Complementary supramolecular drug associates in perfecting the multidrug therapy against multidrug resistant bacteria.

The inappropriate and inconsistent use of antibiotics in combating multidrug-resistant bacteria exacerbates their drug resistance through a few distinct pathways. Firstly, these bacteria can accumulate multiple genes, each conferring resistance to a specific drug, within a single cell. This accumulation usually takes place on resistance plasmids (R). Secondly, multidrug resistance can arise from the heightened expression of genes encoding multidrug efflux pumps, which expel a broad spectrum of drugs from the bacterial cells. Additionally, bacteria can also eliminate or destroy antibiotic molecules by modifying enzymes or cell walls and removing porins. A significant limitation of traditional multidrug therapy lies in its inability to guarantee the simultaneous delivery of various drug molecules to a specific bacterial cell, thereby fostering incremental drug resistance in either of these paths. Consequently, this approach prolongs the treatment duration. Rather than using a biologically unimportant coformer in forming cocrystals, another drug molecule can be selected either for protecting another drug molecule or, can be selected for its complementary activities to kill a bacteria cell synergistically. The development of a multidrug cocrystal not only improves tabletability and plasticity but also enables the simultaneous delivery of multiple drugs to a specific bacterial cell, philosophically perfecting multidrug therapy. By adhering to the fundamental tenets of multidrug therapy, the synergistic effects of these drug molecules can effectively eradicate bacteria, even before they have the chance to develop resistance. This approach has the potential to shorten treatment periods, reduce costs, and mitigate drug resistance. Herein, four hypotheses are presented to create complementary drug cocrystals capable of simultaneously reaching bacterial cells, effectively destroying them before multidrug resistance can develop. The ongoing surge in the development of novel drugs provides another opportunity in the fight against bacteria that are constantly gaining resistance to existing treatments. This endeavour holds the potential to combat a wide array of multidrug-resistant bacteria.

Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

Cocrystallizing and Codelivering Complementary Drugs to Multidrugresistant Tuberculosis Bacteria in Perfecting Multidrug Therapy.

Bacteria cells exhibit multidrug resistance in one of two ways: by raising the genetic expression of multidrug efflux pumps or by accumulating several drug-resistant components in many genes. Multidrug-resistive tuberculosis bacteria are treated by multidrug therapy, where a few certain antibacterial drugs are administered together to kill a bacterium jointly. A major drawback of conventional multidrug therapy is that the administration never ensures the reaching of different drug molecules to a particular bacterium cell at the same time, which promotes growing drug resistivity step-wise. As a result, it enhances the treatment time. With additional tabletability and plasticity, the formation of a cocrystal of multidrug can ensure administrating the multidrug chemically together to a target bacterium cell. With properly maintaining the basic philosophy of multidrug therapy here, the synergistic effects of drug molecules can ensure killing the bacteria, even before getting the option to raise the drug resistance against them. This can minimize the treatment span, expenditure and drug resistance. A potential threat of epidemic from tuberculosis has appeared after the Covid-19 outbreak. An unwanted loop of finding molecules with the potential to kill tuberculosis, getting their corresponding drug approvals, and abandoning the drug after facing drug resistance can be suppressed here. This perspective aims to develop the universal drug regimen by postulating the principles of drug molecule selection, cocrystallization, and subsequent harmonisation within a short period to address multidrug-resistant bacteria.

Mitigation of arsenic toxicity in rice by the co-inoculation of arsenate reducer yeast with multifunctional arsenite oxidizing bacteria.

