Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.     

酱香型白酒机械化制曲发酵细菌群落的演替

利用高通量测序并结合数理统计分析对酱香型白酒机械化制曲发酵过程中细菌群落结构进行分析。结果表明,其发酵过程的优势菌门为Firmicutes、Proteobacteria、Actinobacteria及Cyanobacteria,随着发酵的进行,逐渐由多菌种演替为单一Firmicutes为主导。机械化制曲发酵过程中共检出84 个细菌属,稍高于传统制曲的82 个细菌属;机械化制曲发酵过程优势细菌属共14 个,包括Pantoea、Rhizobium、Lactobacillus、Weissella、Bacillus、Oceanobacillus、Lentibacillus、Kroppenstedtia、Thermoactinomyces、Staphylococcus、Enterobacter、Saccharopolyspora、Pediococcus和Tepidimicrobium,其中Pediococcus和Tepidimicrobium是机械制曲发酵过程特有的优势细菌属,Leuconostoc及Pseudomonas是传统制曲过程特有的优势细菌属,表明机械化制曲与传统制曲过程优势菌具有较高的相似性。通过优势菌与环境因子相关性分析发现,主要功能细菌属Bacillus、Lactobacillus及Weissella都与大曲制曲温度呈正相关,与大曲酸度呈正相关,表明在机械化制曲过程中要合理科学控制相对较高的温度及酸度,既有利于主要功能微生物的生长,又能抑制不耐热、不耐酸杂菌的繁殖。本研究从发酵细菌群落结构上说明酱香型白酒的机械化制曲可以代替人工制曲,为酱香型白酒机械化制曲的理论研究和工程应用奠定了一定科学基础。     

羊羔美酒大曲中乳酸菌多样性及分子鉴定

目的：分离和鉴定羊羔美酒大曲中的乳酸菌,探寻其菌群多样性,为酿酒工艺条件改进提供参考。方法：将酒曲进行梯度稀释、富集培养、平板画线等分离纯化,获取乳酸菌单菌落。采用菌落特征和个体形态特征结合的方法进行乳酸菌形态鉴定。乳酸菌的分子鉴定采用重复序列聚合酶链式反应（repetitive sequence-basedpolymerase chain reaction,Rep-PCR）技术和16S rDNA序列分析法。结果：从羊羔美酒大曲中共得到65 株乳酸菌,形态学分为9 类。用Rep-PCR技术在75%的相似性上将其区分为5 类,经基因序列分析,鉴定为分属于4 个属的乳酸菌,分别为：片球菌属（Pediococcus）、明串珠菌属（Leuconostoc）、乳杆菌属（Lactobacillus）、肠球菌属（Enterococcus）。结论：传统微生物分离技术与现代分子鉴定技术相结合,可快速准确鉴定出羊羔美酒大曲中乳酸菌的菌群组成和多样性。     

A survey of lactic acid bacteria for enzymes of interest to oenology

Isolates from wine-associated genera of lactic acid bacteria (LAB), including Lactobacillus , Oenococcus and Pediococcus , were examined for enzymatic properties that could be of value in the production and processing of wine. Up to 438 combinations of bacterial isolates and substrates were examined. Across the 50 isolates investigated, all were found to hydrolyse esters, with the greatest activity being seen amongst the oenococci followed by the lactobacilli and pediococci. Lipase activity was restricted to three Lactobacillus isolates, while all tested isolates displayed tannase activity. Cellulase, β-glucanase and lichenase activities were observed for almost all Pediococcus and Lactobacillus isolates, while such activities were seen for only a minority of oenococci. No degradation of the xylan substrate was detected. Once confirmed under winelike conditions, our findings will help define potentially interesting genera of LAB for winemaking.     

山西老陈醋和怀仁醋酒精发酵阶段细菌菌群多样性分析

为了探明山西老陈醋酿造工艺和怀仁醋酿造工艺对细菌菌群的影响,该研究利用高通量测序技术对两种工艺酒精发酵阶段样品（分别编号为S和X）的细菌菌群多样性进行分析。结果表明,S样品的细菌菌群多样性更高。两种工艺酒精发酵阶段样品的细菌菌群组成差别较大,与S样品相比,X样品中相对丰度较高的乳杆菌属（Lactobacillus）、魏斯氏菌属（Weissella）、乳球菌属（Lactococcus）均增多,片球菌属（Pediococcus）、明串珠菌属（Leuconostoc）、泛菌属（Pantoea）、链霉菌属（Streptomyces）、短状杆菌属（Brachybacterium）、肠杆菌属（Enterobacter）和非培养细菌（uncultured bacterium）均减少。导致两种工艺酒精发酵阶段样品有显著差异的细菌在S样品中为片球菌属、明串珠菌属、链霉菌属和泛菌属,在X样品中则为乳杆菌属、魏斯氏菌属和乳球菌属。相关性分析结果表明,乳杆菌属、泛菌属、魏斯氏菌属和片球菌属与其他物种联系紧密。     

Diversity of lactic acid bacteria in fermented brines used to make stinky tofu

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.     

Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas

Gundruk, sinki and khalpi are lactic-fermented vegetable products of Sikkim in India, and inziangsang is a fermented leafy vegetable product of Nagaland and Manipur in India. A total of 65 samples of gundruk (25), sinki (12), khalpi (25) and inziangsang (3) were analysed for microbial counts. The population of lactic acid bacteria (LAB) as well as aerobic mesophilic counts were at the level of 10(7) cfu g(-1). Yeasts were detected only in few samples of sinki and khalpi. No moulds were detected. In order to identify the predominating organisms, a total of 269 strains of LAB were isolated from gundruk, sinki, khalpi and inziangsang samples. The phenotypic characteristics of these strains were determined followed by genotyping using RAPD-PCR, repetitive element PCR and species-specific PCR techniques. The major representatives of the LAB involved in these fermentations were identified as Lactobacillus brevis, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactici and Leuconostoc fallax.     

Characterization and heterologous expression of a class IIa bacteriocin,plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.     

A polyphasic approach in order to identify dominant lactic acid bacteria during pasta manufacturing

Using a polyphasic approach, we have isolated and identified, lactic acid bacteria (LAB) in samples directly collected from an artisanal pasta-making manufactory located in Puglia (South Italy). Samples were collected during several steps of pasta processing and LAB were isolated on MRS and M17 plates. Furthermore, strains were grouped in a total of eight species by means of randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing and 16S rDNA sequencing. The majority of strains were identified as belonging to Pediococcus pentosaceus and Enterococcus faecium species. The remaining strains were characterized and assigned to Weissella confusa, Pediococcus acidilactici, Leuconostoc mesenteroides, Leuconostoc citreum, Lactobacillus fermentum and Lactobacillus plantarum species. Strains were identified from all the analysed samples, and growth of LAB was monitored from extrusion to pre-heating steps.     

Characterization and identification of lactic acid bacteria in "morcilla de Burgos"

A total of 176 lactic acid bacteria (LAB) isolated from a typical Spanish blood sausage called "morcilla de Burgos" were identified by means of phenotypic characteristics and 16S rDNA RFLP (ribotyping). LAB were isolated from "morcilla" of different producers and in different storage periods, which includes unpackaged, vacuum and modified atmosphere packaged "morcilla" and vacuum packed and pasteurised "morcilla". The knowledge of specific spoilage bacteria of "morcilla de Burgos" will be useful to design new preservation methods to extend the shelf-life of this product. Identification made according to phenotypic and biochemical characteristics shows the majority of the isolates were heterofermentative LAB (93.2%) and eight different bacterial groups could be distinguished (A-G). Weisella viridescens was the main species detected (42%). In addition, Leuconostoc spp. (23.9%), Weissella confusa (11.4%) and Lactobacillus fructosus (5.7%) species were found. Few strains were phenotypically misidentified as Lactobacillus sanfrancisco, Pediococcus spp., Lactobacillus sakei/curvatus and Carnobacterium spp. and 11 strains remained unknown. Most of the leuconostocs were identified as Leuconostoc mesenteroides and Leuconostoc carnosum species. Ribotyping shows a quite good correlation with phenotypic methods, although it has been possible to identify 15 different clusters. W. viridescens and leuconostocs were also the predominant LAB. Strains identified as W. confusa by phenotypic characteristics were resolved in W. confusa and Weissella cibaria by ribotyping. Neither Carnobacterium piscicola nor Lb. sanfrancisco were identified by means of genotypic method. All Lb. fructosus strains and some more included in different phenotypic groups (17 strains in total) could not be associated with any reference strain (cluster VII).     

酸菜发酵期间细菌群落结构动态变化分析

以自制酸菜为研究对象,利用16S rRNA高通量测序技术检测酸菜发酵期间细菌群落的组成及演替规律。结果表明,发酵第1天的酸菜样本中微生物丰富度和多样性最高,随着发酵的不断进行,微生物丰富度和多样性呈先降后缓慢增加的趋势。细菌群落结构与发酵进程显著相关,因此不同发酵时期优势菌属不同。酸菜发酵期间的主要优势细菌属为乳杆菌属（Lactobacillus）（0.01%～82.32%）、肠杆菌属（Enterobacter）（0～20.60%）、明串珠菌属（Leuconostoc）（1.66%～9.48%）、盐单胞菌属（Halomonas）（0.03%～1.84%）和魏斯氏菌属（Weissella）（0.01%～8.26%）等,其中乳酸菌占据绝对的优势地位。     

Culture media for non-sporulating gram-positive food spoilage bacteria.

