A microbiological study of Hussuwa: a traditional Sudanese fermented food from germinated Sorghum bicolor c.v. feterita

Hussuwa is a traditional Sudanese fermented food. Hussuwa made from Sorghum bicolor variety feterita exists in northern, central and eastern Sudan. The microbiological study indicated that the fermentation was primarily a lactic acid fermentation. The changes in microbial population, acetic acid bacteria, lactic acid bacteria and yeasts during all stages of hussuwa preparation and ripening were studied. The identification of fermented hussuwa microorganisms revealed that the main microorganisms were Lactobacillus saccharolyticum, Gluconobacter oxydans, Acetobacter xylinum and Saccharomyces cerevisiae. The metabolic products were studied in all stages of preparation and the period of ripening of hussuwa. The values of pH decreased as fermentation proceeded, and titratable acidity and volatile fatty acids increased.     

Energy metabolism of a unique acetic acid bacterium, Asaia bogorensis, that lacks ethanol oxidation activity

Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo(3)-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H(+)/O ratio.     

Investigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

     

Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation

AIMS: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. METHODS AND RESULTS: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. CONCLUSIONS: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.     

Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter

Phylogenetic relationships among three genera, Gluconobacter, Acetobacter, and Gluconacetobacter, of acetic acid bacteria (AAB) are still unclear, although phylogenetic analysis using 16S rRNA gene sequence has shown that Gluconacetobacter diverged first from the ancestor of these three genera. Therefore, the relationships among these three genera were investigated by genome-wide phylogenetic analysis of AAB. Contrary to the results of 16S rRNA gene analysis, phylogenetic analysis of 293 enzymes involved in metabolism clearly showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. In addition, we defined 753 unique orthologous proteins among five known complete genomes of AAB, and phylogenetic analysis was carried out using concatenated gene sequences of these 753 proteins. The result also showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. Our results strongly suggest that Gluconobacter was the first to diverge from the common ancestor of Gluconobacter, Acetobacter, and Gluconacetobacter, a relationship that is in good agreement with the physiologies and habitats of these genera.     

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts. Received 4 August 1998/ Accepted in revised form 9 December 1998     

Distribution of Bacterial Contaminants in a South African Lager Brewery

Bacteria isolated from contaminated pitching yeast, fermenting wort and beer samples from a South African lager brewery over a one-year period were tentatively identified by an improved, rapid diagnostic procedure as pediococci (41%), homofermentative lactobacilli (5%), heterofermentative lactobacilli (9%), Acetobacter spp. (7%), Gluconobacter spp. (13%) and Hafnia protea (25%). Pediococci and lactobacilli dominated samples taken from fermentation, storage and 'bright' beer tanks but were absent from pitched wort samples, from collection vessels and the single pitching yeast sample investigated. Acetic acid bacteria and H. protea were widely distributed in collection vessel, fermentation and storage tank samples, and H. protea was isolated from recycled pitching yeast.     

Relationships of Phenylobacterium immobile and purple nonsulfur bacteria on the basis of surface antigens

Abstract Indirect immunofluorescence tests with antisera against whole cells of Phenylobacterium immobile strains revealed a serological relationship to Pseudomonas vesicularis, Aquaspirillum itersonii and Rhodospirillum rubrum , three members of the purple nonsulfur bacteria (group I) and also to Gluconobacter oxydans and Azotobacter vinelandii . Antisera against whole cells of Gluconobacter oxydans and Pseudomonas vesicularis reacted positively with the Phenylobacterium immobile strains, tested. Furthermore, a serological relationship of Gluconobacter oxydans to Acetobacter aceti , and of Pseudomonas vesicularis to Pseudomonas diminuta and Aquaspirillum itersonii could be demonstrated.     

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.     

Quantification of the expression of reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth conditions

Aims:  The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)–alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB).
Methods and Results:  Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adh A was measured in Acetobacter aceti , Acetobacter pasteurianus , Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adh A expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adh A expression.
Conclusions:  The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter .
Significance and Impact of the Study:  Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.     

