Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Isolation and characterisation of lactic acid bacteria from yan-jiang (fermented ginger), a traditional fermented food in Taiwan

BACKGROUND: Yan‐jiang (fermented ginger) is a popular traditional fermented food in Taiwan. The lactic acid bacteria (LAB) microflora in yan‐jiang has not been studied in detail. In this study, LAB from yan‐jiang were isolated, characterised and identified. RESULTS: A total of 176 LAB were isolated; 160 cultures were isolated from yan‐jiang samples and 16 cultures were isolated from raw ginger. These isolates were characterised phenotypically and then divided into nine groups (A to I) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus sakei and Lactococcus lactis subsp. lactis were the major LAB found in the initial 2 days of fermentation without pickled plums; these species were mostly replaced by Weissella cibaria and L. plantarum after 3 days of fermentation. In the fermentation bucket with added pickled plums, W. cibaria was the most abundant LAB found during fermentation. The antibacterial activities of the isolates were determined. Twenty‐four Lc. lactis subsp. lactis and 19 W. cibaria strains showed inhibitory activity against the indicator strain L. sakei JCM 1157^T. CONCLUSION: Results demonstrate that various LAB species were more numerous when fermentation was carried out without pickled plums. LAB also had effects on the aroma and flavour of yan‐jiang. Copyright © 2011 Society of Chemical Industry     

Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Identification of lactic acid bacteria isolated during traditional fura processing in Ghana

Fura is a millet-based spontaneously fermented dumpling produced and consumed in parts of West Africa, particularly Nigeria, Burkina Faso and Ghana. From eight traditional fura production sites in northern Ghana, 862 lactic acid bacteria were isolated and identified to species level using a combination of genotypic and phenotypic methods including (GTG)₅-based PCR fingerprinting and 16S rRNA gene sequencing, multiplex PCR by means of recA gene sequence comparison, conventional morphological characteristics and carbohydrate fermentation profiling. During millet dough fermentation, pH decreased from 5.6–6.4 to 4.1–3.7 and total lactic acid bacteria (LAB) counts increased from 4.4–5.3 to 7.9–9.2 log₁₀ (cfu/g). The initial stages of the fermentation were characterized by co-dominance of homo- and heterofermentative species of Pediococcus acidilactici, Weisella confusa, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus paraplantarum whereas L. fermentum was dominating at the end of the fermentation. L. fermentum was predominant in all fermentations (p < 0.05) and a high uniformity was observed among production sites regarding the dominance of L. fermentum. L. fermentum and W. confusa were isolated in all production sites and almost at all fermentation stages indicating that they are indigenous to traditional fura processing. The other LAB bacteria species which comprised a minor proportion of the total LAB occurred occasionally and in an irregular pattern among the production sites.     

Biodiversity of yeasts, lactic acid bacteria and acetic acid bacteria in the fermentation of "Shanxi aged vinegar", a traditional Chinese vinegar

Shanxi aged vinegar is a famous traditional Chinese vinegar made from several kinds of cereal by spontaneous solid-state fermentation techniques. In order to get a comprehensive understanding of culturable microorganism’s diversity present in its fermentation, the indigenous microorganisms including 47 yeast isolates, 28 lactic acid bacteria isolates and 58 acetic acid bacteria isolates were recovered in different fermenting time and characterized based on a combination of phenotypic and genotypic approaches including inter-delta/PCR, PCR-RFLP, ERIC/PCR analysis, as well as 16S rRNA and 26S rRNA partial gene sequencing. In the alcoholic fermentation, the dominant yeast species Saccharomyces (S.) cerevisiae (96%) exhibited low phenotypic and genotypic diversity among the isolates, while Lactobacillus (Lb.) fermentum together with Lb. plantarum, Lb. buchneri, Lb. casei, Pediococcus (P.) acidilactici, P. pentosaceus and Weissella confusa were predominated in the bacterial population at the same stage. Acetobacter (A.) pasteurianus showing great variety both in genotypic and phenotypic tests was the dominant species (76%) in the acetic acid fermentation stage, while the other acetic acid bacteria species including A. senegalensis, A. indonesiensis, A. malorum and A. orientalis, as well as Gluconobacter (G.) oxydans were detected at initial point of alcoholic and acetic acid fermentation stage respectively.     