The study aimed to explicate the role of microbial co-inoculants for the mitigation of arsenic (As) toxicity in rice. Arsenate (AsV) reducer yeast Debaryomyces hansenii NBRI-Sh2.11 (Sh2.11) with bacterial strains of different biotransformation potential was attempted to develop microbial co-inoculants. An experiment to test their efficacy (yeast and bacterial strains) on plant growth and As uptake was conducted under a stressed condition of 20 mg kg-1 of arsenite (AsIII). A combination of Sh2.11 with an As(III)-oxidizer, Citrobacter sp. NBRI-B5.12 (B5.12), resulted in ∼90% decrease in grain As content as compared to Sh2.11 alone (∼40%). Reduced As accumulation in rice roots under co-treated condition was validated with SEM-EDS analysis. Enhanced As expulsion in the selected combination under in vitro conditions was found to be correlated with higher As content in the soil during their interaction with plants. Selected co-inoculant mediated enhanced nutrient uptake in association with better production of indole acetic acid (IAA) and gibberellic acid (GA) in shoot, support microbial co-inoculant mediated better biomass under stressful condition. Boosted defense response in association with enhanced glutathione-S-transferase (GST) and glutathione reductase (GR), activities under in vitro and in vivo conditions were observed. These results indicated that the As(III) oxidizer-B5.12 accelerated the As detoxification property of the As(V) reducer-Sh2.11. Henceforth, the results confer that the coupled reduction-oxidation process of the co-inoculant reduces the accumulation of As in rice grain. These co-inoculants can be further developed for field trials to achieve higher biomass with alleviated As toxicity in rice.

Melanoma growth in non-chemically modified translucid bacterial nanocellulose hollow and compartimentalized spheres.

BACKGROUND: Bacterial nanocellulose (BNC) has been used as cell support in numerous tissue engineering studies. Its use can be explained based on the fact its structure allows the creation of a required microenvironment for an ideal material, which supports 3D cell culture. Its structure and interconnected pores lead to animal cells adhesion and proliferation, also allowing oxygen and nutrients transportation. METHODS: We developed a new methodology to produce spherical platforms synthesized by Komagataebacter hansenii (ATCC 23769) under dynamic culture conditions in minimal medium. The chemical composition and physical properties of the platforms were evaluated. Then, human melanoma cells (SK-MEL-28) were encapsulated into the platforms and evaluated by metabolic activity, morphology and their ability on adhering to the Hollow Translucid BNC Spheres (BNC-TS-H) and Compartmentalized Translucid BNC Spheres (BNC-TS-C) up to 3 days. RESULTS: BNC-TS-H and BNC-TS-C platforms were produced as translucid spheroid platforms with distinct microenvironment under dynamic fermentation. The chemical and physical characterizations confirmed the platforms composition as BNC. The produced internal microenvironments in spherical platforms are relevant to determine tumor cell fate. In the first 12 h of culture, cells could adhere to nanocellulose microfibers assuming their typical tumorous phenotype in 72 h of culture. CONCLUSION: The dynamic fermentation in minimal medium produced distinct microstructured platforms of BNC-TS-H and BNC-TS-C. The platforms microstructure resulted in microenvironments that enabled distinct cell-cell and cell-matrix interactions. This behavior suggests several applications in tissue engineering. GENERAL SIGNIFICANCE: The method produced translucid BNC sphere platforms with distinct microenvironments for 3D cell culture.

Changes in microbial communities and their predictive functionalities during fermentation of toddy, an alcoholic beverage of India.

Toddy is a traditional mild-alcoholic drink of India, which is produced from fresh palm saps by natural fermentation. We studied the successional changes in bacterial and fungal communities during the natural fermentation (0 h-96 h) of toddy. During fermentation, alcohol content of the fermenting saps increased significantly from 0.6 %±0.15 to 5.6 %±0.02, pH decreased from 6.33 %±0.02-3.93 ± 0.01, volatile and titratable acidity acidity (g/100 mL) increased from 0.17 ± 0.02 (0 h) to 0.48 ± 0.02 (96 h) and 1.30 ± 0.005 (0 h) to 2.47 ± 0.005 (96 h), respectively. Total sugar content and ˚BRIX also decreased during the fermentation. Firmicutes (78.25 %) was the most abundant phylum followed by Proteobacteria (21.57 %). Leuconostoc was the most abundant genus in the early stages of fermentation. However, Lactobacillus and Gluconoacetobacter were found abundant with increase in pH during the later phases of fermentation (72 h-96 h). Ascomycota (99.02 %) was the most abundant fungal phylum. Hanseniaspora was the abundant yeast in the initial stages of fermentation, whereas the population of Saccharomyces increased significantly after 24 h of fermentation. Torulaspora, Lachancea and Starmerella showed their heterogeneous distribution throughout the fermentation. Computational analysis of metagenomes based on KEGG and MetaCyc databases showed different predictive functional profiles such as folate biosynthesis, glutathione metabolism, terpenoids biosynthesis and biosynthesis of amino acids with significant differences between the fresh palm saps and fermenting saps during toddy fermentation.

Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation.

Cocoa beans used for chocolate production are fermented seeds of Theobroma cacao obtained by a natural fermentation process. The flavors and chemical compounds produced during the fermentation process make this step one of the most important in fine chocolate production. Herein, an integrative analysis of the variation of microbial community structure, using a shotgun metagenomics approach and associated physicochemical features, was performed during fermentation of fine cocoa beans. Samples of Forastero variety (FOR) and a mixture of two hybrids (PS1319 and CCN51) (MIX) from Bahia, Brazil, were analyzed at 7 different times. In the beginning (0 h), the structures of microbial communities were very different between FOR and MIX, reflecting the original plant-associated microbiomes. The highest change in microbial community structures occurred at the first 24 h of fermentation, with a marked increase in temperature and acetic acid concentration, and pH decrease. At 24-48 h both microbial community structures were quite homogenous regarding temperature, acetic acid, succinic acid, pH, soluble proteins and total phenols. During 72-96 h, the community structure resembles an acidic and warmer environment, prevailing few acetic acid bacteria. Taxonomic richness and abundance at 72-144 h exhibited significant correlation with temperature, reducing sugars, succinic, and acetic acids. Finally, we recommend that dominant microbial species of spontaneous fine cocoa fermentations should be considered as inoculum in accordance with the farm/region and GMP to maintain a differential organoleptic feature for production of fine chocolate. In our study, a starter inoculum composed of Acetobacter pausterianus and Hanseniaspora opuntiae strains is indicated.

Cheese yeasts.

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH3 from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.

Protein Tagging, Destruction and Infection.

Cells possess protein quality control mechanisms to maintain proper cellular homeostasis. In eukaryotes, the roles of the ubiquitination and proteasome-mediated degradation of cellular proteins is well established. Recent studies have elucidated protein tagging mechanisms in prokaryotes, involving transfer messenger RNA (tmRNA) and pupylation. In this review, newer insights and bioinformatics analysis of two distinct bacterial protein tagging machineries are discussed. The machinery for tmRNAmediated tagging is present in several eubacterial representatives, e.g. Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis etc., but not in two archaeal representatives, such as Thermoplasma acidophilum and Sulfolobus solfataricus. On the other hand, the machinery involving tagging with the prokaryotic ubiquitin-like protein (Pup) is absent in most bacteria but is encoded in some eubacterial representatives, e.g. Mycobacterium tuberculosis and Mycobacterium leprae. Furthermore, molecular details on the relationship between protein tagging and enzymes involved in protein degradation in bacteria during infection are emerging. Several pathogenic bacteria that do not express the major ATP-dependent proteases, Lon and Caseinolytic protease (ClpP), are avirulent. Also, some ATP-independent peptidases, such as PepA and PepN, modulate the infection process. The roles of bacterial proteins involved in tagging and degradation during infection are discussed. These aspects add a new dimension to better understanding of the peculiarities of host-pathogen interactions.

Production of bacterial cellulose with controlled deuterium-hydrogen substitution for neutron scattering studies.

Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media formulation for growth of Acetobacter xylinus subsp. sucrofermentans and Gluconacetobacter hansenii was formulated that supports cellulose production in deuterium (D) oxide. The level of D incorporation can be varied by altering the ratio of deuterated and protiated glycerol used during cell growth in the D2O-based growth medium. Spectroscopic analysis and mass spectrometry show that the level of deuterium incorporation is high (>90%) for the perdeuterated form of bacterial cellulose. The small-angle neutron scattering profiles of the cellulose with different amounts of D incorporation are all similar indicating that there are no structural changes in the cellulose due to substitution of deuterium for hydrogen. In addition, by varying the amount of deuterated glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. The ability to control deuterium content of cellulose extends the range of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose, and its interactions with other biomacromolecules as well as synthetic polymers used for development of composite materials.

Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy Processing Environment.

Microbial contamination in food processing plants can play a fundamental role in food quality and safety. In this study, the microbiota in a dairy plant was studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput amplicon sequencing. Environmental samples from surfaces and tools were studied along with the different types of cheese produced in the same plant. The microbiota of environmental swabs was very complex, including more than 200 operational taxonomic units with extremely variable relative abundances (0.01 to 99%) depending on the species and sample. A core microbiota shared by 70% of the samples indicated a coexistence of lactic acid bacteria with a remarkable level of Streptococcus thermophilus and possible spoilage-associated bacteria, including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus, Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii. Beta-diversity analyses showed a clear separation of environmental and cheese samples based on both yeast and bacterial community structure. In addition, predicted metagenomes also indicated differential distribution of metabolic pathways between the two categories of samples. Cooccurrence and coexclusion pattern analyses indicated that the occurrence of potential spoilers was excluded by lactic acid bacteria. In addition, their persistence in the environment can be helpful to counter the development of potential spoilers that may contaminate the cheeses, with possible negative effects on their microbiological quality.

Changes in flavour and microbial diversity during natural fermentation of suan-cai, a traditional food made in Northeast China.

We measured changes in the main physical and chemical properties, flavour compounds and microbial diversity in suan-cai during natural fermentation. The results showed that the pH and concentration of soluble protein initially decreased but were then maintained at a stable level; the concentration of nitrite increased in the initial fermentation stage and after reaching a peak it decreased significantly to a low level by the end of fermentation. Suan-cai was rich in 17 free amino acids. All of the free amino acids increased in concentration to different degrees, except histidine. Total free amino acids reached their highest levels in the mid-fermentation stage. The 17 volatile flavour components identified at the start of fermentation increased to 57 by the mid-fermentation stage; esters and aldehydes were in the greatest diversity and abundance, contributing most to the aroma of suan-cai. Bacteria were more abundant and diverse than fungi in suan-cai; 14 bacterial species were identified from the genera Leuconostoc, Bacillus, Pseudomonas and Lactobacillus. The predominant fungal species identified were Debaryomyces hansenii, Candida tropicalis and Penicillium expansum.

Growth and adaptation of microorganisms on the cheese surface.

Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Spontaneous cocoa bean fermentation carried out in a novel-design stainless steel tank: influence on the dynamics of microbial populations and physical-chemical properties.

