Isolation and Identification of Lactic Acid Bacteria from Cow Milk and Milk Products.

Lactic acid bacteria (LAB) have long been consumed by people in several fermented foods such as dairy products. A study was conducted on lactating dairy cows to isolate and characterize LAB from dairy products found in and around Bahir-Dar city, North Western Ethiopia. Milk and milk products were randomly collected from dairy farms, milk vending shops, individual households, and supermarkets for bacteriological investigations. A total of sixteen samples were taken from different sources and cultured on different selective media: de Man, Rogosa, and Sharpe (MRS) agar for Lactobacillus spp.; M17 agar for Lactococcus spp.; Rogasa SL agar for Streptococci spp.; and MRS supplemented with cysteine (0.5%) for Bifidobacteria spp. Different laboratory techniques were implemented for LAB isolation and identification. A total of 41 bacterial isolates were grouped under five different genera of LAB and Bifidobacteria spp. were identified based on the growth morphology on the selective media, growth at a different temperature, gas production from glucose, carbohydrate fermentation, and other biochemical tests. LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Bifidobacterium spp. were isolated and identified from raw milk, cheese, and yogurt. Based on the current study, the majority of the LAB (24.38%) was isolated from cheese and yogurt. Among these, Lactobacillus, Lactococcus (21.94%), Streptococcus (19.51%), Leuconostoc (14.64%), Bifidobacteria (12.19%), and Pediococcus (7.31%) spp. were also identified from these products. Furthermore, based on the bacterial load count and different identification methodologies, our study revealed that Lactobacillus spp. were the dominant LAB isolated from milk and milk products. As a result, since there are few studies on the isolation and identification of lactic acid bacteria from dairy products in Ethiopia, more research studies are needed to complete the identification and characterization to species level and their possible role as probiotics.

Probiotic characterization of Pediococcus strains isolated from Iranian cereal-dairy fermented product: Interaction with pathogenic bacteria and the enteric cell line Caco-2.

In present study, we investigated the probiotic potential of three Pediococcus spp. isolated from Iranian traditional fermented cereal-dairy product, Tarkhineh. These 3 strains were identified as Pediococcus acidilactici VKU2, P. acidilactici IAH-5 and P. pentosaceus DHR005 by 16S rRNA gene sequencing. All the strain were found tolerate to pH 3 and 0.3% oxall for 3 h as well as simulated gastric and intestinal juice. P. acidilactici IAH-5 showed the highest cholesterol removal (67.52%), hydroxyl radical scavenging activity (58.32%), hydrophobicity (40.3%) and auto-aggregation (48%). Pediococcus spp. inhibited the growth of tested pathogens (Escherichia coli ATCC 25922, Pseudomonas aeruginosa PTCC 1707, Salmonella typhimurium PTCC 1609, and Staphylococcus aureus ATCC 25923) which the most susceptible strain was S. aureus. In competition assay, P. acidilactici IAH-5 was able to inhibited adhesion of 67.3% of S. typhimurium and in inhibition assay 45.8% of the pathogenic adhesion to Caco-2 cells were decreased. P. acidilactici VKU2 and P. acidilactici IAH-5 showed 16.32 and 12.25% adhesion to simulated epithelial cell line which were also confirmed by scanning electron microscopy. Pediococcus spp. did not showed DNase production or hemolytic activity which confirm its safety aspects. Our findings suggested that the P. acidilactici IAH-5 has the best properties with probiotic features and cholesterol assimilation for its application as novel bio-therapeutic and bio-preservation agents.

Reduction of Trimethylamine Off-Odor by Lactic Acid Bacteria Isolated from Korean Traditional Fermented Food and Their In Situ Application.

