characterization of lactic acid bacteria from fresh Chinese traditional rice wines using denaturing gradient gel electrophoresis

Food Science and Biotechnology - Lactic acid bacteria (LAB) are a prevalent bacterial group in rice wine maturation that contributes to flavor, texture, and nutritive value. To better understand...     

蜂蜜对酸奶生产中乳酸菌生长和产酸能力的影响

在脱脂乳粉制成的全乳固体含量为12%(w/v) 的复原乳中,分别添加5%(w/v)的蔗糖、果糖或蜂蜜,并以不添加甜味剂的复原乳作为对照,经过70℃、15min灭菌,冷却至室温,分别接种唾液链球菌嗜热亚种、德氏乳杆菌保加利亚亚种和嗜酸乳杆菌,接种量5%(v/v),培养温度37℃,分别在培养初始和经过24h培养时取样,测定其乳酸菌活性、pH值和乳酸含量。结果表明,添加量为5%(w/v)的蜂蜜对于3种乳酸菌均没有抑制作用,是酸奶生产的合适甜味剂。     

荆州地区鲊广椒乳酸菌多样性解析及其分离株发酵特性的评价

运用纯培养技术从荆州地区鲊广椒中分离纯化乳酸菌,并采用分子生物学方法并对其进行同源性分析和系统发育树的构建,同时使用电子舌和电子鼻技术对乳酸菌分离株纯种发酵制备鲊广椒的滋味品质进行了评价。结果表明:从10个鲊广椒样品中共分离出22株疑似乳酸菌,分为4个属,8个种,其中优势菌为Lactobacillus plantarum(植物乳杆菌)。通过电子舌分析发现,添加乳酸菌的多数鲊广椒样品其酸味均有较明显的提升。通过电子鼻分析发现,传感器W5S、W1S、W1W、W2S和W2W对添加L. plantarum和肠球菌制备的鲊广椒样品响应值明显较低,而传感器W1C、W3C和W5C呈现出相反的趋势。通过主成分分析发现,L.plantarum HBUAS52151和L. plantarum HBUAS52166发酵制备的鲊广椒样品具有较好的品质。由此可见,荆州地区鲊广椒乳酸菌具有较高的多样性,且添加乳酸菌进行鲊广椒纯种发酵可明显改善产品的滋味和气味品质。     

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 ± 2.5 °C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.     

Ultrafiltration flux of acid whey obtained by lactic acid fermentation

Acid whey was prepared from reconstituted ultra-low heat skimmed milk powder fermented with Lactobacillus helveticus, clarified by centrifugation, pH adjusted and ultrafiltered (cut-off limit 10 or 30 kDa) at 50 °C to a volume reduction ratio of 10. Centrifugation primarily removed the serum albumin and, to a lesser extent, immunoglobulins, considered the most critical membrane foulants during ultrafiltration of acid whey. Below pH 3.9 the flux improvement was directly dependent on pH lowering. At pH 3.9 a deviation point in the relationship between pH and flux of acid whey was observed. Above this pH a severe flux decline was detected and at pH 4.6 the fermented acid whey was non-filterable. Largest flux increase was obtained at a transmembrane pressure of 2.0 bar and 50 °C. Under these conditions the permeation flux achieved using a 30 kDa membrane was about 34 ± 5 kg h⁻¹m⁻², comparable with that for sweet whey.     

Hydrogen production by fermentation using acetic acid and lactic acid

Microbial hydrogen production from sho-chu post-distillation slurry solution (slurry solution) containing large amounts of organic acids was investigated. The highest hydrogen producer, Clostridium diolis JPCC H-3, was isolated from natural environment and produced hydrogen at 6.03+/-0.15 ml from 5 ml slurry solution in 30 h. Interestingly, the concentration of acetic acid and lactic acid in the slurry solution decreased during hydrogen production. The substrates for hydrogen production by C. diolis JPCC H-3, in particular organic acids, were investigated in an artificial medium. No hydrogen was produced from acetic acid, propionic acid, succinic acid, or citric acid on their own. Hydrogen and butyric acid were produced from a mixture of acetic acid and lactic acid, showing that C. diolis. JPCC H-3 could produce hydrogen from acetic acid and lactic acid. Furthermore, calculation of the Gibbs free energy strongly suggests that this reaction would proceed. In this paper, we describe for the first time microbial hydrogen production from acetic acid and lactic acid by fermentation.     

