Diversity of bacteria and yeast in the naturally fermented cotton seed and rice beverage produced by Brazilian Amerindians

Microorganisms associated with the fermentation of cotton seed and rice were studied using a combination of culture-dependent and -independent methods. Samples of the cotton seed and rice beverage were collected every 8 h during the fermentation process for analysis of the microbiota present over 48 h. The lactic acid bacteria (LAB) population reached values of approximately 8.0 log cfu/mL. A total of 162 bacteria and 81 yeast isolates were identified using polyphasic methods. LAB (Lactobacillus plantarum, Lactobacillus vermiforme, Lactobacillus paracasei) were the most frequently isolated bacteria. Bacillus subtilis was present from 16 h until the end of the fermentation process. A decrease in pH value from 6.92 (0 h) to 4.76 (48 h) was observed, and the concentration of lactic acid reached 24 g/L at the end of the fermentation process. DGGE (denaturing gradient gel electrophoresis) was performed to determine the dynamics of the communities of bacteria and yeast, and the analysis revealed a predominance of LAB throughout the fermentation process. No changes were observed in the yeast community. The yeast species detected were Candida parapsilosis, Candida orthopsilosis, Clavispora lusitaniae and Rhodotorula mucilaginosa. Our studies indicate that the DGGE technique combined with a culture-dependent method is required to discern the dynamics in the fermentation of cotton seed and rice.     

Effects of pectinolytic yeast on the microbial composition and spoilage of olive fermentations

This study resulted in the identification of pectinolytic yeasts in directly brined Sicilian-style green olive fermentations and examination of the influence of those yeasts on the microbial composition and quality of fermented olives. Firstly, defective olives processed in Northern California from 2007 to 2008 and characterized by high levels of mesocarp tissue degradation were found to contain distinct yeast and bacterial populations according to DNA sequence-based analyses. Strains of (pectinolytic) Saccharomyces cerevisiae, Pichia manshurica, Pichia kudriavzevii, and Candida boidinii isolated from directly brined olives were then inoculated into laboratory-scale olive fermentations to quantify the effects of individual yeast strains on the olives. The pH, titratable acidity, and numbers of lactic acid bacteria (LAB) and yeasts varied between the fermentations and fermentations inoculated with P. kudriavzevii and C. boidinii promoted the development of LAB populations. Olive tissue structural integrity declined significantly within 30, 74, and 192 days after the inoculation of pectinolytic S. cerevisiae, P. manshurica and C. boidinii, respectively. In comparison, tissue integrity of olives in control fermentations remained intact although pectinolytic yeasts were present. Notably, pectinolytic yeasts were not found in fermentations inoculated with (non-pectinolytic) P. kudriavzevii and olives exposed to a 1:1 ratio of P. kudriavzevii and P. manshurica exhibited no significant tissue defects. This study showed that pectinolytic yeast are important components of directly brined green olive fermentations and damage caused by pectinolytic yeasts might be prevented by other microbial colonists of the olives.     

Heat shock effects on the viability of Cronobacter sakazakii during the dehydration,fermentation, and storage of lactic cultured milk products

In the present study, the viability of heat-shocked and non-shocked Cronobacter sakazakii, a foodborne pathogen, after drying and during the fermentation as well as storage of lactic cultured milk was evaluated. It was found that heat shock increased the viability of C. sakazakii. The pure culture of C. sakazakii, regardless of heat shock, grew rapidly in skim milk with a viable population of ca. 8.59–8.70 log cfu/ml after ca. 48 h of cultivation. Thereafter, the viable population of C. sakazakii remained stable. While in the mix culture with Streptococcus thermophilus or Lactobacillus bulgaricus, a marked reduction in the viable population of C. sakazakii was noted after 24 h of cultivation in skim milk. Nevertheless, at the end of fermentation, the heat-shocked C. sakazakii had a viable population of 5.93–6.01 log cfu/ml, which is significantly higher (P < 0.05) than that of non-shocked cells of 4.96–4.99 log cfu/ml. While the presence of C. sakazakii did not affect the growth of lactic acid bacteria in skim milk. Additionally, heat shock was found to enhance the survival of C. sakazakii after freeze-drying or spray-drying and during the storage of the lactic fermented milk products (pH 4.3) at 5 °C for 48 h.     

Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs

In this study, four different laboratory scale gluten-free (GF) sourdoughs were developed from buckwheat or teff flours. The fermentations were initiated by the spontaneous biota of the flours and developed under two technological conditions (A and B). Sourdoughs were propagated by continuous back-slopping until the stability was reached. The composition of the stable biota occurring in each sourdough was assessed using both culture-dependent and -independent techniques. Overall, a broad spectrum of lactic acid bacteria (LAB) and yeasts species, belonging mainly to the genera Lactobacillus, Pediococcus, Leuconostoc, Kazachstania and Candida, were identified in the stable sourdoughs. Buckwheat and teff sourdoughs were dominated mainly by obligate or facultative heterofermentative LAB, which are commonly associated with traditional wheat or rye sourdoughs. However, the spontaneous fermentation of the GF flours resulted also in the selection of species which are not consider endemic to traditional sourdoughs, i.e. Pediococcus pentosaceus, Leuconostoc holzapfelii, Lactobacillus gallinarum, Lactobacillus vaginalis, Lactobacillus sakei, Lactobacillus graminis and Weissella cibaria. In general, the composition of the stable biota was strongly affected by the fermentation conditions, whilst Lactobacillus plantarum dominated in all buckwheat sourdoughs. Lactobacillus pontis is described for the first time as dominant species in teff sourdough. Among yeasts, Saccharomyces cerevisiae and Candida glabrata dominated teff sourdoughs, whereas the solely Kazachstania barnetti was isolated in buckwheat sourdough developed under condition A. This study allowed the identification and isolation of LAB and yeasts species which are highly competitive during fermentation of buckwheat or teff flours. Representatives of these species can be selected as starters for the production of sourdough destined to GF bread production.     

Evolution of fermenting microbiota in tarhana produced under controlled technological conditions

The purpose of this study was to evaluate the evolution of lactic acid bacteria (LAB) and yeasts during the fermentation of tarhana produced with some pasteurised ingredients and carried out at 30 and 40 °C. The chemical parameters were those typical for tarhana production. Coliform bacteria were not detected during fermentation, while LAB and yeasts were in the range 10⁷-10⁸ colony forming units (CFU) g⁻¹. Plate counts showed an optimal development of both fermenting microbial groups and the differences in cell concentrations were not significant (P > 0.05). LAB were isolated during fermentation and grouped on the basis of phenotypic and polymorphic characteristics. LAB isolates were identified by a combined genetic approach consisting of 16S/23S rRNA intergenic spacer region (ITS) and partial 16S rRNA gene sequencing as Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus brevis. Hence, the pasteurisation of the vegetable ingredients, excluded wheat flour, enhanced the hygienic conditions of tarhana without influencing the normal evolution of LAB. However, the fermentation at 40 °C favoured pediococci, while the production at 30 °C was mainly characterised by lactobacilli. Yeasts, identified by the restriction fragment length polymorphism (RFLP) of the 5.8S ITS rRNA gene, were mainly represented by the species Saccharomyces cerevisiae in both productions.     

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.     

Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP

Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC⁺) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.     

Effects of electron-beam and gamma irradiation treatments on the microbial populations, respiratory activity and sensory characteristics of Tuber melanosporum truffles packaged under modified atmospheres

The effects of electron-beam or gamma irradiation (doses of 1.5 kGy and 2.5 kGy of either one) on the microbial populations, respiratory activity and sensory characteristics of Tuber melanosporum packaged under modified atmospheres were monitored immediately after treatment, and subsequently every seven days during 35 days of storage at 4 °C. Treatments with 1.5 and 2.5 kGy reduced the total mesophilic aerobes counts respectively by 4.3 and 5.6 log cfu/g for electron-beam treatment, and by 6.4 and 6.6 log cfu/g for gamma irradiation. Other microbial groups studied (Pseudomonas genus, Enterobacteriaceae family, lactic acid bacteria, mesophilic aerobic spores, molds and yeasts) were not detected after the treatments. A decrease in the respiratory activity was detected in all the irradiated batches, indicating that the carbon dioxide levels were lower and the oxygen levels higher than those of the non-irradiated ones. Two species of yeasts, Candida sake and Candida membranifaciens var. santamariae, survived the irradiation treatments and became the dominant microbial populations with counts of up to 7.0 log cfu/g. The growth of these microorganisms was visible on the surface of irradiated truffles from day 21 onwards, affecting the flavor and the general acceptability of the ascocarps. Moreover, a watery exudate was detected in the treated truffles from the third week onwards, so the application of irradiation treatments in doses equal to or above 1.5 kGy did not preserve the quality characteristics of T. melanosporum truffles beyond 28 days.     

