Isolation Of Acetic Acid Bacteria Research Articles

Microbial characterization of Sichuan Baoning vinegar: lactic acid bacteria, acetic acid bacteria and yeasts.

Sichuan Baoning vinegar, a typical representative of Sichuan bran vinegar, is a famous traditional fermented food made from cereals in China. At present, there are few studies on microbial characterization of culturable microorganisms in solid-state fermentation of Sichuan bran vinegar. To comprehensively understand the diversity of lactic acid bacteria, acetic acid bacteria and yeasts, which play an important role in the fermentation of Sichuan bran vinegar, traditional culture-dependent methods combined with morphological, biochemical, and molecular identification techniques were employed to screen and identify these isolates. A total of 34 lactic acid bacteria isolates, 39 acetic acid bacteria isolates, and 48 yeast isolates were obtained. Lactic acid bacteria were dominated by Enterococcus durans, Leuconostoc citreum, Lactococcus lactis, and Lactiplantibacillus plantarum, respectively. Latilactobacillus sakei was the first discovery in cereal vinegar. Acetic acid bacteria were mainly Acetobacter pomorum and A. pasteurianus. The dominant yeast isolates were Saccharomyces cerevisiae, in addition to four non-Saccharomyces yeasts. DNA fingerprinting revealed that isolates belonging to the same species exhibited intraspecific diversity, and there were differences between phenotypic and genotypic classification results. This study further enriches studies on cereal vinegar and lays a foundation for the development of vinegar starters.

Screening of Acetic Acid Bacteria Isolated from Various Sources for Use in Kombucha Production

The objective of this study was to isolate and identify strains of Acetobacter suitable for use in the development of a complex microbial culture for producing Kombucha and to examine the fermentation characteristics for selection of suitable strains. A medium supplemented with calcium carbonate was used for isolation of acetic acid bacteria from 22 various sources. Colonies observed in the clear zone resulting from decomposition of calcium carbonate by acid produced by microorganisms were collected. Identification of the collected strains was based on biological and morphological characteristics, and the results of base sequence analysis. A total of 37 strains were identified, including six species in the Acetobacter genus: Acetobacter pasteurianus, Acetobacter orientalis, Acetobacter cibinongensis, Acetobacter pomorum, Acetobacter ascendens, and Acetobacter malorum, as well as one species in the Gluconobacter genus, Gluconobacter oxydans. Among thirty-seven strains, seven strains of acetic acid bacteria with exceptional acid and alcohol tolerance were selected, and an evaluation of their fermentation characteristics according to fermentation temperature and period was performed. The results showed a titratable acidity of 1.68% for the Acetobacter pasteurianus SFT-18 strain, and an acetic acid bacteria count of 9.52 log CFU/mL at a fermentation temperature of 35 °C. The glucuronic acid and gluconate contents for the Gluconobacter oxydans SFT-27 strain were 10.32 mg/mL and 25.49 mg/mL, respectively.

Quality and Functional Characterization of Acetic Acid Bacteria Isolated from Farm-Produced Fruit Vinegars

Acetic acid bacteria (AAB) form a bacterial film on the surface of alcoholic solutions and ferment ethanol to acetic acid while also producing bioactive compounds. To discover functional AAB for industrial use, we isolated and selected strains from farm-produced vinegars using a CaCO3-containing medium. The seven isolated strains belonged to Acetobacter cerevisiae and Acetobacter pasteurianus. These strains were tolerant to ethanol concentrations up to 12% (v/v). Acidification was seen for GHA 7, GYA 23, JGB 21-17, and GHA 20 strains at a growth temperature of 40 °C. The seven AAB isolates had strong antibacterial activity against Staphylococcus aureus. Antioxidant activity, as assessed using the DPPH and ABTS assays, was two- and four-fold higher than that for the negative control (1% acetic acid), respectively. We also observed 91.3% inhibition of angiotensin-converting enzyme activity for the KSO 5 strain, which was higher than that for the positive control, 0.1% captopril (76.9%). All strains showed complete inhibition of α-glucosidase, except JGB 21-17 and GHA 7, which showed 98.3% inhibition. Our work suggests the usefulness of the selected strains as seed strains for the highly efficient production of functional vinegar and illustrates the identification of useful functional characteristics on a scientific basis.