Microbial Succession and Metabolite Changes during Long‐Term Storage of Kimchi

Kimchi is often stored for a long period of time for a diet during the winter season because it is an essential side dish for Korean meals. In this study pH, abundance of bacteria and yeasts, bacterial communities, and metabolites were monitored periodically to investigate the fermentation process of kimchi for 120 d. Bacterial abundance increased quickly with a pH decrease after an initial pH increase during the early fermentation period. After 20 d, pH values became relatively stable and free sugars were maintained at relatively constant levels, indicating that kimchi fermentation by lactic acid bacteria (LAB) was almost completed. After that time, a decrease in bacterial abundance and a growth in Saccharomyces occurred concurrently with increased free sugar consumption and production of glycerol and ethanol. Finally, after 100 d, the growth of Candida was observed. Community analysis using pyrosequencing revealed that diverse LAB including Leuconostoc citreum, Leuconostoc holzapfelii, Lactococcus lactis, and Weissella soli were present during the early fermentation period, but the LAB community was quickly replaced with Lactobacillus sakei, Leuconostoc gasicomitatum, and Weissella koreensis as the fermentation progressed. Metabolite analysis using ¹H‐NMR showed that organic acids (lactate, acetate, and succinate) as well as bioactive substances (mannitol and gamma‐aminobutyric acid (GABA)) were produced during the kimchi fermentation, and Leuconostoc strains and Lactobacillus sakei were identified as the producers of mannitol and GABA, respectively. Practical Application In this study, we have shown that the growth inhibition of yeasts including Saccharomyces and Candida is necessary to extend the shelf life of kimchi in long‐term storage. Additionally, we have shown that a mixed culture of Leuconostoc strains and Lactobacillus sakei is necessary to produce kimchi that contains both mannitol and gamma‐aminobutyric acid.     

Phenotypic and genotypic identification of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages

Aims: A survey on lactic acid bacteria isolated from a small-scale facility producing traditional dry sausage. A first report on LAB in raw material, products and processing equipment.Methods and Results: A total of 88 isolates were identified phenotypically and genotypically. Phenotypic characterization at the species level indicated that 36 (40.9%) were Lactobacillus sakei, 27 (30.68%) were Enterococcus faecium, 14 (15.90%) were “Enterococcus seriolicida”, 10 (11.36%) were Enterococcus faecalis and 1 (1.13%) was Leuconostoc mesenteroides subsp. mesenteroides/dextranicum 2. In general, genotypic identification correlated well with phenotypic classification since it indicated 36 Lb. sakei, 22 Ec. faecium, 16 Lactococcus garvieae (senior synonym of Ec. seriolicida), 11 Vagococcus carniphilus and 1 Ln. mesenteroides subsp. mesenteroides. Two isolates were assigned as Enterococcus spp.Conclusions: While Ec. faecium, Vc. carniphilus and Lc. garvieae were the predominant species isolated from processing equipment and raw material, Lb. sakei species became the predominant species during fermentation of the product.Significance and Impact of the Study: This study constitutes the first step in the designing process of LAB starter cultures endogenous to the small-scale facility in order to protect the typical organoleptic characteristics of its traditional dry sausage. Otherwise, the presence of undesirable micro-organisms in the processing equipment emphasizes the necessity of improving the general hygienic conditions of sausage production in this facility.     

