Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine

Background: Hong Qu glutinous rice wine (HQGRW) is brewed under non-aseptic fermentation conditions, so it usually has a relatively high total acid content. The aim of this study was to investigate the dynamics of the bacterial communities and total acid during the fermentation of HQGRW and elucidate the correlation between total acid and bacterial communities. Results: The results showed that the period of rapid acid increase during fermentation occurred at the early stage of fermentation. There was a negative response between total acid increase and the rate of increase in alcohol during the early fermentation stage. Bacterial community analysis using high-throughput sequencing technology was found that the dominant bacterial communities changed during the traditional fermentation of HQGRW. Both principal component analysis (PCA) and hierarchical clustering analysis revealed that there was a great difference between the bacterial communities of Hong Qu starter and those identified during the fermentation process. Furthermore, the key bacteria likely to be associated with total acid were identified by Spearman's correlation analysis. Lactobacillus, unclassified Lactobacillaceae, and Pediococcus were found, which can make significant contributions to the total acid development (| r| N 0.6 with FDR adjusted P b 0.05), establishing that these bacteria can associate closely with the total acid of rice wine. Conclusions: This was the first study to investigate the correlation between bacterial communities and total acid during the fermentation of HQGRW. These findings may be helpful in the development of a set of fermentation techniques for controlling total acid.

Pediococcus acidolactici and Pediococcus pentosaceus isolated from a rainbow trout ecosystem have probiotic and ABF1 adsorbing/degrading abilities in vitro.

Probiotics are being used in biological control of bacterial pathogens, as an alternative to antibiotics, to improve health and production parameters in fish farming. Fish farming production is severely affected by aflatoxins (AFs), which are a significant problem in aquaculture systems. Aflatoxins exert substantial impact on production, causing disease with high mortality and a gradual decline of reared fish stock quality. Some aspects of aflatoxicosis in fish, particularly its effects on the gastrointestinal tract, have not been well documented. The aim of the present study was to evaluate probiotic properties of lactic acid bacterial (LAB) strains isolated from rainbow trout intestine and feed. Moreover, AFB1-binding and/or degrading abilities were also evaluated to assess their use in the formulation of feed additives. Growth at pH 2, the ability to co-aggregate with bacterial pathogens, inhibition of bacterial pathogens, and determination of the inhibitory mechanism were tested. Aflatoxin B1 (AFB1) adsorption and degradation ability were also tested. All strains were able to maintain viable (107 cells ml-1) at pH 2. Pediococcus acidilactici RC001 and RC008 showed the strongest antimicrobial activity, inhibiting all the pathogens tested. The strains produced antimicrobial compounds of different nature, being affected by different treatments (catalase, NaOH and heating), which indicated that they could be H2O2, organic acids or proteins. All LAB strains tested showed the ability to coaggregate pathogenic bacteria, showing inhibition percentages above 40%. Pediococcus acidilactici RC003 was the one with the highest adsorption capacity and all LAB strains were able to degrade AFB1 with percentages higher than 15%, showing significant differences with respect to the control. The ability of some of the LAB strains isolated in the present work to compete with pathogens, together with stability against bile and gastric pH, reduction of bioavailability and degradation of AFB1, may indicate the potential of LAB for use in rainbow trout culture.

Biodiversity and technological potential of lactic acid bacteria isolated from spontaneously fermented amaranth sourdough.

