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.     

Genera and species in acetic acid bacteria

Taxonomic studies of acetic acid bacteria were historically surveyed. The genus Acetobacter was first introduced in 1898 with a single species, Acetobacter aceti. The genus Gluconobacter was proposed in 1935 for strains with intense oxidation of glucose to gluconic acid rather than oxidation of ethanol to acetic acid and no oxidation of acetate. The genus "Acetomonas" was described in 1954 for strains with polar flagellation and no oxidation of acetate. The proposals of the two generic names were due to confusion, and "Acetomonas" was a junior subjective synonym of Gluconobacter. The genus Acetobacter was in 1984 divided into two subgenera, Acetobacter and Gluconoacetobacter. The latter was elevated to the genus Gluconacetobacter in 1998. In the acetic acid bacteria, ten genera are presently recognized and accommodated to the family Acetobacteraceae, the Alphaproteobacteria: Acetobacteer, Gluconobacter, Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania, Saccharibacter, Neoasaia and Granulibacter. In contrast, the genus Frateuria, strains of which were once named 'pseudacetic acid bacteria', was classified into the Gammaproteobacteria. The genus Gluconacetobacter was phylogenetically divided into two groups: the Gluconacetobacter liquefaciens group and the Gluconacetobacter xylinus group. The two groups were discussed taxonomically.     

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 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).     

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.     

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.     

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.     

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.     

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。     

The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus

The oxidation of ethanol to acetic acid is the most characteristic process in acetic acid bacteria.Gluconacetobacter diazotrophicus is rather unique among the acetic acid bacteria as it carries out nitrogen fixation and is a true endophyte, originally isolated from sugar cane. Aside its peculiar life style, Ga. diazotrophicus, possesses a constitutive membrane-bound oxidase system for ethanol. The Alcohol dehydrogenase complex (ADH) of Ga. diazotrophicus was purified to homogeneity from the membrane fraction. It-exhibited two subunits with molecular masses of 71.4 kDa and 43.5 kDa. A positive peroxidase reaction confirmed the presence of cytochrome c in both subunits. Pyrroloquinoline quinone (PQQ) of ADH was identified by UV-visible light and fluorescence spectroscopy. The enzyme was purified in its full reduced state; potassium ferricyanide induced its oxidation. Ethanol or acetaldehyde restored the full reduced state. The enzyme showed an isoelectric point (pI) of 6.1 and its optimal pH was 6.0. Both ethanol and acetaldehyde were oxidized at almost the same rate, thus suggesting that the ADH complex of Ga. diazotrophicus could be kinetically competent to catalyze, at least in vitro, the double oxidation of ethanol to acetic acid.     

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.     

开菲尔粒中主要组成菌的分离鉴定及微囊化纯培养混合发酵指标分析

开菲尔粒是一个复杂的微生物共生体系,包含很多有益生作用的微生物。本文研究了一种开菲尔粒的主要组成菌并制成发酵剂。通过形态学特征初步分离纯化得出:该开菲尔粒样品主要由两株酵母菌、三株乳酸菌以及两株醋酸菌构成。经16S r DNA序列分析进一步确定其种属,得出其分别为Kluyveromyces marxianus和Pichia kudriavzevii、Lactobacillus pontis和Lactobacillus kefiri、Acetobacter lovaniensis和Acetobacter cibinongensis。采用分离出的菌株纯培养微囊化之后进行混合发酵,得到具有优良稳定发酵性能的混合发酵剂,测定结果显示发酵乳的营养成分、挥发性成分和抑菌性与原粒对比十分接近。      

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.     

Characterization of acetic acid bacteria in "traditional balsamic vinegar"

This study evaluated the glucose tolerance of acetic acid bacteria strains isolated from Traditional Balsamic Vinegar. The results showed that the greatest hurdle to acetic acid bacteria growth is the high sugar concentration, since the majority of the isolated strains are inhibited by 25% of glucose. Sugar tolerance is an important technological trait because Traditional Balsamic Vinegar is made with concentrated cooked must. On the contrary, ethanol concentration of the cooked and fermented must is less significant for acetic acid bacteria growth. A tentative identification of the isolated strains was done by 16S-23S-5S rDNA PCR/RFLP technique and the isolated strains were clustered: 32 strains belong to Gluconacetobacter xylinus group, two strains to Acetobacter pasteurianus group and one to Acetobacter aceti.     

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.     

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.     

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.     

Effect of synthetic microbial community on nutraceutical and sensory qualities of kombucha

To mass-produce nutritional kombucha with quality consistency, it is necessary to design synthetic microbial community (SMC) starters with clear bacterial compositions. In this study, a three-species SMC, including Acetobacter pasteurianus, Gluconacetobacter xylinus and Zygosaccharomyces bailii, was designed. Acetic acid bacteria (AAB) in SMC produced a higher concentration of organic acids synergistically compared to each strain inoculated alone. Gluconic acid was the most abundant organic acid in kombucha fermented by SMC, which contributed to the pleasant sour taste of the beverage. AAB could also oxidise the alcohol produced by Z. bailii, making kombucha a soft beverage. Total phenol and flavonoid concentrations increased during fermentation, which enhanced the health benefits of kombucha. The sensory evaluation demonstrated tsshat sensory attributes of SMC fermented kombucha were quite similar to those fermented by the symbiotic culture of bacteria and yeast (SCOBY). Therefore, SMC could be utilised in the industrial mass production of kombucha for better quality control and product consistency.     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

诺丽自然发酵汁中醋酸菌的分离鉴定及发酵特性

为分析诺丽自然发酵汁中醋酸菌的多样性,丰富诺丽中醋酸菌种属信息,采用传统分离培养和16S rRNA基因序列分析相结合的方法,对诺丽自然发酵汁中的醋酸菌进行分离鉴定和发酵特性研究。结果表明,从不同发酵阶段诺丽自然发酵汁的67 个分离菌株中鉴定出了24 株醋酸菌,分别为Acetobacter fabarum（9 株）、Acetobacter syzygii（7 株）、Acetobacter pasteurianus（2 株）、Acetobacter tropicalis（1 株）、Acetobacter lambici（1 株）和Gluconobacter japonicus（4 株）,其中A. syzygii和A. fabarum为诺丽自然发酵过程中的优势菌种。在发酵性能方面,A. tropicalis N21性能最优,产酸量可达28.92 g/L,在40 ℃高温中仍能良好生长且产酸量为14.85 g/L,乙醇体积分数为7%时,产酸量略有下降,但依然可达23.60 g/L,其耐高温和耐乙醇能力均高于其他菌株。