Spontaneous cocoa bean fermentations carried out in a novel-design 40-kg-capacity stainless steel tank (SST) was studied in parallel to traditional Brazilian methods of fermentation in wooden boxes (40-kg-capacity wooden boxes (WB1) and 600-kg-capacity wooden boxes (WB2)) using a multiphasic approach that entailed culture-dependent and -independent microbiological analyses of fermenting cocoa bean pulp samples and target metabolite analyses of both cocoa pulp and cotyledons. Both microbiological approaches revealed that the dominant species of major physiological roles were the same for fermentations in SST, relative to boxes. These species consisted of Saccharomyces cerevisiae and Hanseniaspora sp. in the yeast group; Lactobacillus fermentum and L. plantarum in the lactic acid bacteria (LAB) group; Acetobacter tropicalis belonging to the acetic acid bacteria (AAB) group; and Bacillus subtilis in the Bacillaceae family. A greater diversity of bacteria and non-Saccharomyces yeasts was observed in box fermentations. Additionally, a potentially novel AAB belonging to the genus Asaia was isolated during fermentation in WB1. Cluster analysis of the rRNA genes-PCR-DGGE profiles revealed a more complex picture of the box samples, indicating that bacterial and yeast ecology were fermentation-specific processes (wooden boxes vs. SST). The profile of carbohydrate consumption and fermentation products in the pulp and beans showed similar trends during both fermentation processes. However, the yeast-AAB-mediated conversion of carbohydrates into ethanol, and subsequent conversion of ethanol into acetic acid, was achieved with greater efficiency in SST, while temperatures were generally higher during fermentation in wooden boxes. With further refinements, the SST model may be useful in designing novel bioreactors for the optimisation of cocoa fermentation with starter cultures.

Isolation and identification of the microbiota of Danish farmhouse and industrially produced surface-ripened cheeses.

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.

Function-specific accelerations in rates of sequence evolution suggest predictable epistatic responses to reduced effective population size.

Changes in effective population size impinge on patterns of molecular evolution. Notably, slightly deleterious mutations are more likely to drift to fixation in smaller populations, which should typically also lead to an overall acceleration in the rates of evolution. This prediction has been validated empirically for several endosymbiont and island taxa. Here, we first show that rate accelerations are also evident in bacterial pathogens whose recent shifts in virulence make them prime candidates for reduced effective population size: Bacillus anthracis, Bordetella parapertussis, Mycobacterium leprae, Salmonella enterica typhi, Shigella spp., and Yersinia pestis. Using closely related genomes to analyze substitution rate dynamics across six phylogenetically independent bacterial clades, we demonstrate that relative rates of coding sequence evolution are biased according to gene functional category. Notably, genes that buffer against slightly deleterious mutations, such as chaperones, experience stronger rate accelerations than other functional classes at both nonsynonymous and synonymous sites. Although theory predicts altered evolutionary dynamics for buffer loci in the face of accumulating deleterious mutations, to observe even stronger rate accelerations is surprising. We suggest that buffer loci experience elevated substitution rates because the accumulation of deleterious mutations in the remainder of the genome favors compensatory substitutions in trans. Critically, the hyper-acceleration is evident across phylogenetically independent clades, supporting the hypothesis that reductions in effective population size predictably induce epistatic responses in genes that buffer against slightly deleterious mutations.

Production of indole by wine-associated microorganisms under oenological conditions.

A high concentration of indole has been linked to 'plastic-like' off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 microg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 microg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.

Improvement of the quality and abatement of the biogenic amines of grass carp muscles by fermentation using mixed cultures.

BACKGROUND: To improve the quality of processed grass carp (Ctenopharyngodon idellus) products and control the accumulation of hazardous substances therein, minced grass carp slices were salted for 6 h at room temperature and then inoculated with mixed starter cultures of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen and Monascus anka and fermented for 12 h at 30 degrees C. The changes in some characteristics and biogenic amine contents of the fermented muscles were investigated. RESULTS: During the 12 h fermentation at 30 degrees C, muscles inoculated with mixed starter cultures showed a rapid decrease in pH from 6.0 to 5.1 and suppression of the growth of enterobacteria and pseudomonads. The fermented muscles exhibited better colour, appearance, flavour and overall acceptability than the control (P < 0.05). The changes in non-protein nitrogen and free amino acid contents of the fermented muscles and in their sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles indicated that severe hydrolysis of muscle proteins occurred during fermentation. The accumulation of biogenic amines in the muscles was efficiently reduced by fermentation with mixed starter cultures. CONCLUSION: Fermentation with mixed starter cultures of L. casei, S. lactis, S. cerevisiae Hansen and M. anka significantly improved the characteristics of grass carp muscles and controlled the accumulation of biogenic amines.

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

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.