The spoilage association especially of protein-rich foods can be dominated by Gram-positive bacteria, notably lactic acid bacteria (LAB) which affect vacuum packaged refrigerated processed meats and some dairy products. New food ecosystems are being created by novel packaging and processing technologies, resulting in spoilage associations differing from those previously reported. In addition, improvement in identification methods, allow the detection and isolation of 'novel' bacterial groups, e.g., Carnobacterium spp. This review considers the genera Aerococcus, Brevibacterium, Brochothrix, Carnobacterium, Kurthia, Lactobacillus, Leuconostoc, Microbacterium, Micrococcus, Pediococcus and Propionibacterium. Strictly selective procedures, including incubation temperature and atmosphere, are not yet available for the genera Aerococcus, Brevibacterium, Microbacterium and Micrococcus, and only with some limitations for Kurthia and Propionibacterium. On the other hand, a causative role in food spoilage has not been established clearly for all those groups, some of which may be 'opportunistic' in their behaviour. The LAB groups Lactobacillus, Leuconostoc and Pediococcus ('LLP-Group') often share similar habitats and show similar physiological behaviour on a number of elective and selective media. Modifications to increase selectivity have been based mainly on de Man, Rogosa and Sharpe (MRS) or Rogosa agar, and include pH reduction, supplementation with chemical preservatives (e.g., sorbic acid and nitrate) and the use of reduced atmospheres or suboptimal incubation temperatures. Carnobacteria differ from other LAB in their non-aciduric nature, and selective plating procedures use high-pH media (pH 8-9) by which competitors (mainly lactobacilli) are eliminated.     

Identification and antimicrobial susceptibility of lactic acid bacteria from retail fermented foods

One important safety criterion of using lactic acid bacteria (LAB) in food applications is to ensure that they do not carry transferable antimicrobial resistance (AR) determinants. In this study, 63 LAB belonging to six genera, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, and Pediococcus, were recovered from 28 retail fermented food products in Maryland, identified to species with 16S-23S rRNA spacer PCRs, and characterized for antimicrobial susceptibility against eight antimicrobials. Besides intrinsic resistance to ciprofloxacin or vancomycin in some lactobacilli, tetracycline resistance was observed in two Streptococcus thermophilus isolates from one cheese and one sour cream sample and was associated with the presence of a nonconjugative tet(S) gene. The results indicated a low level of AR among naturally occurring and starter LAB cultures in fermented dairy and meat products in the United States; therefore, the probability for foodborne LAB to serve as reservoirs of AR is low. Further studies involving a larger sample size are needed to assess the potential risk of AR gene transfer from LAB in fermented food products.     

Biodiversity of Bacteriocin-Producing Lactic Acid Bacteria from Mexican Regional Cheeses and their Contribution to Milk Fermentation

The aim of this work was to examine the biodiversity of bacteriocin-producing lactic acid bacteria from homemade cheeses produced in Veracruz (México) and assess their contribution as adjunct cultures in dairy products. Ninety-three presumptive bacteriocinogenic strains were detected by direct antagonism assays and 29 of them were active against Enterococcus faecalis NRRL-B537, Listeria innocua 062 AST, or Listeria monocytogenes ATCC19115 by the well diffusion test using cell-free supernatants, adjusted to pH 6.0 to exclude inhibition by organic acids. Positive isolates were identified by partial sequencing of the 16s rDNA as Pediococcus acidilactici (four isolates), Enterococcus faecium (17 isolates), Lactobacillus plantarum (six isolates) and Lactobacillus fermentum (two isolates). RAPD-PCR discriminated seven groups with a 50% similarity and revealed the presence of the same isolates. The coding genes for the synthesis of plantaricin EF, plantaricin JK, plantaricin N, plantaricin NC8 and the inducing peptide plantaricin A were detected by PCR in L. plantarum. Similarly, enterocin P and pediocin PA-1 genes were amplified from Enterococcus and Pediococcus genomic DNA, respectively. Overall, co-culturing of bacteriocin producing Lactobacillus and Pediococcus strains with the dairy starter Lactococcus lactis IPLA947 did not interfere with milk acidification. Lactose consumption, acidification rate and production of lactic acid were unchanged. Nonetheless, higher levels of acetic acid, ethanol and succinic acid were detected depending on the strain. Our results demonstrate the diversity of bacteriocinogenic species in homemade Mexican cheeses which may be used as adjunct cultures to enhancing safety of this well-appreciated cheese while providing a richer range of metabolites.     