Genetic diversity of exopolysaccharides from acetic acid bacteria isolates originating from apple cider vinegars

Acetic acid bacteria (AAB) produce acetic acid but are also gaining importance as safe microorganisms for producing extracellular polysaccharides (EPSs). The best-known homopolysaccharides among them are cellulose and levan. In addition, acetic acid bacteria also produce heteropolysaccharides, water-soluble acetans. Isolates from the broth of organic and conventional apple cider vinegar production were screened for biofilm production. Phenotypic and genomic diversity of EPS-producing isolates was assessed. The diversity of phenotypically different EPSs of apple cider vinegar isolates was investigated at the gene level for the following novel strains: Komagataeibacter (K.) melomenusus SI3083, K. oboediens SI3053, K. pomaceti SI3133, and Gluconacetobacter (Ga.) entanii SI2084. Strain K. melomenusus SI3083 possesses cellulose operons bcs1, bcs2, and bcs4 together with the type I acetan cluster in the absence of the levan operon, strain K. oboediens SI3053 has the operons bcs1, bcs2, bcs3, and bcs4, the levan operon, and the acetan cluster (type I), and the strains K. pomaceti SI3133 and Ga. entanii SI2084 both contain recently described novel ace-type II cluster in addition to the incomplete operon bcs1. A comparison of the genetic diversity of these EPSs to those of the reference strains suggests that the studied EPSs are not species-descriptive. The results of this study deepen our understanding of the genetic variability of the EPS genes in AAB, thereby enabling us to better characterize and exploit the various insoluble and soluble exopolysaccharides produced by AAB for biotechnological applications in the future

Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia.

Microbial activity involved in the cocoa beans fermentation process is essential to maintain and improve the organoleptic and nutritional qualities of chocolate; therefore, the aim of this investigation was to search and select microbial isolates with the potential to improve the quality of cocoa beans. Fermentation experimentswere conducted on farms located in Maceo (Antioquia), San Vicente de Chucurí (Santander), and Rivera and Algeciras (Huila), Colombia. Yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and mesophilic aerobic microorganisms were obtained from different fermentation batches. The growth of these microorganismswas tested in six treatments as follows: 50% cocoa pulp agar (CPA), high concentrations of glucose (10%), ethanol (5%), and acetic acid (7%), an acidic pH of 3.0, and a high temperature of 50oC for 24 h. The isolates with the highest growth were identified by 18S and 16S rRNA gene analysis, revealing a high diversity ofspecies associated with cocoa fermentation, including eight species of yeasts (Debaryomyces hansenii, Meyerozyma guillermondii, Wickerhanomyces anomalus, Pichia guillermondii, Pichia kudriavzevii, Trichosporon asahii, Candida parapsilosis, and Pichia manshurica), six species of LAB (Pediococcus acidilactici, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus farraginis, Lactobacillus rhamnosus, and Leuconostoc mesenteroides), four species of AAB (Gluconobacter japonicus, Acetobacter tropicalis, Acetobacter pasteurianus, and Acetobacter malorum/tropicalis), and three species of Bacillus spp. (Bacillusaryabhattai /megaterium, Bacillus subtilis, and Bacillus coagulans). In general, microbial populations increased in cocoa batches after 12 h of fermentation and decreased after 84-96 h. All the yeast isolates grew in 10% glucose and CPA, 85.7% in 5% ethanol, and 95% at a pH of 3.0. All the yeast isolates were affectedby 7% acetic acid and incubation at 50oC for 24 h. Eighty-five percent of the LAB grew in 10% glucose, 100% in 5% ethanol, 42.8% in CPA, 64% at a pH of 3.0, and 35.7% grew after being exposed to 50oC for 24 h; all were affected by 7% acetic acid. As for the AAB, 100% grew in 10% glucose, 71% in 7% ethanol, 100% grew in CPA, in 7% acetic acid, and at a pH of 3.0, while 100% were affected by incubation at 50oC. Three yeast isolates, W. anomalus, D. hansenii and M. guillermondii, three LAB isolates, P. acidilactici, L. brevis, and L. plantarum, and three AAB isolates, A. tropicalis, A. pasteurianus and G. japonicus, were selected as promising strains to be used in a microbial starter culture for cocoa bean fermentation to improve the organoleptic quality of cocoa.