Influence of preprocessing methods and fermentation of adzuki beans on γ-aminobutyric acid (GABA) accumulation by lactic acid bacteria

The effects of pre-processes (immersing, germinating, and cold shock) and fermentation conditions of adzuki beans on γ-aminobutyric acid (GABA) accumulation using mixed cultures of Lactococcus lactis and Lactobacillus rhamnosus were investigated in this study. Among the preprocessing methods, cold shock treatment resulted in the highest observed GABA content (201.2 mg/100 g); a 150-fold increase compared to the non-treated adzuki beans. The LAB strains grew rapidly in cold-treated adzuki bean substrates and reached 10⁸ cfu/ml after 24 h of fermentation at 30 °C. After optimization, the GABA yield reached 68.2 mg/100 ml; a 20-fold increase compared to the non-fermentation yield. The viable cell counts of LAB remained above 10⁸ cfu/ml after 28 days of storage at 4 °C. Our results suggest that the combination of cold shock pretreatment and fermentation by LAB may be used for the preparation of adzuki beans with high GABA content, which can then be used as a natural resource of functional foods.     

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~ 10² to 10⁵-fold. Seven yeasts examined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by ~ 10³ to 10⁶-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10⁶-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.     

Lactic acid bacteria and yeasts isolated from the starter doughs for Chinese steamed buns in Thailand

Isolation of lactic acid bacteria (LAB) and yeasts from the starter dough of Chinese steamed buns from four different commercial sources in Thailand was carried out. Thirty-one lactic acid bacteria and eight yeast strains were isolated. Total counts of LAB were from 1.8 × 10⁴ to 10⁸ colonies/g sample, whilst yeasts were very low from 10 to 2.3 × 10² colonies/g sample. The pH values of all starter doughs ranged from 3.36 to 3.52 and the Total Titratable Acidity (TTA) varied from 11.1 to 17.0 ml of 0.1 N NaOH/10 g dough. All LAB isolates were identified as Lactobacillus. The phenotypic characteristics were used to cluster all the LAB isolates into two major groups (Group A and Group B), with the B group subdivided into four groups. Phylogenetic analysis, based upon partial 16S rRNA gene sequences, showed that isolates of Group A, which all contained meso-diaminopimelic acid in their cell wall and produced dl-lactic acid, were closely related to Lactobacillus plantarum, whilst the strains of Group B that produced l-lactic acid were closely related to Lactobacillus casei. For yeasts, eight isolates based on the D1/D2 domain sequences of 26S rRNA were identified as Candida tropicalis, Pichia stipitis, Candida parapsilosis, Issatchenkia orientalis and Saccharomyces cerevisiae.     

Microbiological and physicochemical characterisation of green table olives of Halkidiki and Conservolea varieties processed by the Spanish method on industrial scale

The microbiological and physicochemical changes of industrially fermented Halkidiki and Conservolea green table olives were determined. Samples were analysed to monitor the population of lactic acid bacteria (LAB), yeasts and Enterobacteriaceae, together with changes in pH, acidity, salinity, colour, lactic acid, acetic acid and ethanol. LAB and yeast species diversity was evaluated at the beginning (1 day), middle (75 days) and final (135 days) stages of fermentation by RAPD-PCR genomic fingerprinting. Results revealed vigorous lactic acid processes as indicated by the dominance of LAB over yeasts. No Enterobacteriaceae could be detected after 30 days. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) dominated in the beginning of fermentation in both varieties. In the end, Lactiplantibacillus pentosus (formerly Lactobacillus pentosus) and Pediococcus ethanolidurans prevailed in Halkidiki and Conservolea varieties, respectively. As for yeasts, Kluyveromyces lactis/marxianus and Pichia manshurica prevailed at the onset of fermentation in Halkidiki and Conservolea varieties, whereas in the end Pichia membranifaciens dominated in both varieties.     