Evolution of Acetic Acid Bacteria During Fermentation and Storage of Wine

Acetic acid bacteria were present at all stages of wine making, from the mature grape through vinification to conservation. A succession of Gluconobacter oxydans, Acetobacter pasteurianus, and Acetobacter aceti during the course of these stages was noted. Low levels of A. aceti remained in the wine; they exhibited rapid proliferation on short exposure of the wine to air and caused significant increases in the concentration of acetic acid. Higher temperature of wine storage and higher wine pH favored the development and metabolism of these species.     

Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).     

Antagonism Between Osmophilic Lactic Acid Bacteria and Yeasts in Brine Fermentation of Soy Sauce

Brine fermentation by osmophilic lactic acid bacteria and yeasts for long periods of time is essential to produce a good quality of shoyu (Japanese fermented soy sauce). It is well known that lactic acid fermentation by osmophilic lactic acid bacteria results in the depression of alcoholic fermentation by osmophilic yeasts, but the nature of the interaction between osmophilic lactic acid bacteria and yeasts in brine fermentation of shoyu has not been revealed. The inhibitory effect of osmophilic lactic acid bacteria on the growth of osmophilic yeasts was investigated. It was recognized that osmophilic shoyu yeasts such as Saccharomyces rouxii and Torulopsis versatilis were inhibited by a metabolite produced by osmophilic lactic acid bacteria (belonging to Pediococcus halophilus) in brine fermentation of shoyu. The primary inhibitor was considered to be acetic acid, although lactic acid was slightly inhibitory.     

Successive Microbial Populations in Calimyrna Figs

Smyrna-type (Calimyrna) figs have essentially sterile internal tissue until visited by the pollinating fig wasp, Blastophaga psenes, which introduces a specific microflora consisting of Candida guilliermondii var. carpophila and Serratia plymuthica. This flora persists and develops in numbers throughout the ripening period until maturity of the fruit. These organisms do not cause spoilage. The presence of C. guilliermondii var. carpophila appears to increase the attractiveness of the fruit to drosophilae. Drosophila (mainly D. melanogaster) carry spoilage yeasts and bacteria on their exterior body parts, and introduce these organisms during ovipositing in the fruit cavity. The spoilage yeasts consist almost entirely of apiculate yeasts (Hanseniaspora valbyensis, H. uvarum, and Kloeckera apiculata) and of Torulopsis stellata, which cause active fermentative spoilage. Spoilage bacteria (primarily Acetobacter melanogenus) are also introduced with the yeasts. Organic acids are produced by these yeasts as well as by the Acetobacter. A number of minor spoilage yeasts were also identified.     

Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts

AIMS: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. METHODS AND RESULTS: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases beta-glucanases, lichenases, beta-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clarified grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except beta-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. CONCLUSION: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.     

Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.     

Identification of Acetobacter liquefaciens as Causal Agent of Pink-Disease of Pineapple Fruit

Two bacterial strains causing pink-disease of pineapple were identified as Acetobacter liquefaciens and compared with 8 other Acetobacter liquefaciens, 10 Gluconobacter oxydans and 7 Frateuria aurantia strains. The similarieties and differences between these bacteria are discussed.     

Regulation of aspartokinase and homoserine dehydrogenase in acetic acid bacteria

The regulation of aspartokinase and homoserine dehydrogenase has been studied in three Acetobacter and two Gluconobacter species. Both enzymes were regulated by feedback inhibition. Aspartokinase was inhibited by L-threonine and concertedly inhibited by L-threonine plus L-lysine. The homoserine dehydrogenase was NADP-specific and was inhibited by L-threonine. Separation of the two enzymes by ammonium sulphate fractionation was possible in Acetobacter peroxydans, A. rancens and Gluconobacter melanogenus but not in A. liquefaciens or G. oxydans.     

Cloning of Escherichia coli lacZ and lacY genes and their expression in Gluconobacter oxydans and Acetobacter liquefaciens

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.