Trimethylamine (TMA) is a well-known off-odor compound in fish and fishery products and is a metabolic product of trimethylamine N-oxide (TMAO) generated by the enzymatic action of microorganisms. The off-odor is a factor that can debase the value of fish and fishery products. The present study aimed to remove TMA using lactic acid bacteria (LAB). A total of fifteen isolates exhibiting the TMA reduction efficacy were isolated from Korean traditional fermented foods. Among these isolates, five LAB isolates (Lactobacillus plantarum SKD 1 and 4; Lactobacillus paraplantarum SKD 15; Pediococcus stilesii SKD 11; P. pentosaceus SKD 14) were selected based on their high TMA reduction efficacy. In situ reduction of TMA efficacy by the LAB cell-free supernatant was evaluated using a spoiled fish sample. The results showed effective TMA reduction by our selected strains: SKD1 (45%), SKD4 (62%), SKD11 (60%), SKD14 (59%), and SKD15 (52%), respectively. This is the first study on TMA reduction by the metabolic activity of LAB and in situ reduction of TMA using cell-free supernatant of LAB. The present finding suggests an economically useful and ecofriendly approach to the reduction of TMA.

Persistence and ß-glucan formation of beer-spoiling lactic acid bacteria in wheat and rye sourdoughs.

Some beverage-spoiling lactic acid bacteria (LAB) produce capsular ß-glucans from UDP-glucose, which is accompanied by cell network formation causing viscosity increases of liquids. This feature of certain LAB is feared in breweries but could be useful for structural and nutritional improvement of baked goods, provided that these LAB are suited for the manufacture of sourdoughs. The aim of this study was to investigate the persistence and ß-glucan formation of the brewery isolates Levilactobacillus (L.) brevis TMW 1.2112 and Pediococcus (P.) claussenii TMW 2.340 in wheat and rye sourdoughs. Both the wild-type strains and the respective ß-glucan-deficient mutants were dominant in wheat and rye sourdoughs and acidified them to characteristic pH ranges. The formation of ß-glucan capsules during sourdough fermentations was stable in L. brevis TMW 1.2112 in contrast to P. claussenii TMW 2.340. Wheat sourdoughs fermented with the ß-glucan producing L. brevis TMW 1.2112 cells were significantly more viscous than doughs fermented by the P. claussenii TMW 2.340 cells and the applied mutant strains. In conclusion, L. brevis TMW 1.2112 and P. claussenii TMW 2.340 were suited and persistent wheat and rye sourdough starters, while the in situ ß-glucan formation in sourdoughs was hardly detectable in case of P. claussenii.

Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine

Background: Hong Qu glutinous rice wine (HQGRW) is brewed under non-aseptic fermentation conditions, so it usually has a relatively high total acid content. The aim of this study was to investigate the dynamics of the bacterial communities and total acid during the fermentation of HQGRW and elucidate the correlation between total acid and bacterial communities. Results: The results showed that the period of rapid acid increase during fermentation occurred at the early stage of fermentation. There was a negative response between total acid increase and the rate of increase in alcohol during the early fermentation stage. Bacterial community analysis using high-throughput sequencing technology was found that the dominant bacterial communities changed during the traditional fermentation of HQGRW. Both principal component analysis (PCA) and hierarchical clustering analysis revealed that there was a great difference between the bacterial communities of Hong Qu starter and those identified during the fermentation process. Furthermore, the key bacteria likely to be associated with total acid were identified by Spearman's correlation analysis. Lactobacillus, unclassified Lactobacillaceae, and Pediococcus were found, which can make significant contributions to the total acid development (| r| N 0.6 with FDR adjusted P b 0.05), establishing that these bacteria can associate closely with the total acid of rice wine. Conclusions: This was the first study to investigate the correlation between bacterial communities and total acid during the fermentation of HQGRW. These findings may be helpful in the development of a set of fermentation techniques for controlling total acid.

Isolation, characterization and identification of antigenotoxic and anticancerous indigenous probiotics and their prophylactic potential in experimental colon carcinogenesis.