Microorganisms associated with Maari, a Baobab seed fermented product

A microbiological study was carried out on Baobab fermented seeds (Maari) obtained from 4 different production sites in Burkina Faso (Mansila, Toulfé, Ouagadougou and Gorgadji).A total of 390 representative isolates comprising 251 aerobic mesophilic bacteria (AMB) and 139 lactic acid bacteria (LAB) were isolated and identified to species level using a combination of pheno- and genotypic methods including conventional morphological analysis, carbohydrate fermentation profiling, rep-PCR ((GTG)₅-fingerprinting) and 16S rRNA gene sequencing.The fermentation of Baobab seeds was initiated by the AMB identified as Bacillus subtilis (82% of AMB isolates) and Staphylococcus sciuri (18% of AMB isolates). No lactic acid bacteria were isolated at the beginning of the process. After 24 h fermentation time, Enterococcus faecium appeared in the fermenting seeds and remained until the end of the fermentation, as the predominant LAB.In Maari collected from retail outlets the AMB count ranged from 6.7 log10 CFU/g to 10 log10 CFU/g while the LAB load ranged from 4.4 log10 CFU/g to 9.9 log10 CFU/g. The AMB were identified as belonging to genus Bacillus (12 species), Staphylococcus (3 species) and one species of Aerococcus, Macrococcus, Leifsonia, Kurthia, Proteus, Acinetobacter and Globicatella, respectively. A putatively novel, previously undescribed Corynebacterium sp. was also found. E. faecium was the dominant LAB in all investigated retail samples except one sample dominated by Pediococcus acidilactici.     

Analysis of volatile compounds produced by different species of lactobacilli in rye sourdough using multiple headspace extraction

Profiles of volatile organic compound (VOC) produced by nine individual lactic acid bacteria (LAB) during rye sourdough fermentation were compared by automated SPME and GC/MS‐Tof. The dough samples were inoculated with individual strains, placed inside the headspace vials and incubated during next 24 h. The production or loss of VOC‐s was followed by adsorbing volatiles onto 85‐m Car/PDMS fibre in every 4 h. Volatile profiles differed among LAB species and divided LAB into two main groups – hetero‐ and homofermentative. Hetrofermentative LAB (Lactobacillus brevis; Leuconostoc citreum; Lactobacillus vaginalis, Lactobacillus panis) showed high production of acetic acid, CO₂, ethanol, ethylacetate, producing also hexyl acetate, ethyl hexanoate and isopentyl acetate. Whereas homofermentative LAB species (Lactobacillus helveticus; Lactobacillus casei; Lactobacillus sakei; Lactobacillus curvatus) produced a considerable amount of 2,3‐butanedione. Production of l ‐leucine methyl ester was unique for Lb. sakei, Lb. casei and Lb. curvatus strains. Lb. helveticus was the only LAB that produced benzaldehyde.     

Isolation of halophilic lactic acid bacteria from traditional Chinese fermented soybean paste and assessment of the isolates for industrial potential

Lactic acid bacteria (LAB) from traditional Chinese fermented soybean paste were isolated and identified. A total of 61 LAB were selectively obtained from 32 homemade Chinese soybean pastes. The isolated LAB were divided into two groups by their salt tolerance, 28 halophilic LAB and 33 non-halophilic LAB. Phenotypic analysis showed that these LAB belonged to four genera and 13 species. Tetragenococcus halophilus was the predominant species in the identified strains. Four species of LAB were firstly isolated from fermented soybean food product, Lactobacillus panis, Lactobacillus pentosus, Lactobacillus vaccinostercus and Lactobacillus collinoides. T. halophilus T5 showed vigorous growth and fast acidification in high salt concentration. The volatile compounds of mixed microorganism soybean paste with T. halophilus T5, Zygosaccharomyces and Torulopsis candida, during the different fermentation stage were higher in number than those of normal soybean paste at same processing procedure.     

