Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria

Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)₅-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.     

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

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

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan–MGB probes

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10²–10³ cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S–23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.     

Changes in sour rotten grape berry microbiota during ripening and wine fermentation

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.     

Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.     

Directional isolation of ethanol-tolerant acetic acid bacteria from industrial fermented vinegar

The aim of this study was to isolate indigenous ethanol-tolerant acetic acid bacteria (AAB) from industrial fermented vinegar. As routine phenotypic methods for AAB isolation and identification appear to be very time-consuming and not so accurate, we adopted a two-step isolation strategy in the present study. In the preliminary screening step, GYEC agar plates with 3–10 % (v/v) ethanol as selective stress were utilized to recover potential AAB strains with ethanol tolerance from vinegar samples. In the rescreening process, acetic acid bacterial genus’ specific adhA gene was amplified as an effective DNA target for directional detection of real AAB so that non-AAB isolates could be eliminated rapidly. In this way, an AAB pure isolate named ET-7-3 with 7 % (v/v) ethanol tolerance was successfully isolated and further identified to Acetobacter pasteurianus according to 16S rDNA sequencing and phylogenetic analysis. Summarizing, by the use of ethanol as selective stress combined with molecular identification method, ethanol-tolerant AAB could be isolated from industrial fermented vinegar with both efficiency and accuracy.     

The gyrase B gene as a molecular marker to resolve interspecific relationships within the Acetobacter pasteurianus group and a novel target for species-specific PCR

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays.     

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations,underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.     

Evaluation of Bacterial Diversity in Palm Wine by 16S rDNA Analysis of Community DNA

Bacterial diversity and fermentation dynamics in palm wine, a traditional alcoholic fermented beverage, collected from upright palm trees from Idiaba community, Abeokuta, Ogun State, Nigeria were evaluated by DNA based method using the 16S rDNA of the microbial community to verify and complement previous reports, improve our understanding and document yet unreported, uncultured microbial diversity associated with palm wine. The 16S rRNA gene fragments were amplified from microbial community and genomic DNA of isolates, by Polymerase Chain Reaction (PCR) using universal primers; and sequenced. The partial sequences were identified by comparison with sequences deposited in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). This analysis revealed that 32 community clones were identified as Lactobacillus sp, Lactobacillus casei strain zhang, Lactobacillusplantarum, Leuconostoc mesenteriodes ssp dextranicum, Leuconcostoc lactis, Pediococcusparvulus strain Bpe-299, Acetobacter pomorum, Acetobacter pasteurianus, Gluconobacter oxydans, Acinetobacter calcoaceticus, Enterobacterium bacterium, Acidovorax sp, Comamonas sp, Bacillus subtilis, Staphylococcuspiscifermentans and uncultured bacteria clone D1-78. The results showed that bacterial diversity in the palm wine sample is dominated by Lactobacillus and Leuconostoc species as reported by previous workers and uncultured bacteria clone D1-78 (1 clone) was detected for the first time in palm wine.     

Rapid molecular methods for enumeration and taxonomical identification of acetic acid bacteria responsible for submerged vinegar production

The aim of the present study was to search for a rapid and reliable method to enumerate viable acetic acid bacteria (AAB) and to identify to genera and species level AAB isolates from vinegars in full acetic fermentation elaborated by the submerged method from cider, wine and spirit ethanol in industrial bioreactors. Results showed that the rapid epifluorescence staining method using the LIVE/DEAD BacLight bacterial viability kit and direct counts in Neubauer chamber rendered consistent and reliable data for viable cell counts of bacteria in all the studied vinegars. A linear correlation was shown between viable cell counts and fermentation rates. The highest fermentation rates and viable cell counts were found in cider vinegars, whereas spirit vinegars showed the lowest values for both parameters. Eighty-four AAB pure isolates were recovered from 41 different vinegar samples and were submitted to DNA extraction. PCR amplification of the 16S–23S intergenic spacer region of rDNA and subsequent sequencing were carried out to identify isolates to species level. Results showed that Gluconacetobacter europaeus was the predominant cultivable species, appearing in 79% of the total isolates. This was the unique species found in spirit vinegars, and this is the first time that AAB from spirit vinegars are taxonomically identified. Ga. europaeus was as well the predominant cultivable species in white wine vinegars. Cider vinegars presented the highest variability of species: Ga. europaeus (35.3% appearance among cultivable isolates), Ga. xylinus (35.3%), Acetobacter pasteurianus (17.6%) and Ga. hansenii (11.8%). Red wine vinegars showed cultivable isolates of the species Ga. xylinus (71.4%) and Ga. europaeus (28.6%). Summarising, both described methods for AAB enumeration and taxonomical identification proved to be fast and reliable methods, and results revealed Ga. europaeus as the cultivable major species in vinegars in full fermentation conducted by the submerged method, suggesting that Ga. europaeus strains can constitute excellent starter cultures for the elaboration of vinegars by the submerged method.     