Colorectal cancer, the third most commonly diagnosed cancer, is a lifestyle disease where diet and gut microbiome contribute intricately in its initiation and progression. Prophylactic bio-interventions mainly probiotics offer an alternate approach towards reducing or delaying its progression. Therefore, the present study was designed wherein a robust protocol for the isolation, characterization, and identification of indigenous probiotics having antigenotoxic and anticancerous activity was followed along with their prophylactic potential assessment in early experimental colorectal carcinogenesis. Among forty-six isolated lactic acid bacterial strains, only three were selected on the basis of antigenotoxicity against N,N-Dimethyl dihydrazine dihydrochloride and 4-Nitroquinoline 1-oxide and probiotic attributes. All three selected probiotic strains exhibited anticancerous potential as is evident by the reduced Aberrant Crypt Foci, reduced fecal pH, enhanced fecal lactic acid bacteria and altered fecal enzymes (ß-glucuronidase, nitroreductase, ß-glucosidase) that modulated gut microbiota and microenvironment resulting into restored histoarchitecture of the colon. The results are a clear indicator of the prophylactic potential of selected indigenous probiotics which may be used as an alternative prophylactic biological therapy against colon carcinogenesis particularly in highly susceptible individuals.

Bacterial community and composition in Jiang-shui and Suan-cai revealed by high-throughput sequencing of 16S rRNA.

Fermented vegetables have a long history in many cultures. Jiang-shui and Suan-cai are two of the most well-known instances in North China. They are made by a process of natural lactic acid fermentation. However, they have the different characteristics, i.e. acidity, taste and flavor, which are influenced by the specific bacterial community. Therefore, we used high-throughput sequencing methods to identify the bacterial community structure of Jiang-shui and Suan-cai in this study. Subsequently, we figured out the bacterial differences of these two products using the statistical analysis. Firmicutes and Proteobacteria were the dominant phyla in both Jiang-shui and Suan-cai. However, Lactobacillus was the main genus in Jiang-shui samples, whereas both Lactobacillus and Pediococcus were the major genera in the Suan-cai samples. At the species level, Lactobacillus amylolyticus, Lactobacillus fermentum and Lactobacillus pontis were the major species in Jiang-shui samples, while Pediococcus parvulus, Lactobacillus coryniformis, Lactobacillus pentosus and Lactobacillus parabrevis were the dominant species in the Suan-cai samples. These results suggested that Jiang-shui and Suan-cai had their own unique bacterial community, leading to the specific characteristics. Furthermore, the bacterial communities of both fermented vegetables varied at different locations. This study revealed the flora present in the Jiang-shui and the Suan-cai, providing a deep insight of the microbial species of Chinese fermented vegetables and guidance for the production of the Jiang-shui and the Suan-cai.

Extracellular Proteolytic Activity and Amino Acid Production by Lactic Acid Bacteria Isolated from Malaysian Foods.

Amino acids (AAs) are vital elements for growth, reproduction, and maintenance of organisms. Current technology uses genetically engineered microorganisms for AAs production, which has urged the search for a safer food-grade AA producer strain. The extracellular proteolytic activities of lactic acid bacteria (LAB) can be a vital tool to hydrolyze extracellular protein molecules into free AAs, thereby exhibiting great potential for functional AA production. In this study, eight LAB isolated from Malaysian foods were determined for their extracellular proteolytic activities and their capability of producing AAs. All studied LAB exhibited versatile extracellular proteolytic activities from acidic to alkaline pH conditions. In comparison, Pediococcus pentosaceus UP-2 exhibited the highest ability to produce 15 AAs extracellularly, including aspartate, lysine, methionine, threonine, isoleucine, glutamate, proline, alanine, valine, leucine, tryptophan, tyrosine, serine, glycine, and cystine, followed by Pediococcus pentosaceus UL-2, Pediococcus acidilactici UB-6, and Pediococcus acidilactici UP-1 with 11 to 12 different AAs production detected extracellularly. Pediococcus pentosaceus UL-6 demonstrated the highest increment of proline production at 24 h of incubation. However, Pediococcus acidilactici UL-3 and Lactobacillus plantarum I-UL4 exhibited the greatest requirement for AA. The results of this study showed that different LAB possess different extracellular proteolytic activities and potentials as extracellular AA producers.

Biopreservation potential of antimicrobial protein producing Pediococcus spp. towards selected food samples in comparison with chemical preservatives.

