Comparison of the Effects of Microbial Inoculants on Fermentation Quality and Microbiota in Napier Grass (Pennisetum purpureum) and Corn (Zea mays L.) Silage.

Forage preservation for livestock feeding is usually done by drying the plant material and storing it as hay or ensiling it into silage. During the ensiling process, the pH in the system is lowered by the activities of lactic acid-producing bacteria (LAB), inhibiting the growth of spoilage microorganisms and maintaining the quality of the ensiled product. To improve this process, inoculation of LAB could be used as starter cultures to shorten the ensiling time and control the fermentation process. Here, we compared fermentation quality and bacterial dynamics in two plant materials, whole-plant corn (Zea mays L.) and Napier grass (Pennisetum purpureum), with and without starter inoculation. The efficacy of Lactobacillus plantarum, L. brevis, and Pediococcus pentosaceus as starter cultures were also compared in the ensiling system. In whole-plant corn, pH decreased significantly, while lactic acid content increased significantly on Day 3 in both the non-inoculated and LAB-inoculated groups. Prior to ensiling, the predominant LAB bacteria were Weissella, Enterococcus, and Lactococcus, which shifted to Lactobacillus during ensiling of whole-plant corn in both the non-inoculated and LAB inoculated groups. Interestingly, the epiphytic LAB associated with Napier grass were much lower than those of whole-plant corn before ensiling. Consequently, the fermentation quality of Napier grass was improved by the addition of LAB inoculants, especially L. plantarum and a combination of all three selected LAB strains showed better fermentation quality than the non-inoculated control. Therefore, the different abundance and diversity of epiphytic LAB in plant raw materials could be one of the most important factors determining whether LAB starter cultures would be necessary for silage fermentation.

Development of bacteria identification array to detect lactobacilli in Thai fermented sausage.

To improve the quality and safety of food products, there is a need in the food industry for a reliable method for simultaneously monitoring multiple bacterial strains. Microarray technology is a high-throughput screening approach that can provide an alternative for bacteria detection. A total of 164 bacteria-specific probes were designed from 16S rRNA gene sequences to target 12 bacteria species, including lactic acid bacteria and selected food pathogens. After fabrication onto aminosilane-coated slides, hybridization conditions of the array were optimized for high specificity and signal intensities. The array was applied to detect 12 bacteria individually and was specific to all (Lactobacillus plantarum group, L. fermentum, L. brevis, L. delbrueckii, L. casei, L. sakei, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes) except L. animalis. Multiplex detection using mixed bacteria populations was evaluated and accurate detection was obtained. The feasibility of using the array to detect the target bacteria in food was evaluated with Thai fermented sausages (Nham). Meat samples were collected on days 2, 3 and 7 after natural fermentation, L. plantarum-inoculated fermentation and L. brevis-inoculated fermentation before applying to the array. The naturally-fermented Nham contained L. sakei, L. delbrueckii, L. plantarum and L. fermentum. The L. plantarum-inoculated Nham showed a similar lactic acid bacteria population but the positive signal level for L. plantarum was higher than with natural fermentation. The L. brevis-inoculated Nham contained L. brevis, L. plantarum, L. delbrueckii and L. fermentum. The array was used to monitor bacteria population dynamics during the fermentation process. The naturally-fermented and L. brevis-inoculated samples showed lower positive signal levels of L. plantarum on day 2, but signals gradually increased on days 3 and 7 of the fermentation. In contrast, the L. plantarum-started fermentation showed a higher positive signal level on day 2 than the natural and L. brevis-inoculated samples, and the positive signal level remained high on days 3 and 7. The bacteria identification array was proven to be useful as an alternative method to detect and monitor target bacteria populations during food fermentation.

Systematic identification of Lactobacillus plantarum auxotrophs for fermented Nham using genome-scale metabolic model.