A culture-dependent and -independent approach for the identification of lactic acid bacteria associated with the production of nem chua,a Vietnamese fermented meat product

In this study, the diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented meat, was described using a combination of culture-dependent and culture-independent methods. A total of two hundred seventy-three LAB isolates were subjected to a polyphasic identification approach combining (GTG)₅-PCR fingerprinting and phenylalanyl-tRNA synthase α subunit (pheS) and RNA polymerase α subunit (rpoA) gene sequence analysis. LAB associated with nem chua were identified as Lactobacillus pentosus (21%), Lactobacillus plantarum (29.7%), Lactobacillus brevis (5%), Lactobacillus paracasei (0.4%), Lactobacillus fermentum (0.7%), Lactobacillus acidipiscis (0.4%), Lactobacillus farciminis (23%), Lactobacillus rossiae (0.4%), Lactobacillus fuchuensis (0.7%), Lactobacillus namurensis (0.4%), Lactococcus lactis (0.4%), Leuconostoc citreum (9.5%), Leuconostoc fallax (1%), Pediococcus acidilactici (1%), Pediococcus pentosaceus (4%), Pediococcus stilesii (1%), Weissella cibaria (0.7%) and Weissella paramesenteroides (0.7%). Furthermore, PCR-DGGE was also applied as a culture-independent method in this study. Results indicated the presence of species of which no isolates were recovered, i.e. Lactobacillus helveticus/crispatus, Lactococcus garvieae and Vagococcus sp. Conversely, not all isolated bacteria were detected by PCR-DGGE. Principal component and discriminant analysis disclosed correlations between the different production locations and certain isolated LAB species and strains and/or DGGE bands suggesting possible influences of locally prevailing production practices on the nem chua LAB microbiota.     

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.     

不同盐质量分数泡白菜发酵过程中乳酸菌群落结构的变化

设计3种不同盐质量分数的泡白菜,包括低盐(2%)、中盐(5%)、高盐(8%),利用可培养方法结合多种分子生物学手段研究其发酵过程中乳酸菌的群落结构。结果表明:盐质量分数越低,乳酸菌增长速率越快。本研究从3种不同盐质量分数泡白菜发酵过程中分离筛选得到了563株乳酸菌,并采用16S rDNA测序、多重聚合酶链式反应、聚合酶链反应-限制性片段长度多态性、API 50CH等多种不同的鉴定方法进行鉴定,鉴定结果表明这563株乳酸菌属于5个属11个种。结果表明,低盐泡白菜发酵前期的优势菌为Lactococcus lactis、Lactobacillus pentosus和Leuconostoc,发酵后期则由Lactobacillus pentosus主导;中高盐泡白菜发酵前期由Lactobacillus pentosus和Weissella主导,发酵后期主要由Lactobacillus pentosus完成。     

The microbiology of South African traditional fermented milks

A total of 15 samples of traditional fermented milk were collected from individual households in South Africa and Namibia. Lactic acid bacteria dominated the microflora of these samples, especially the genera Leuconostoc, Lactococcus and Lactobacillus. Other groups identified included pyogenic streptococci and enterococci. The dominant lactococci species was Lactococcus lactis subsp. lactis. Eighty-three percent of the leuconostoc isolates were identified as Leuconostoc mesenteroides subsp. dextranicum. Other species identified included Leuconostoc citreum, Leuconostoc lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus plantarum.     

Source Tracking and Succession of Kimchi Lactic Acid Bacteria during Fermentation

This study aimed at evaluating raw materials as potential lactic acid bacteria (LAB) sources for kimchi fermentation and investigating LAB successions during fermentation. The bacterial abundances and communities of five different sets of raw materials were investigated using plate‐counting and pyrosequencing. LAB were found to be highly abundant in all garlic samples, suggesting that garlic may be a major LAB source for kimchi fermentation. LAB were observed in three and two out of five ginger and leek samples, respectively, indicating that they can also be potential important LAB sources. LAB were identified in only one cabbage sample with low abundance, suggesting that cabbage may not be an important LAB source. Bacterial successions during fermentation in the five kimchi samples were investigated by community analysis using pyrosequencing. LAB communities in initial kimchi were similar to the combined LAB communities of individual raw materials, suggesting that kimchi LAB were derived from their raw materials. LAB community analyses showed that species in the genera Leuconostoc, Lactobacillus, and Weissella were key players in kimchi fermentation, but their successions during fermentation varied with the species, indicating that members of the key genera may have different acid tolerance or growth competitiveness depending on their respective species.     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.