Identification of yeast and acetic acid bacteria isolated from the fermentation and acetification of persimmon (Diospyros kaki)

Persimmon (Diospyros kaki) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomyces cerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non-Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant strain isolated throughout, while the commercial strain of S. cerevisiae was the most common strain isolated from the inoculated fermentations. The main non-Saccharomyces strains isolated included Pichia guilliermondii, Hanseniaspora uvarum, Zygosaccharomyces florentinus and Cryptococcus sp. A distinct succession of AAB was observed during the acetification process. Acetobacter malorun was abundant during the initial and mid-stages, while Gluconacetobacter saccharivorans was the main species during the final stages of these acetifications. Four additional AAB species, Acetobacter pasteurianus, Acetobacter syzygii, Gluconacetobacter intermedius and Gluconacetobacter europaeus, were also detected. We observed 28 different AAB genotypes, though only 6 of these were present in high numbers (between 25%–60%), resulting in a high biodiversity index.     

Potentially probiotic acetic acid bacteria isolation and identification from traditional dairies microbiota

Among probiotics, acetic acid bacteria (AAB) can be used as preservative agents because of their high fermentation and acidification activities. This study aimed to isolate, identify and biologically characterise Acetobacter strains from traditional Iranian dairy products. Acetobacter strains were identified by catalase assay, Gram staining, and combined repetitive sequence‐based PCR [(GTG)₅‐PCR fingerprints] and 16S rDNA gene sequencing. We identified eight strains belonging to four species, including Acetobacter aceti, Acetobacter indonesiensis, Acetobacter cibinongensis and Acetobacter syzygii. The molecular techniques could be used as an effective and rapid alternative tool to identify and characterise dairy‐associated AAB. Primary probiotic assessments, including low pH and high bile salt tolerance tests, antagonistic activity test against pathogens, and antibiotic susceptibility confirmed the probiotic properties of these AAB, particularly A. cibinongensis 34L strain, which was isolated from curd. Therefore, this strain can be introduced as novel candidate probiotics that could be used in the food industry.     

Metallic content of wines from the Canary Islands (Spain). Application of artificial neural networks to the data analysis

Eleven elements, K, Na, Ca, Mg, Fe, Cu, Zn, Mn, Sr, Li and Rb, were determined in dry and sweet wines bearing the denominations of origin of El Hierro, La Palma and Lanzarote islands (Canary Islands, Spain). Analyses were performed by flame atomic absorption spectrophotometry, with the exceptions of Li and Rb for which flame atomic emission spectrophotometry was used. The content in copper and iron did not present risks of cases. All samples presented a copper and zinc content below the maximum amount recommended by the Office International de la Vigne et du Vin (OIV) for these elements. Significant differences in the metallic content were found among the different islands. Thus, Lanzarote presented the highest mean content in sodium and lithium and the lowest mean content in rubidium, and La Palma presented the highest mean content in strontium and rubidium. Sweet wines from La Palma, elaborated as naturally sweet with over-ripe grapes, presented mean contents significantly higher with regard to dry wines from the same island in the majority of the analysed elements. Cluster analysis and Kohonen self-organising maps showed differences in wines according to the island of origin and the ripening state of the grapes. Back-propagation artificial neural networks showed better prediction ability than stepwise linear discriminant analysis.     