In this study, a systematic strategy is presented, which identifies auxotrophic starters for the popular Thai fermented sausage product, called Nham, using a genome-scale metabolic model. A published genome-scale model of Lactobacillus plantarum WCFS1 is adopted for studying the L. plantarum BCC9546 characteristics cultured on Simulated Nham Broth. Single gene deletion analysis is performed to determine the genes essential for cell growth. Strains lacking such essential genes are considered potential auxotrophic mutants. Then, metabolite supplement analysis is introduced to determine a list of metabolites supplements for each mutant required to restore its growth. Herein, 9 potential auxotrophs are proposed for use in Nham fermentation, along with their metabolite supplements. Simulation studies showed that the secreted fluxes of organic acids, as well as amino-derived flavor compounds of these auxotrophs, are similar to those of the wild-type, indicating that Nham fermented by these auxotrophs would have similar tastes and flavors as Nham fermented by the wild-type. These proposed auxotrophs and corresponding nutritional supplements will be useful for the design of auxotroph starter culture utilized for Nham production in the laboratory. The systematic strategy presented here will facilitate the analysis and development of auxotroph starters used in the food industry.

Draft genome sequence of Arthrospira platensis C1 (PCC9438).

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Insights into the phylogeny and metabolic potential of a primary tropical peat swamp forest microbial community by metagenomic analysis.

A primary tropical peat swamp forest is a unique ecosystem characterized by long-term accumulation of plant biomass under high humidity and acidic water-logged conditions, and is regarded as an important terrestrial carbon sink in the biosphere. In this study, the microbial community in the surface peat layer in Pru Toh Daeng, a primary tropical peat swamp forest, was studied for its phylogenetic diversity and metabolic potential using direct shotgun pyrosequencing of environmental DNA, together with analysis of 16S rRNA gene library and key metabolic genes. The community was dominated by aerobic microbes together with a significant number of facultative and anaerobic microbial taxa. Acidobacteria and diverse Proteobacteria (mainly Alphaproteobacteria) constituted the major phylogenetic groups, with minor representation of archaea and eukaryotic microbes. Based on comparative pyrosequencing dataset analysis, the microbial community showed high metabolic versatility of plant polysaccharide decomposition. A variety of glycosyl hydrolases targeting lignocellulosic and starch-based polysaccharides from diverse bacterial phyla were annotated, originating mostly from Proteobacteria, and Acidobacteria together with Firmicutes, Bacteroidetes, Chlamydiae/Verrucomicrobia, and Actinobacteria, suggesting the key role of these microbes in plant biomass degradation. Pyrosequencing dataset annotation and direct mcrA gene analysis indicated the presence of methanogenic archaea clustering in the order Methanomicrobiales, suggesting the potential on partial carbon flux from biomass degradation through methanogenesis. The insights on the peat swamp microbial assemblage thus provide a valuable approach for further study on biogeochemical processes in this unique ecosystem.

Monitoring Lactobacillus plantarum BCC 9546 starter culture during fermentation of Nham, a traditional Thai pork sausage.

The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditional Thai fermented pork sausage ensures product quality and consistency. However, no direct evidence has confirmed the growth of this starter during Nham fermentation. In order to investigate its role during Nham fermentation, LpBCC9546 was genetically modified to distinguish it from the natural microflora in Nham. LpBCC9546 was transformed with a recombinant plasmid pRV85 to produce the recombinant strain LpG11, which is resistant to erythromycin and emits green fluorescence. LpG11 was used as a starter culture for Nham fermentation, and its growth was monitored by plating on a selective medium and assay of fluorescent activity. During Nham fermentation the numbers of LpG11 increased ten fold during the first 12 h of fermentation, reaching maximum numbers of between 10(7) and 10(8) cfu g(-1) after 24 h, and then declining after 60 h to 10(5) cfu g(-1) at 168 h. The growth of LpG11 starter culture during Nham fermentation was very similar to that of the untransformed LpBCC9546, although after a prolonged period of fermentation the recombinant LpG11 bacteria appeared to lose the plasmid, or were outgrown by naturally present L. plantarum. The acidity, texture and color of fermented Nham inoculated with recombinant LpG11 or untransformed LpBCC9546 were similar. These results indicated that the recombinant L. plantarum strain LpG11 is a suitable starter culture for Nham fermentation, and that the ability to monitor its growth directly during Nham fermentation could be exploited to further improve Nham production.

DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations.

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.