UNLABELLED: Spontaneous fermented sourdoughs prepared from amaranth flour were investigated for the presence of autochthonous lactic acid bacteria (LAB) predominating microbiota. The doughs were fermented with daily backslopping on a laboratory scale at 30°C for 10 days. LAB counts ranged from 2·60 to 8·54 log CFU g(-1) with a pH declined from 6·2 to 3·8 throughout fermentation. The combined use of randomly amplified polymorphic DNA (RAPD)-PCR analysis and sequence analysis of 16S rRNA was applied for LAB intraspecies differentiation and taxonomic identification, respectively. Enterococcus, Pediococcus and Lactobacillus species were present in amaranth sourdoughs (AS). After the first refreshment step, Lactobacillus plantarum dominated AS until the end of fermentation. In coincidence, when DGGE analysis was performed, the occurrence of a progressive change in bacterial communities allowed the selection of Lact. plantarum as a dominant species. Moreover, technological, functional and safety characteristics of representative RAPD-biotypes were investigated. Lact. plantarum CRL1898 was selected as a potential candidate for gluten-free amaranth sourdough starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is an increasing interest in ancient noncereal gluten-free (GF) crops such as amaranth, due to their reported nutritional and health benefits. However, the use of these grains is still limited to traditional foods and bread making processes that are not yet well standardized. Results on the dynamics of autochthonous lactic acid bacteria (LAB) microbiota during laboratory spontaneous amaranth sourdoughs (AS) fermentation will contribute to overcome challenges for GF-fermented products development. In addition, knowledge about LAB diversity involving Enterococcus, Pediococcus and Lactobacillus species, with Lactobacillus plantarum predominating during AS fermentation, and their technological and functional properties provides the basis for the selection of autochthonous strains as starters cultures for novel gluten-free bakery products with enhanced nutritional, sensory and/or safety quality.

Pediococcus acidilactici isolated from the rumen of lambs with rumen acidosis, 16S rRNA identification and sensibility to monensin and lasalocid.

A lactic-acid producing bacterium was isolated from the rumen of lambs with rumen acidosis. The cells were gram-positive, nonmotile, nonsporing, catalase negative spherical, 1.5-2.0 µm in diameter, and occur in pairs and tetrads. Analysis of 16S ribosomal RNA indicated that the rumen bacterium was a strain of Pediococcus acidilactici with 99% of nucleotide homology. This bacterium was sensible to monensin and lasalocid at the unique dose tested of 300 ppm. The concentration of lactic acid and DM degradation decreased (P<0.05) when monensin or lasalocid were added to the culture media after 24, 48 and 72 h of incubation. In contrast, total VFA concentration and pH were higher (P<0.05) in the culture media added with the ionophores. Up to now S. bovis is considered the main ruminal bacterium related with rumen acidosis, but the importance of P. acidilactici should be also reconsidered in experimental studies focused on the control rumen acidosis.

Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples.

We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20 min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20 min.

Culture-independent analysis of lactic acid bacteria diversity associated with mezcal fermentation.

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.

Transport of glycerol by Pediococcus pentosaceus isolated from wine.

Pediococcus pentosaceus N(5)p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis-Menten relationship with an observed apparent K(m) of 33 microM and a V(max) of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl(2) and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide-m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process.

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 degrees C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1 mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process / Exploración del uso de agentes antimicrobianos naturales y de campos eléctricos pulsantes para el control de bacterias contaminantes durante el proceso de elaboración de cerveza

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 °C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Diferentes antimicrobianos naturales disminuyeron la viabilidad de bacterias contaminantes aisladas en etapas críticas del proceso de producción de cerveza. En un extracto de malta, el agregado de 1 mg/ml de quitosano y de 0,3 mg ml de lúpulo permitió reducir la viabilidad de Escherichia coli a 0,7 y 0,1%, respectivamente, al cabo de 2 horas de incubación a 4 °C. El agregado de 0,0002 mg/ml de nisina, 0,1 mg/ml de quitosano o de 0,3 mg/ml de lúpulo inhibió selectivamente (10.000 veces más) el crecimiento de Pediococcus sp. respecto de la levadura de cerveza en un cultivo mixto. El agregado de 0,1 mg/ml de quitosano permitió disminuir la viabilidad de una cepa bacteriana termorresistente, Bacillus megaterium, hasta niveles no detectables. Por otra parte, el agregado de nisina, quitosano y lúpulo aumentó la estabilidad microbiológica durante el almacenamiento de cervezas inoculadas con Lactobacillus plantarum y Pediococcus sp. aislados de mosto de cerveza. La aplicación de campos eléctricos pulsantes (CEP) (3 pulsos de 8kV/cm) aumentó el efecto antimicrobiano de la nisina y del lúpulo, pero no el del quitosano. Los resultados obtenidos indicarían que el uso de antimicrobianos naturales en forma individual o en combinación con CEP puede constituir un procedimiento efectivo para el control de la contaminación bacteriana durante el proceso de elaboración y almacenamiento de la cerveza.