The present study elucidates biopreservation potential of an antimicrobial protein; bacteriocin, producing Pediococcus spp. isolated from dairy sample and enhancement of their shelf life in comparison with two chemical preservatives. The antimicrobial protein producing Pediococcus spp. was isolated from selected diary samples and characterised by standard microbiology and molecular biology protocols. The cell free supernatant of Pediococcus spp. was applied on the selected food samples and monitored on daily basis. Antimicrobial potential of the partially purified protein from this bacterium was tested against clinical isolates by well diffusion assay. The preservation efficiency of bacteriocin producing isolate at various concentrations was tested against selected food samples and compared with two chemical preservatives such as sodium sulphite and sodium benzoate. The bacteriocin was partially purified and the microbiological qualities of the biopreservative treated food samples were assessed. The present study suggested that 100 µg/l of bacteriocin extract demonstrated antimicrobial potential against E. coli and Shigella spp. The treatment with the Pediococcus spp. showed enhanced preservation at 15 mL/kg of selected samples for a period of 15 days in comparison with sodium sulphite and sodium benzoate. The microbiological quality of food samples treated with biopreservative showed lesser total bacterial count (CFU/g) in comparison with the food samples applied with chemicals (p ≤ 0.05). Thus, the present study suggests that bacteriocin producing Pediococcus probably provides enhanced shelf life to the selected food samples and can be used as biopreservatives.

Dipstick Assay for Rapid Detection of Beer Spoilage Organisms.

Background: Beer spoilage caused by wild yeast and bacteria is a major concern to both commercial and home brewers. Objective: To address this problem, Molecular Epidemiology Inc. (MEI, Seattle, WA) has developed a beer spoilage organism detection kit consisting of an enrichment media (BSE) and a multiplex PCR DNA dipstick that simultaneously detects these organisms within 2 h following enrichment. Methods: The kit was tested by using samples obtained from breweries located in the Greater Seattle area. Samples were spiked with the target microbes, when necessary, and used for assessing the performance characteristics of the DNA dipstick assay. Microbial enumerations were performed as per the standard microbiological plating methods. The suitability of the BSE medium to support the growth of beer spoilage microbes was compared with the industry-approved NBB-C medium (Dohler, Darmstadt, Germany). Results: Inclusivity (a panel of 50 isolates) and Exclusivity (a panel of 92 isolates) testing indicated that the dipstick assay can exclusively detect the indicated target beer spoilage microbes. When compared with the NBB-C medium (Dohler, Darmstadt, Germany) approved by the European Brewers Convention for beer spoilage organisms, the BSE medium supported faster growth of critical spoilage lactic acid bacteria such as Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Conclusions: The beer spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the brewing process, thus offering a convenient, cost effective, and faster test system for brewers interested in monitoring the quality of their product.

Composition of lactic acid bacteria during spontaneous curly kale (Brassica oleracea var. sabellica) fermentation.

The present work is the first report on spontaneous fermentation of curly kale and characteristics of autochthonous lactic acid bacteria (LAB). Our results indicate that curly kale fermentation is the new possibility of the technological use of this vegetable. Bacteria representing ten different species were isolated from three phases of curly kale fermentation and identified by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Among them, four species were identified as Lactobacillus spp. (Lb. plantarum 332, Lb. paraplantarum G2114, Lb. brevis R413, Lb. curvatus 154), two as Weissella spp. (W. hellenica 152, W. cibaria G44), two as Pediococcus spp. (P. pentosaceus 45AN, P. acidilactici 2211), one as Leuconostoc mesenteroides 153, and one as Lactococcus lactis 37BN. The functional properties of isolates, i.e. acid, NaCl and bile salt tolerance, enzyme activities, adhesion to hydrocarbons, and antibiotic resistance, were examined. Among the tested strains, Lb. plantarum 332, Lb. paraplantarum G2114, P. pentosaceus 2211, and Lb. brevis R413 exhibited the best hydrophobicity value and high tolerance to bile salts, NaCl, and low pH.