Bacterial community dynamics,lactic acid bacteria species diversity and metabolite kinetics of traditional Romanian vegetable fermentations

BACKGROUND: Artisanal vegetable fermentations are very popular in Eastern European countries. Fresh vegetables undergo a spontaneous fermentation in the presence of salt, which is mainly carried out by lactic acid bacteria (LAB). RESULTS: Culture‐dependent and culture‐independent analyses of end‐samples of various spontaneous vegetable fermentations carried out in houses of the Chiodju region (central Romania) revealed Lactobacillus plantarum and Lactobacillus brevis as the most frequently isolated LAB species. Leuconostoc mesenteroides and Leuconostoc citreum were also found. Furthermore, the community dynamics of spontaneous cauliflower and mixed‐vegetable (green tomatoes, carrots and cauliflower) fermentations revealed three steps: an initial phase characterised by the presence of Enterobacteriaceae and a wide LAB species diversity, encompassing Weissella species; a second phase from day 3 onwards wherein L. citreum and Lb. brevis occurred; and a final phase characterised by the prevalence of Lb. brevis and Lb. plantarum. Metabolite target analysis revealed that glucose and fructose were mostly depleted at the end of fermentation. The main products of carbohydrate metabolism were lactic acid, acetic acid, ethanol and small amounts of mannitol, indicating heterolactate fermentation. CONCLUSION: Given their prevalence at the end of vegetable fermentations, Lb. brevis and Lb. plantarum appear to be good candidate starter cultures for controlled vegetable fermentation processes. © 2012 Society of Chemical Industry     

Halotolerance and Survival Kinetics of Lactic Acid Bacteria Isolated from Jalapeño Pepper (Capsicum annuum L.) Fermentation

The microbiota associated with spontaneous fermentation of vegetables in a saline substrate may represent an important group of bacteria in the food industry. In this work, the lactic acid bacteria (LAB) Weissella cibaria, Lactobacillus plantarum, Lactobacillus paraplantarum, and Leuconostoc citreum were identified by partial 16S rRNA gene sequence analysis. In addition, entophytic bacteria such as Pantoea eucalypti, Pantoea anthophila, Enterobacter cowanii, and Enterobacter asburiae were detected, but they were irrelevant for the fermentation process and were inhibited after 12 h of fermentation when the pH decreased from 6.5 to 4.9. Moreover, 2 species of yeast were isolated and identified as Hanseniaspora pseudoguilliermondii and Kodamaea ohmeri by their partial 26S rRNA gene sequence. The growth of LAB was evaluated at different sodium chloride contents. L. citreum was the most halotolerant species followed by L. plantarum and W. cibaria with a concentration index to obtain a 50% population reduction (IC₅₀) of 7.2%, 6.6%, and 5.2%, respectively. Furthermore, the growth of LAB and Escherichia coli O157:H7 was evaluated in the presence of the main phenylpropanoids from chilli peppers such as p‐coumaric and ferulic acid. It was determined that LAB can grow in both acids at 4 mM, unlike E. coli O157:H7, whose growth is inhibited in the presence of these acids.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

Isolation and characterization of lactic acid bacteria from jiang‐sun (fermented bamboo shoots), a traditional fermented food in Taiwan

BACKGROUND: In this study, lactic acid bacteria (LAB) were isolated, characterized and identified from jiang‐sun (fermented bamboo shoots; a traditional fermented food in Taiwan). Samples were collected at seven time intervals from a fixed fermenting bucket during the fermentation process of jiang‐sun and its initial ingredients, dochi (fermented soybeans) and bamboo shoots. RESULTS: A total of 57 LAB cultures were isolated; 42 cultures were isolated from jiang‐sun samples and 15 cultures were isolated from dochi and bamboo shoots. These isolates were characterized phenotypically and then divided into three groups (A–C) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Alteration of microbial populations during the fermentation process was observed. While various LAB were found in the dochi and bamboo shoots, it was mostly replaced by Lactobacillus plantarum after 1 day of fermentation. Furthermore, the antibacterial activities of the isolates were determined, and one Enterococcus faecium strain showed inhibitory activity against all the indicator strains. CONCLUSION: Results suggest that L. plantarum is the main LAB present during the fermentation of jiang‐sun. To our knowledge, this is the first report describing the distribution and varieties of LAB that exist in the jiang‐sun fermentation process. Copyright © 2010 Society of Chemical Industry     

Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava and use of competitive strains as starters

