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.     

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.     

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.     

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.     

Phenotypic and genotypic characterization of foodborne bacteria isolated from Sinop Province,Turkey

In this study, genotypic and phenotypic characteristics of the strains, antibiotic susceptibility, and antibiotic resistance genes of the Escherichia coli, Bacillus cereus, Listeria spp., and Salmonella spp. isolated from various animal foods in Sinop province were determined. Resistance percentages of the isolated strains to the antibiotics penicillin, vancomycin, clindamycin, tetracycline, ampicillin/sulbactam, gentamicin, ciprofloxacin, and chloramphenicol were 87.8, 71.9, 69.7, 63.4, 27.5, 23.1, 20.9, and 13.4%, respectively. Antibiotic resistance genes tetA, bla_CMY-2, and cat1 were found in 45, 21.25, and 6.25% of all the strains, respectively. The virulence genes were also investigated in the isolated in E. coli strains; the eae gene was found in 13.1% of the strains, while the stx1 was not identified in any strains. As a result, we can say that SDS-PAGE method may provide discrimination at species level; however, surveys at subspecies level demand molecular approaches such as ERIC and (GTG)₅-PCR. In addition, we can suggest that molecular techniques may be especially helpful in the rapid identification of multidrug-resistant and isolates with virulence genes. This study has revealed that the investigated bacteria in animal foods is a significant reservoir of resistance genes.     

新疆特色干酪中乳酸菌的分离鉴定

从新疆不同牧区采集工艺不同的干酪制品,对其中的乳酸菌进行分离纯化、生理生化性质试验和16S rRNA分析.结果表明,分离、纯化出的104株乳酸菌种,有82株为乳杆菌属(Lactobacillus),12株为肠球菌属(Enterococcus),10株为魏斯氏菌属(Weissella).利用16S rRNA序列同源分析和系统发育树分析对具有不同生理生化特性的代表菌株进行了分子鉴定,鉴定结果为TNM-2与干酪乳杆菌(Lactobacillus casei)、Y5-4与食窦魏斯氏菌(Weissella cibaria)、NS2-2与植物乳杆菌(Lactobacillus plantarum)的同源性达到100％,NM-2与瑞士乳杆菌(Lactobacillus helveticus)、Y1-1与马乳酒乳杆菌(Lactobacillus kefiranofaciens)、WG与耐久肠球菌(Enterococcus durans)的同源性达到99％.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Fingerprint of lactic acid bacteria population in beef carpaccio is influenced by storage process and seasonal changes

We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log₁₀ cfu g⁻¹ to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log₁₀ cfu g⁻¹ to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.     

Metabolism of lactic acid in fermented cucumbers by Lactobacillus buchneri and related species,potential spoilage organisms in reduced salt fermentations

Recent evidence suggests that Lactobacillus buchneri may play an important role in spoilage-associated secondary fermentation of cucumbers. Lactic acid degradation during fermented cucumber spoilage is influenced by sodium chloride (NaCl) concentration, pH, and presence of oxygen. Objectives were to evaluate these factors on lactic acid utilization by L. buchneri, and to compare the biochemical changes to those which occur during fermented cucumber spoilage. Effects of NaCl (0, 2, 4, and 6% w/w), pH (3.8 vs 5.0), and aerobic environment were investigated using fermented cucumber media (FC) inoculated with spoilage microorganisms. At pH 3.8, L. buchneri degraded lactic acid in all NaCl concentrations. The highest rate of lactic acid utilization occurred in FC with 2% NaCl (P < 0.05). Lactic acid utilization was nearly identical under aerobic and anaerobic conditions, indicating that oxygen does not influence lactate metabolism by L. buchneri. Lactic acid utilization was accompanied by increases in acetic acid and 1,2-propanediol, and Lactobacillus rapi was able to convert 1,2-propanediol to propionic acid and propanol. L. buchneri initiated spoilage in a wide range of environmental conditions that may be present in commercial cucumber fermentations, and L. rapi may act syntrophically with L. buchneri to produce the commonly observed spoilage metabolites.     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.     

Microbiological and organoleptic characteristics of beef trim and ground beef treated with acetic acid, lactic acid, acidified sodium chlorite, or sterile water in a simulated commercial processing environment to reduce Escherichia coli O157:H7 and Salmonella

The objective of this study was to validate the effectiveness of acetic and lactic acids (2% and 5%), acidified sodium chlorite (1000 ppm), and sterile water in reducing Escherichia coli O157:H7 and Salmonella Typhimurium in inoculated beef trim in a simulated processing environment. Samples were collected to assess microbial characteristics at three processing points. Results from this study indicate that all treatments, including sterile water, reduced pathogen concentrations (P < 0.05) of both E. coli O157:H7 and Salmonella Typhimurium in ground beef up to 0.5 and 0.6 log by 24 h, respectively. In some cases, there were no significant differences between the antimicrobial treatments and the sterile water using this application method. Triangle sensory test results of non-inoculated beef indicated there were no differences (P < 0.05) in the means of correct responses between controls or antimicrobial treatments at 6 or 24 h. While interventions are important for beef trim, use of the interventions must be validated under industry conditions to ensure proper effectiveness.     

Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora

In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures.     

Rosmarinic acid,scutellarein 4′-methyl ether 7-O-glucuronide and (16S)-coleon E are the main compounds responsible for the antiacetylcholinesterase and antioxidant activity in herbal tea of Plectranthus barbatus (“falso boldo”)

Plectranthus barbatus, known as “falso boldo” in Brazil, is used in herbal tea or cooked as a vegetable. Infusions and decoctions of leaves from P. barbatus were analysed for their inhibition of acetylcholinesterase and their antioxidant activity. The decoction showed high inhibition activity (31% inhibition with 0.5 mg of extract/ml) and also high antioxidant activity (IC₅₀ = 45.8 ± 0.5 μg of dry extract/ml in the DPPH test; IC₅₀ = 69.8 ± 3.1 μg of dry extract/ml in the β-carotene–linoleic acid test). Rosmarinic acid, scutellarein 4′-methyl ether 7-O-glucuronide and (16S)-coleon E were the main constituents identified. These compounds have antiacetylcholinesterase activity. Rosmarinic acid and the scutellarein derivative have IC₅₀ = 440 μg/ml and 1 mg/ml, respectively. One milligram per millilitre of (16S)-coleon E showed 61% inhibition of the enzyme. Other Plectranthus species, P. ecklonii, P. fructicosus, P. lanuginosus and P. verticillatus, were also analysed and the results obtained correlated with the content in rosmarinic acid.