Lactobacillus backii and Pediococcus damnosus isolated from 170-year-old beer recovered from a shipwreck lack the metabolic activities required to grow in modern lager beer.

In 2010, bottles of beer containing viable bacteria of the common beer-spoilage species Lactobacillus backii and Pediococcus damnosus were recovered from a shipwreck near the Åland Islands, Finland. The 170-year quiescent state maintained by the shipwreck bacteria presented a unique opportunity to study lactic acid bacteria (LAB) evolution vis-a-vis growth and survival in the beer environment. Three shipwreck bacteria (one L. backii strain and two P. damnosus strains) and modern-day beer-spoilage isolates of the same two species were genome sequenced, characterized for hop iso-α-acid tolerance, and growth in degassed lager and wheat beer. In addition, plasmid variants of the modern-day P. damnosus strain were analyzed for the effect of plasmid-encoded genes on growth in lager beer. Coding content on two plasmids was identified as essential for LAB growth in modern lager beer. Three chromosomal regions containing genes related to sugar transport and cell wall polysaccharides were shared by pediococci able to grow in beer. Our results show that the three shipwreck bacteria lack the necessary plasmid-located genetic content to grow in modern lager beer, but carry additional genes related to acid tolerance and biofilm formation compared to their modern counterparts.

Use of hop extract as antifungal ingredient for bread making and selection of autochthonous resistant starters for sourdough fermentation.

Aiming at meeting the consumers' demand in terms of bio-preservation, the potential of the combination of the lactic acid bacteria fermentation and the addition of hop extract as natural preservative in breadmaking, was exploited. The antifungal properties of a hop (Humulus lupulus) extract were investigated, showing a significant inhibition of the hyphal growth of Aspergillus parasiticus, Penicillium carneum, Penicillium polonicum, Penicillium paneum, Penicillium chermesinum, Aspergillus niger, Penicillium roqueforti. Lactic acid bacteria belonging to species of Enterococcus feacium, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus curvatus, Pediococcus pentosaceus, and Pediococcus acidilactici were isolated from hop and subjected to selection based on kinetics of growth and acidification. The sourdough (hS) enriched with hop extract (hE), started with three selected strains, had phenols concentration and antioxidant activity higher than those obtained in the same condition but without the hE. Hop-sourdough used in breadmaking delayed the fungal growth (14 days), giving a bread characterized by free aminoacids concentration, antioxidant and phytase activities higher than bread started only with baker's yeast, with or without the addition of hE. Specific volume and cell-total area of the bread containing hE improved, and its sensory profile was characterized by typical sourdough attributes, and a moderate bitter/herbaceous perception.

Pediococcus acidolactici and Pediococcus pentosaceus isolated from a rainbow trout ecosystem have probiotic and ABF1 adsorbing/degrading abilities in vitro.

Probiotics are being used in biological control of bacterial pathogens, as an alternative to antibiotics, to improve health and production parameters in fish farming. Fish farming production is severely affected by aflatoxins (AFs), which are a significant problem in aquaculture systems. Aflatoxins exert substantial impact on production, causing disease with high mortality and a gradual decline of reared fish stock quality. Some aspects of aflatoxicosis in fish, particularly its effects on the gastrointestinal tract, have not been well documented. The aim of the present study was to evaluate probiotic properties of lactic acid bacterial (LAB) strains isolated from rainbow trout intestine and feed. Moreover, AFB1-binding and/or degrading abilities were also evaluated to assess their use in the formulation of feed additives. Growth at pH 2, the ability to co-aggregate with bacterial pathogens, inhibition of bacterial pathogens, and determination of the inhibitory mechanism were tested. Aflatoxin B1 (AFB1) adsorption and degradation ability were also tested. All strains were able to maintain viable (107 cells ml-1) at pH 2. Pediococcus acidilactici RC001 and RC008 showed the strongest antimicrobial activity, inhibiting all the pathogens tested. The strains produced antimicrobial compounds of different nature, being affected by different treatments (catalase, NaOH and heating), which indicated that they could be H2O2, organic acids or proteins. All LAB strains tested showed the ability to coaggregate pathogenic bacteria, showing inhibition percentages above 40%. Pediococcus acidilactici RC003 was the one with the highest adsorption capacity and all LAB strains were able to degrade AFB1 with percentages higher than 15%, showing significant differences with respect to the control. The ability of some of the LAB strains isolated in the present work to compete with pathogens, together with stability against bile and gastric pH, reduction of bioavailability and degradation of AFB1, may indicate the potential of LAB for use in rainbow trout culture.