Study of green Sicilian table olive fermentations through microbiological,chemical and sensory analyses

The production of five different green table olive cultivars was studied by a combined strategy consisting of chemical, microbiological and sensory analyses. Cultivable microflora of samples collected during processing was monitored by plate counts on seven synthetic culture media. In all samples Enterobacteriaceae, Pseudomonaceae, staphylococci, lactic acid bacteria and spore-forming bacteria were undetectable. Yeasts and moulds were countable from the day 42 (2 log CFU/ml) till the end of fermentation (6 log CFU/ml). The use of three different approaches for microorganism detection, including a culture-independent methodology, revealed the presence of barely three yeast species during the entire fermentation period: Candida parapsilosis, Pichia guilliermondii and Pichia kluyveri. Biochemical features of technological interest were evaluated for 94 strains in order to investigate their potential role in fermentation of green Sicilian table olives.     

Evaluating the individual effects of temperature and salt on table olive related microorganisms

Statistical modelling techniques were used in the present study to assess the individual effects of temperature and NaCl concentration on the growth of 10 lactic acid bacteria and 6 yeast strains mostly isolated from different forms of table olive processing and belonging to the species Lactobacillus pentosus, Lactobacillus plantarum, Saccharomyces cerevisiae, Wickerhamomyces anomalus and Candida boidinii. The mathematical models obtained in synthetic laboratory media show that yeasts, except for C. boidinii, were more resistant to a high salt concentration than lactic acid bacteria, with an MIC value ranging from 163.5 (S. cerevisiae) to 166.9 g/L (W. anomalus); while for L. pentosus and L. plantarum this parameter ranged from 110.6 to 117.6 g/L, respectively. With regards to temperature, lactic acid bacteria showed a slight trend towards supporting higher temperature values than yeasts, with the exception of S. cerevisiae. The maximum temperatures for growth of L. pentosus and L. plantarum were 41.9 and 43.0 °C, respectively; while for W. anomalus and C. boidinii they were 38.2 and 36.5 °C. The optimum temperatures for growth were also higher for L. pentosus and L. plantarum (35.5 and 32.9 °C), compared to W. anomalus and C. boidinii (29.3 and 26.9 °C, respectively). Additional experiments carried out in natural olive brines confirmed previous results, showing that high NaCl concentrations clearly favoured yeast growth and that at high temperatures LAB slightly overcame yeasts. Results obtained in this paper could be useful for industry for a better control of both table olive fermentation and packaging.     

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.     

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24 h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48 h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.     

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.     

Lactic acid bacteria isolated from ethnic preserved meat products of the Western Himalayas

We used culture- and molecular-biology-based methods to investigate the diversity of lactic acid bacteria (LAB) in the ethnic chevon (goat) meat products chartayshya, jamma and arjia of the Western Himalayas. In six chartayshya, six jamma and four arjia samples, LAB were the predominant microbial component involved in the fermentation of these samples, and the total LAB population in arjia (7.8 ± 0.1 log cfu g⁻¹; mean ± SD) was significantly higher (P < 0.05) than in chartayshya (6.9 ± 0.1 log cfu g⁻¹) and jamma (7.5 ± 0.1 log cfu g⁻¹). We identified 53 LAB samples by 16S rRNA and phenylalanyl-tRNA synthase (pheS) genes sequencing. The LAB isolates were identified as Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Leuconostoc citreum, Leuconostoc mesenteroides, Pediococcus pentosaceus, and Weissella cibaria. These results revealed that there is a high level of diversity of LAB in the Himalayan ethnic preserved meat products.     

Impact of ecological factors on the stability of microbial associations in sourdough fermentation

The limits for the stability of the microbial association 1 (Lactobacillus sanfranciscensis and Candida humilis) and association 2 (Lactobacillus reuteri, Lactobacillus johnsonii and Issatchenkia orientalis) during sourdough fermentation were evaluated by investigating the effects of the ecological factors substrate, refreshment time, temperature, amount of backslopping and competing species in different combinations on their growth. Sourdoughs were fermented in 28 batches under different conditions using the associations and possible competing strains as starters. The dominating microbiota was characterized by bacteriological culture, rRNA gene sequence analysis and RAPD-PCR. Association 1 was found to be competitive in doughs with rye and wheat flour at temperatures between 20 and 30 °C, refreshment times of 12 and 24 h, amounts of backslopping dough from 5 to 20% and against all competing lactic acid bacteria and yeasts. The processing parameters for the competitiveness of the association 2 were temperatures of 35-40 °C, refreshment times of 12-24 h and the substrates rye bran, wheat and rye flour, but not in every case. Issatchenkia orientalis could only grow when enough oxygen was available. Its cell counts fell rapidly under the limit of detection when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 h. In conclusion, the results demonstrated that the two associations were remarkably stable under most of the investigated process conditions.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.