蔬菜乳酸菌腌渍发酵过程亚硝酸盐变化研究

研究了蔬菜腌渍发酵过程中添加乳酸菌纯培养液对亚硝酸盐含量变化的影响。实验结果表明,接种乳酸菌能降低蔬菜腌渍发酵过程中亚硝酸盐含量。4组乳酸菌腌渍发酵实验中,接种混合菌种(干酪乳杆菌∶鼠李糖乳杆菌∶植物乳杆菌=1∶1∶1)对蔬菜湿腌发酵时菜料和菜汤的亚硝酸盐含量降低效果最佳,接种植物乳杆菌对蔬菜干腌发酵时菜料亚硝酸盐含量降低作用最显著。     

Comparison of lactic acid bacteria diversity during the fermentation of Tarhana produced at home and on a commercial scale

Food Science and Biotechnology - In this study, lactic acid bacteria diversity during the fermentation of homemade and commercially prepared Tarhana, a traditional fermented cereal food from...     

Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages

The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.     

Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis

We established a novel system using denaturing gradient gel electrophoresis (DGGE) to quickly identify bacteria known to be responsible for spoilage in meat processing plants and meat products. We extracted bacterial DNA from swabbed samples at various locations in the plant and from meat products and performed PCR amplification, targeting 16S rDNA from the dominant organisms. The amplification products were subjected to DGGE, and the contaminating bacteria in the meat products and the plant were analyzed. This analysis indicated that lactic acid bacteria and spoilage-causing bacteria are widely distributed within the meat processing plant. We developed molecular size markers to identify the dominant organisms obtained from the plant and meat products. The establishment of the present method allows quick and simple identification of bacteria causing the possible deterioration of products and contamination and thus permits constant monitoring of any harmful bacteria within meat processing plants.     

Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels

To speed up research on the usefulness and selection of bacterial starter cultures for cocoa bean fermentation, a benchmark cocoa bean fermentation process under natural fermentation conditions was developed successfully. Therefore, spontaneous fermentations of cocoa pulp-bean mass in vessels on a 20 kg scale were tried out in triplicate. The community dynamics and kinetics of these fermentations were studied through a multiphasic approach. Microbiological analysis revealed a limited bacterial species diversity and targeted community dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation, as was the case during cocoa bean fermentations processes carried out in the field. LAB isolates belonged to two main (GTG)₅-PCR clusters, namely Lactobacillus plantarum and Lactobacillus fermentum, with Fructobacillus pseudofilculneus occurring occasionally; one main (GTG)₅-PCR cluster, composed of Acetobacter pasteurianus, was found among the AAB isolates, besides minor clusters of Acetobacter ghanensis and Acetobacter senegalensis. 16S rRNA-PCR-DGGE revealed that L. plantarum and L. fermentum dominated the fermentations from day two until the end and Acetobacter was the only AAB species present at the end of the fermentations. Also, species of Tatumella and Pantoea were detected culture-independently at the beginning of the fermentations. Further, it was shown through metabolite target analyses that similar substrate consumption and metabolite production kinetics occurred in the vessels compared to spontaneous cocoa bean fermentation processes. Current drawbacks of the vessel fermentations encompassed an insufficient mixing of the cocoa pulp-bean mass and retarded yeast growth.     

乳酸菌发酵柿汁的制备工艺

介绍了以柿子汁为原料制备乳酸菌发酵饮料的工艺流程,并对原材料和发酵产品的一些营养指标进行了测量。实验表明,乳酸菌发酵的最佳条件是发酵温度30℃、接种量6%、菌种AS1.1482∶6038=2∶1。产品既保留了部分柿子汁的原有风味,又具有乳酸发酵形成的特殊风味,营养价值较高,有一定的保健作用。     

乳酸发酵香椿芽新工艺研究

探讨了香椿芽接种乳酸菌,低盐快速发酵的新工艺。通过对香椿芽乳酸发酵过程中亚硝酸盐含量的动态观察,在确保香椿芽卫生质量和风味的基础上,借助于接种乳酸菌使腌渍时间从传统方法的20d以上降至10d左右,食盐浓度从8%～10%或更高降至4%,亚硝峰出现从传统方法的第15～19d提前至第7～9d,且峰值从传统方法的7mg/kg以上降至1mg/kg以下。通过正交试验得出优化发酵条件为:食盐4%、蔗糖2%、乳酸菌接种量1%、发酵温度30℃。