Genus-specific profile of acetic acid bacteria by 16S rDNA PCR-DGGE

An effective method for grouping acetic acid bacteria (AAB) genera was defined and evaluated as a tool for preliminary screening of the major AAB species involved in vinegar production. Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, Neoasaia, Saccharibacter, Frateuria and Kozakia AAB strains were screened on the basis of the 16S rDNA sequences using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique. The DGGE profile of all the strains tested, consisted of one single band of approximately 330 bp for each strain and allowed their clustering. The results obtained clearly reflected in silico phylogenetic analysis of the AAB species used in this study, in fact, the species with a higher 16S rDNA sequence homology showed a similar electrophoretic profile. In particular almost all the species belonging to the genus Gluconacetobacter showed a DGGE pattern nearly identical and well distinct from all the other AAB genera. Furthermore by PCR-DGGE it was possible to clearly group the species more frequently recovered from vinegar fermentation which are mainly distributed in the genera Acetobacter, Gluconobacter and Gluconacetobacter.     

A new resource from traditional wines: characterisation of the microbiota of “Vino Santo” grapes as a biocontrol agent against Botrytis cinerea

The microflora of grapes involved in the production of a traditional Italian straw wine, “Vino Santo Trentino”, was evaluated as a biocontrol agent against Botrytis cinerea, one of the main diseases affecting fruit and grapes. The microbiota was described using plate counts and genotypic characterisation (sequencing of 16S rRNA for bacteria and 26s rRNA for yeast), allowing identification of yeasts belonging to the Hanseniaspora, Metschnikowia, Cryptococcus and Issatchenkia genera and bacteria (Bacillus, Microbacterium, Acetobacter and Gluconobacter spp.). The distribution of these species is related to the extent of B. cinerea infection. 7 isolates were able to halt the growth of B. cinerea in antagonistic cultures grown in Petri plates, using both synthetic growth and grape juice media. Technological characterisation of potential biocontrol agents, performed with the help of flow cytometry and HPLC-ECD, demonstrated that these microorganisms did not represent a risk for wine production due to their low resistance to ethanol, low pH and the absence of off-flavours. This ensures that the biocontrol agents disappear during winemaking and excludes a negative impact on the quality of wines. In conclusion, the microflora associated with dried grapes is a precious source of biocontrol agents against B. cinerea, both in terms of preventing disease in the vineyard and in control of the grape drying process for the production of straw wines.     

醋酸菌的分类进展

醋酸菌是好氧型革兰氏阴性菌,能以氧气为终端电子受体,氧化糖类、糖醇类和醇类生成相应的糖醇、酮和有机酸。从1898年Beijerinck定义第一个醋酸菌属-醋酸杆菌属(Acetobacter)以来,截止2014年初,已报道的醋酸菌共16个属,共84个种。该文总结了醋酸菌的分离、分类、鉴定与保藏方法,并对醋酸菌命名原则、种属名称演变以及醋酸菌的种属特征进行了归纳。     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.     

The microbial diversity of water kefir

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.     

Application of culture culture-independent molecular biology based methods to evaluate acetic acid bacteria diversity during vinegar processing

Acetic acid bacteria (AAB) are considered fastidious microorganisms because they are difficult to isolate and cultivate. Different molecular approaches were taken to detect AAB diversity, independently of their capacity to grow in culture media. Those methods were tested in samples that originated during traditional vinegar production. Bacterial diversity was assessed by analysis of 16S rRNA gene, obtained by PCR amplifications of DNA extracted directly from the acetification container. Bacterial composition was analyzed by RFLP-PCR of 16S rRNA gene, Temporal Temperature Gradient Gel Electrophoresis (TTGE) separation of amplicons containing region V3-V5 of 16S rRNA gene and cloning of those amplicons. TTGE bands and clones were grouped based on their electrophoretic pattern similarity and sequenced to be compared with reference strains. The main microorganism identified in vinegar was Acetobacter pasteurianus, which at the end of the acetification process was considered to be the only microorganism present. The diversity was the highest at 2% acetic acid, where indefinite species of Gluconacetobacter xylinus/europaeus/intermedius were also present.