Microbial Diversity of Acetic Acid Producing Bacteria from Protein-Rich Residues

Background: Acetic acid bacteria (AAB) are concrete sets of organism which act as precursor for acetic acid production. Acetic acid is a colorless liquid with strong pungent and sour smell. It is synthesized from oxidation of ethanol by AAB. Vast studies have been made from sugary sources in the isolation of AAB.
 Aim: The needs to study and utilize our protein-rich residues (PRR) for AAB presence spurn this study.
 Place and Duration of Study: Department of Microbiology, University of Port Harcourt, between June and December 2018.
 Methodology: The samples (beans, groundnut and powdered milk) used in this study were surface-sterilized, homogenized, pre-enriched (in balsam medium) and serially diluted with inoculum size (0.1ml) inoculated on sterilized glucose yeast peptone agar, Mannitol agar and low glycemic index (LGI) media and incubated at 30oC for 48 h using the spread plate technique. A total of 11 bacterial isolates were obtained and screened for acetic acid production in brain heart infusion and yeast glucose ethanol acetic acid broth at 30oC for 14 days and positive isolates were identified by titration method. AAB isolates with the highest acetic acid concentration were selected for molecular identification and further studies.
 Results: Two Acetic acid bacteria identified in this study were Acetobacter and Gluconobacter. The result of this study indicated that Acetobacter had acetic acid concentration of 3.6g/100ml while Gluconobacter had 1.8g/100 ml. However, molecular identification highlighted Acetobacter as Bacillus cereus with Genbank accession number MK 332142; whereas Gluconobacter was Stenotrophomonas maltophilia MK 332143. The neighbor-joining phylogenetic tree and bioinformatics revealed B. cereus and S. maltophilia as 97% and 96% similarity index, 854 and 883 nucleotide sequencing letters as well as 450 and 410 base pairs.
 Conclusion: This finding implied that “S. maltophilia” and “B. cereus” are predominant Acetic acid bacteria in spoilt beans and groundnut; and can act as potential strains with industrial importance to man and environment.

Isolation and selection of microorganisms in cocoa fermentation

The objectives of this study were to investigate the change of microorganisms involved in cocoa (Theobroma cacao) fermentation and then to isolate, characterize and select the important microorganisms in cocoa fermentation. The results showed that microbial quantities continuously changed during cocoa fermentation and the highest quantity of dominant microorganisms at different stages of fermentation process as 8.03 log cfu/g of yeast, 6.34 log cfu/g of mold, 7.77 log cfu/g of lactic acid bacteria, 7.87 log cfu/g of acetic acid bacteria, 7.25 log cfu/g of Bacillus, and 10.93 log cfu/g of the total aerobic bacteria. There were nine yeast isolates belonging 5 genera of Saccharomyces, Kluyveromyces, Brettanomyces, Candida and Cystofilobasidium; 9 mould isolates belonging to 2 genera of Rhizopus and Aspergillus; 11 acetic acid bacteria isolates belonging to Acetobacter; and 13 spore-forming bacterial isolates belonging to Bacillus isolates. Three isolates of yeast (CY-1a, CY-1b, CY-2a) belonging to Kluyveromyces possessed the high fermentative capacity and 4 Acetobacter isolates (CAAB-1d, CAAB-1a, CAAB-1e and CAAB-2d) produced high amounts of acetic acid.

ISOLATION AND IDENTIFICATION OF BACTERIAL CELLULOSE PRODUCERS WITH POTENTIAL FOR MEDICINE AND BIOTECHNOLOGY

Recent achievements in investigations of microbial polysaccharides have highlighted their importance in the development of new classes of biomaterials. Some polysaccharides of microbial origin are already widely used in various medical applications, including hyaluronic acid, dextran, alginate, and scleroglucan. Substantial attention has more recently focused on bacterial cellulose as a promising and effective biopolymer.
 In this study, based on knowledge of the ecological niches of acetic acid bacteria, we isolated cellulose-producing bacteria using the fruits of grapes of different varieties and Kombucha. The most effective biocellulose producers were screened, and their biochemical properties were determined. A total of 17 acetic acid bacteria isolates were obtained, and three strains capable of producing a cellulose biofilm were selected, which were identified according to the direct nucleotide sequence of the 16S rRNA gene fragment and biochemical properties.

Isolation of Acetic Acid Bacteria and Preparation of Starter Culture for Apple Cider Vinegar Fermentation

Vinegars are commonly used as food condiments and preservatives. Apple cider vinegar (ACV) is also used in the Ayurvedic pharmaceutical industry because of its medicinal properties. Since specifically selected starter cultures for commercial vinegar production are not readily available, apple juice supplemented with sugar is commonly inoculated with a microbiologically undefined culture obtained from the previous batch of ACV. The present work focuses on the isolation of yeasts and acetic acid bacteria from ACV and the preparation of a starter culture. ACV was produced in a bench scale bioreactor using a traditional fermentation process wherein an acetic acid concentration of 3.8% was obtained after three weeks. Several acetic acid bacteria (AAB) were isolated from ACV using selective media. Microscopy revealed the cultures to be gram negative to gram variable short rods. The growth pattern of the isolates on differential media and biochemical tests suggested the presence of Acetobacter and Gluconobacter species. Ten potent isolates were selected for starter culture preparation. Two consortia were formulated with five AAB isolates in each along with a yeast isolate and used for ACV production, wherein an acetic acid concentration of 4.2% - 4.9% was obtained in 10 - 12 days. Thus, these two starter cultures with locally isolated AAB can be used for the commercial production of apple cider vinegar.

Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa

Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamics were determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolates were identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45°C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeasts were screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarum and C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7–V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S–23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

Acetobacter sicerae sp. nov., isolated from cider and kefir, and identification of species of the genus Acetobacter by dnaK, groEL and rpoB sequence analysis.

Five acetic acid bacteria isolates, awK9_3, awK9_4 ( = LMG 27543), awK9_5 ( = LMG 28092), awK9_6 and awK9_9, obtained during a study of micro-organisms present in traditionally produced kefir, were grouped on the basis of their MALDI-TOF MS profile with LMG 1530 and LMG 1531(T), two strains currently classified as members of the genus Acetobacter. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences as well as on concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB indicated that these isolates were representatives of a single novel species together with LMG 1530 and LMG 1531(T) in the genus Acetobacter, with Acetobacter aceti, Acetobacter nitrogenifigens, Acetobacter oeni and Acetobacter estunensis as nearest phylogenetic neighbours. Pairwise similarity of 16S rRNA gene sequences between LMG 1531(T) and the type strains of the above-mentioned species were 99.7%, 99.1%, 98.4% and 98.2%, respectively. DNA-DNA hybridizations confirmed that status, while amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) data indicated that LMG 1531(T), LMG 1530, LMG 27543 and LMG 28092 represent at least two different strains of the novel species. The major fatty acid of LMG 1531(T) and LMG 27543 was C18 : 1ω7c. The major ubiquinone present was Q-9 and the DNA G+C contents of LMG 1531(T) and LMG 27543 were 58.3 and 56.7 mol%, respectively. The strains were able to grow on D-fructose and D-sorbitol as a single carbon source. They were also able to grow on yeast extract with 30% D-glucose and on standard medium with pH 3.6 or containing 1% NaCl. They had a weak ability to produce acid from d-arabinose. These features enabled their differentiation from their nearest phylogenetic neighbours. The name Acetobacter sicerae sp. nov. is proposed with LMG 1531(T) ( = NCIMB 8941(T)) as the type strain.

Acetobacter strains isolated during the acetification of blueberry (Vaccinium corymbosum L.) wine

Highbush blueberries (Vaccinium corymbosum L.) are known to have positive health benefits. The production of blueberry vinegar is one method to preserve this seasonal fruit and allow extended consumption. In this study, blueberry wine acetification was performed with naturally occurring micro-organisms and with an inoculated Acetobacter cerevisiae strain. Acetifications were carried out in triplicate using the Schützenbach method. The successful spontaneous processes took up to 66% more time than the processes involving inoculation. The isolation of acetic acid bacteria (AAB) and the analysis of these AAB using molecular methods allowed the identification of the main genotypes responsible of the blueberry acetification. Although the Acet. cerevisiae strain was the predominant strain isolated from the inoculated process samples, Acetobacter pasteurianus was isolated from samples for both processes and was the only species present in the spontaneous acetification samples. To the best of our knowledge, this is the first report describing the identification and variability of AAB isolated during blueberry acetification. The isolated Acet. pasteurianus strains could be used for large-scale blueberry vinegar production or as a starter culture in studies of other vinegar production methods.

Microbiological and Physicochemical Characterization of Small-Scale Cocoa Fermentations and Screening of Yeast and Bacterial Strains To Develop a Defined Starter Culture

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.

Diversity of acetic acid bacteria present in healthy grapes from the Canary Islands

The identification of acetic acid bacteria (AAB) from sound grapes from the Canary Islands is reported in the present study. No direct recovery of bacteria was possible in the most commonly used medium, so microvinifications were performed on grapes from Tenerife, La Palma and Lanzarote islands. Up to 396 AAB were isolated from those microvinifications and identified by 16S rRNA gene sequencing and phylogenetic analysis. With this method, Acetobacter pasteurianus, Acetobacter tropicalis, Gluconobacter japonicus and Gluconacetobacter saccharivorans were identified. However, no discrimination between the closely related species Acetobacter malorum and Acetobacter cerevisiae was possible. As previously described, 16S-23S rRNA gene internal transcribed spacer (ITS) region phylogenetic analysis was required to classify isolates as one of those species. These two species were the most frequently occurring, accounting for more than 60% of the isolates. For typing the AAB isolates, both the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and (GTG)5-PCR techniques gave similar resolution. A total of 60 profiles were identified. Thirteen of these profiles were found in more than one vineyard, and only one profile was found on two different islands (Tenerife and La Palma).

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.

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.

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.

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) – PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.

Application of Chemical Dyes as Colour Indicator for Selective Isolation of Acetic Acid Bacteria

Application of Chemical Dyes as Colour Indicator for Selective Isolation of Acetic Acid Bacteria