The adaptability of lactic acid bacteria (LAB) and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava was investigated using PCR-DGGE and bacteriological culture combined with rRNA gene sequence analysis. Sourdoughs were prepared either from flours of the cereals wheat, rye, oat, barley, rice, maize, and millet, or from the pseudocereals amaranth, quinoa, and buckwheat, or from cassava, using a starter consisting of various species of LAB and yeasts. Doughs were propagated until a stable microbiota was established. The dominant LAB and yeast species were Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus spicheri, Issatchenkia orientalis and Saccharomyces cerevisiae. The proportion of the species within the microbiota varied. L. paralimentarius dominated in the pseudocereal sourdoughs, L. fermentum, L. plantarum and L. spicheri in the cassava sourdough, and L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs. S. cerevisiae constituted the dominating yeast, except for quinoa sourdough, where I. orientalis also reached similar counts, and buckwheat and oat sourdoughs, where no yeasts could be detected. To assess the usefulness of competitive LAB and yeasts as starters, the fermentations were repeated using flours from rice, maize, millet and the pseudocereals, and by starting the dough fermentation with selected dominant strains. At the end of fermentation, most of starter strains belonged to the dominating microbiota. For the rice, millet and quinoa sourdoughs the species composition was similar to that of the prior fermentation, whereas in the other sourdoughs, the composition differed.     

Identification of the lactic acid bacteria in Kimchi according to initial and over-ripened fermentation using PCR and 16S rRNA gene sequence analysis

This paper aimed to identify the lactic acid bacteria species involved in kimchi fermentation at different fermentation periods by using culture-independent 16S rRNA gene clone libraries, and develop polymerase chain reaction for the detection of lactic acid bacteria (LAB). We investigated 6 commercially produced kimchi samples, including kimchi at an initial stage of fermentation and kimchi that was fermented to an over-ripened stage. The results of our study show that the analysis with cultureindependent 16S rRNA gene clone libraries could successfully identify 134 clones, 11 species, including Weissella, Lactobacillus, Pediococcus, and Leuconostoc from the 6 commercial kimchi samples. Weisella koreensis and Lactobacillus brevis were the predominant LAB in the initial stage of kimchi fermentation at 4°C (pH 4.96–5.27 and acidity 0.81–0.88), and Leuconostoc gelidum and Lactobacillus sakei subsp. sakei may play an important role in kimchi fermentation at the over-ripened stage (pH 3.61–3.91 and acidity 1.70–1.79).     

A Review of Hop Resistance in Beer Spoilage Lactic Acid Bacteria

Hop bitter acids play a major role in enhancing the microbiological stability of beer. However, beer spoilage lactic acid bacteria (LAB) are able to grow in beer by exhibiting strong hop resistance. Recently two hop resistance genes, horA and horC, have been identified in beer spoilage Lactobacillus brevis ABBC45. The horA gene was shown to encode an ATP dependent multidrug transporter that extrudes hop bitter acids out of bacterial cells. In contrast, the product of the horC gene confers hop resistance by presumably acting as a proton motive force (PMF)‐dependent multidrug transporter. Strikingly, the homologs of horA and horC genes were found to be widely and almost exclusively distributed in various species of beer spoilage LAB strains, indicating these two hop resistance genes are excellent species‐independent genetic markers for differentiating the beer spoilage ability of LAB. Furthermore the nucleotide sequence analysis of horA and horC homologs revealed that both genes are essentially identical among distinct beer spoilage species, indicating horA and horC have been acquired by beer spoilage LAB through horizontal gene transfer. Taken collectively, these insights provide a basis for applying horA and horC to the species‐independent determination of beer spoilage LAB, including yet uncharacterized species. In addition to the hop resistance mechanisms mediated by multidrug transporters, proton translocating ATPase and the ATP production system were shown to contribute to the hop resistance mechanisms in beer spoilage LAB by generating PMF and ATP that are necessary for survival in beer.     

Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider

A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc⁺ (2% of strains) and especially odc⁺ (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines.     

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (< 1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.