Discrimination of wine lactic acid bacteria by Raman spectroscopy.

Species of Lactobacillus, Pediococcus, Oenococcus, and Leuconostoc play an important role in winemaking, as either inoculants or contaminants. The metabolic products of these lactic acid bacteria have considerable effects on the flavor, aroma, and texture of a wine. However, analysis of a wine's microflora, especially the bacteria, is rarely done unless spoilage becomes evident, and identification at the species or strain level is uncommon as the methods required are technically difficult and expensive. In this work, we used Raman spectral fingerprints to discriminate 19 strains of Lactobacillus, Pediococcus, and Oenococcus. Species of Lactobacillus and Pediococcus and strains of O. oeni and P. damnosus were classified with high sensitivity: 86-90 and 84-85%, respectively. Our results demonstrate that a simple, inexpensive method utilizing Raman spectroscopy can be used to accurately identify lactic acid bacteria isolated from wine.

Sourdough-type propagation of faba bean flour: Dynamics of microbial consortia and biochemical implications.

The microbial ecology of faba bean sourdoughs obtained from an Italian (Ita) and a Finnish (Fi) cultivar, belonging respectively to Vicia faba major and V. faba minor groups, was described by 16S rRNA gene pyrosequencing and culture-dependent analysis. The sourdoughs were propagated with traditional backslopping procedure throughout 14days. Higher microbial diversity was found in the sourdough deriving from V. faba minor (Fi), still containing residual hulls after the milling procedure. After 2days of propagation, the microbial profile of Ita sourdough was characterized by the dominance of the genera Pediococcus, Leuconostoc and Weissella, while the genera Lactococcus, Lactobacillus and Escherichia, as well as Enterobacteriaceae were present in Fi sourdoughs. Yeasts were in very low cell density until the second backslopping and were not anymore found after this time by plate count or pyrosequencing analysis. Among the lactic acid bacteria isolates, Pediococcus pentosaceus, Leuconostoc mesenteroides and Weissella koreensis had the highest frequency of occurrence in both the sourdoughs. Lactobacillus sakei was the only lactobacillus isolated from the first to the last propagation day in Fi sourdough. According to microbiological and acidification properties, the maturity of the sourdoughs was reached after 5days. The presence of hulls and the different microbial composition reflected on biochemical characteristics of Fi sourdoughs, including acidification and phenolic compounds. Moreover, proteolysis in Fi sourdough was more intense compared to Ita. The microbial dynamic of the faba bean sourdoughs showed some differences with the most studied cereal sourdoughs.

Reassessment of the succession of lactic acid bacteria in commercial cucumber fermentations and physiological and genomic features associated with their dominance.

A compositional re-assessment of the microbiota present in commercial cucumber fermentation using culture independent and dependent methods was conducted, with emphasis on lactic acid bacteria (LAB). Two commercial cucumber fermentation tanks were monitored by measuring pH, dissolved oxygen and temperature, and used as sources of samples for microbial plating, genomic DNA extraction and measurement of organic acids and carbohydrates by HPLC. Six additional commercial tanks were included to identify the dominant microorganisms using molecular methods. A comparative analysis of the publically available genome sequences corresponding to the LAB found in cucumber fermentations was completed to gain an understanding of genomic features possibly enabling dominance. Analyses of the microbiota suggest Lactobacillales prevail in cucumber fermentations, including in order of prevalence Lactobacillus pentosus, Lb. plantarum, Lb. brevis, Weissella spp., Pediococcus ethanolidurans, Leuconostoc spp. and Lactococcus spp. It was observed that Lb. pentosus and Lb. plantarum have comparatively larger genomes, higher gene counts, uniquely distribute the ribosomal clusters across the genome as opposed to close to the origin of replication, and possess more predicted amino acids prototrophies and selected biosynthesis related genes. It is theorized that Lb. pentosus and Lb. plantarum dominance in cucumber fermentations is the result of their genetic make-up.