Gluten breakdown by lactobacilli and pediococci strains isolated from sourdough.

AIMS: To evaluate the growth and metabolic activity of lactobacilli and pediococci strains in a gluten base medium (GBM), formulated for a proper selection of proteolytic strains to be used in sourdough fermentation. METHODS AND RESULTS: Proteolytic activity by lactic acid bacteria (LAB) was evaluated by SDS-PAGE and by the amino acids released determined by reversed-phase high-performance liquid chromatography. Only 13 LAB (nine lactobacilli and four pediococci), among the 42 evaluated were able to utilize gluten as nitrogen source and to grow in GBM. Pediococcus pentosaceus CRL 797 showed a similar proteolytic activity to lactobacilli strains. In the majority of the cultures, basic amino acid group increased (c. 80% after 12 h incubation) mainly due to the release of ornithine, a flavour precursor of bread. Lysine, a limiting essential amino acid in wheat flour, increased by 150% in cultures of P. pentosaceus CRL 797. CONCLUSIONS: This study allows selecting P. pentosaceus CRL 797 and L. plantarum CRL 759 as potential starter culture for type III sourdough fermentation. It is shown for the first time that pediococci strains isolated from sourdough are proteolytically active on gluten. SIGNIFICANCE AND IMPACT OF THE STUDY: The physiological studies on gluten breakdown by LAB will contribute to the better selection of strains to produce breads with enhanced organoleptic characteristics.

Phenotypic and genotypic characterization of Pediococcus strains isolated from human clinical sources.

Seventy-two strains of pediococci isolated from human clinical sources were characterized by conventional physiological tests, chromogenic enzymatic tests, analysis of whole-cell protein profiles (WCPP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of chromosomal DNA restriction profiles by pulsed-field gel electrophoresis (PFGE). Conventional tests allowed identification of 67 isolates: 52 strains were identified as Pediococcus acidilactici, 15 strains were identified as Pediococcus pentosaceus, and 5 strains were not identified because of atypical reactions. Analysis of WCPP identified all isolates since each species had a unique WCPP. By the WCPP method, the atypical strains were identified as P. acidilactici (two strains) and P. pentosaceus (three strains). The chromogenic substrate test with o-nitrophenyl-beta-D-glucopyranoside differentiated all 54 strains of P. acidilactici (negative reactions) and 13 (72%) of 18 strains of P. pentosaceus (positive reactions). Isolates of both species were shown to be nonclonal as revealed by the genetic diversity when chromosomal DNA was analyzed by PFGE. Using WCPP as the definitive identification procedure, P. acidilactici (28 of 54 strains; 51.8%) was more likely than P. pentosaceus (4 of 18 strains; 22.3%) to be isolated from blood cultures.

Characterization of bacteriocin produced by Pediococcus pentosaceus from wine.

Twenty strains of Pediococcus pentosaceus isolated from wine were examined for production of bacteriocins. Only two of them showed inhibitory activity, Ped. pentosaceus N4p against the indicator strains of the same species and N5p against 19 strains of the three genera of lactic acid bacteria from wine. The antimicrobial substance from N5p strains was removed by membrane (0.2 micron) filtration, destroyed by organic solvents and proteolytic enzymes. It was stable for 60 min at 100 degrees C. The bacteriocin was produced early in the growth cycle and its production was maximum after 48 h of culture in tomato juice medium at an initial pH of 6.5. The bactericidal effect was observed.