Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing.

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/µL to 87 ng/µL. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices.

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast-starch-glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

Molecular characterization of lactic acid bacteria and in situ amylase expression during traditional fermentation of cereal foods.

Lactic acid bacteria play an important role in traditional fermented foods consumed in different countries. Study of their taxonomic structure and diversity is necessary for starter culture selection, improved safety and nutritional enhancement. To achieve these objectives, microbial genomic typing methods were used to study genetic differences of autochthonous bacteria and their distribution in two traditional African fermented cereal foods. A total of 85 predominant bacterial species were isolated from ogi and kunu-zaki obtained from Northern and Southern geographical region of Nigeria. They were identified using combination of 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) based on rpoA, pheS and atpA genes as well as M13-PCR gel fingerprints. The results showed that Lactobacillus fermentum was the most frequently isolated species in ogi (71.4%) and kunu-zaki (84.5%). Other species of lactic acid bacteria (LAB) identified were Lactobacillus plantarum, Streptococcus gallolyticus subsp. macedonicus and Pediococcus pentosaceus. Non lactic acid bacteria isolated from these foods were species belonging to the Bacillus and Staphylococcus. Non-metric multidimensional scaling (nMDS) analysis of the M13-PCR fingerprints for LAB strains showed clonal diversity among strains of the same species. In vitro and in situ expression of amylase gene during fermentation by amylolytic L. plantarum ULAG11 was detected, indicating the potential usefulness of such species for development of starter cultures and for controlled fermentation processes.

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance.

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species.

Incidence and characterization of Bacillus cereus isolated from traditional fermented meals in Nigeria.

The aim of this study was to examine the presence of Bacillus cereus in fermented meals used in food seasoning in Nigeria. The microbial profiles of iru and ogiri, two Nigerian fermented vegetable proteins, were examined for presence of B. cereus. In the 50 samples tested, B. cereus was detected in all the samples, with the level of detection ranging from log 6.3 to log 8.3 g(-1) sample. Phenotypic characteristics of the B. cereus isolates showed that all of them could not ferment many sugars, most especially mannitol, but they utilized propionate citrate as a source of carbon and grew anaerobically. The isolates do not produce gas from glucose but hydrolyzed starch, casein, and gelatin. API-50CHB combined with API-20E identified the isolates as B. cereus. The diarrheal enterotoxin was detected by a reversed passive latex agglutination test kit. Results showed no significant difference in toxin production between ogiri and iru B. cereus isolated from different sources; all the isolates also demonstrated positive hemolytic activity. The API-ZYM enzyme profile showed that the strains have poor hydrolytic enzyme potential; hence, their possible contributions to the fermentation of vegetable protein is doubtful. This study established the proliferation of B. cereus in fermented protein meal and determined the diarrheal toxin production potential of the organism.