Isolation of lactic acid-forming bacteria from biogas plants.

Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative multiplex system and a real-time quantitative PCR could be developed for most isolates, realized by the selection of specific primers. Their role in biogas plants was discussed on the basis of the quantitative results and on physiological data of the isolates.

Rapid and accurate identification of species of the genus Pediococcus isolated from Korean fermented foods by matrix-assisted laser desorption/ionization time-of-flight MS with local database extension.

Pediococci are halophilic lactic acid bacteria, within the family Lactobacillaceae, which are involved in the fermentation of various salted and fermented foods, such as kimchi and jeotgal. In this study, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS method was developed for the rapid identification of species of the genus Pediococcus. Of the 130 Pediococcus spectra aligned with the Biotyper taxonomy database, 122 isolates (93.9 %) yielded log scores <1.7, which means they were not identifiable. After registering the spectra of 11 reference strains of the genus Pediococcus, all of the isolates were correctly identified, of which 84 (64.6 %) and 46 (35.4 %) were identified at the species and genus level, respectively. In comparing food origins, no relationship was found between the bacterial characteristics and food environment. We were able to produce a Biotyper system for identification of members of the genus Pediococcus with locally extended Pediococcus reference strains. The MALDI-TOF MS method is fast, simple and reliable for discriminating between species in the genus Pediococcus and therefore will be useful for quality control in determining the spoilage of alcoholic beverages or in the production of fermented food.

Monitoring of wheat lactic acid bacteria from the field until the first step of dough fermentation.

The present work was carried out to retrieve the origin of lactic acid bacteria (LAB) in sourdough. To this purpose, wheat LAB were monitored from ear harvest until the first step of fermentation for sourdough development. The influence of the geographical area and variety on LAB species/strain composition was also determined. The ears of four Triticum durum varieties (Duilio, Iride, Saragolla and Simeto) were collected from several fields located within the Palermo province (Sicily, Italy) and microbiologically investigated. In order to trace the transfer of LAB during the consecutive steps of manipulation, ears were transformed aseptically and, after threshing, milling and fermentation, samples of kernels, semolinas and doughs, respectively, were analysed. LAB were not found to dominate the microbial communities of the raw materials. In general, kernels harboured lower levels of microorganisms than ears and ears than semolinas. Several samples showing no development of LAB colonies acidified the enrichment broth suggesting the presence of LAB below the detection limit. After fermentation, LAB loads increased consistently for all doughs, reaching levels of 7.0-7.5 Log CFU/g on M17. The values of pH (5.0) and TTA (5.6 mL NaOH/10 g of dough) indicated the occurrence of the acidification process for several doughs. LAB were phenotypically and genotypically differentiated by randomly amplified polymorphic DNA (RAPD)-PCR into eight groups including 51 strains belonging to the species Lactobacillus brevis, Lactobacillus coryniformis, Lactobacillus plantarum, Lactococcus lactis, Lactococcus garvieae, Enterococcus casseliflavus, Enterococcus faecium, Leuconostoc citreum, and Pediococcus pentosaceus. Lactobacilli constituted a minority the LAB community, while lactococci represented more than 50% of strains. Lower LAB complexity was found on kernels, while a richer biodiversity was observed in semolinas and fermented doughs. For broader microbiota characterisation in doughs before fermentation, the 16S rRNA gene fragment profiling was conducted on the unfermented doughs using MiSeq Illumina. LAB group was represented by Enterococcus, Lactococcus and members of Leuconostocaceae family whose relative abundances differed according to both geographical area and variety of wheat. The culture-independent approach confirmed that pediococci and lactobacilli constituted low abundance members of the semolina LAB microbiota and that although some strains may pass from wheat ear to fermented doughs, most are likely to come from other sources.