In-Vitro Synergestic Effects Between Bifidobacterium Pseudocatenulatum G4 and Inulin on Human Gastrointestinal Tract Microbial Composition

The eagerness in finding the most effective probiotic strain has attracted many investigations. Bifidobacterium pseudocatenulatum G4, strain isolated from free-living infant was reported to have characteristics as probiotic candidate. Meanwhile, inulin is a known natural source of carbon that can act as a prebiotic substance. The consumption of probiotic, prebiotic, and its combination (synbiotic) was reported to have the ability to alter microbial composition in human gastrointestinal tract (GIT). In this study, the effects of B. pseudocatenulatum G4 (probiotic), inulin (prebiotic) and its combination (synbiotic) towards the human GIT microbial composition were evaluated in vitro. The effects of inulin incorporated in chocolate products as one of its ingredients were also tested. Real-time PCR assay with selected genus- and species-specific primers were used as a tool in identification and enumeration of selected bacterial strain in fermentation of mixture of bacteria from human faecal sample while dilution and plate count technique was used to enumerate the bacterial cell in fermentation of pure culture bacteria. The morphology of the tested Bifidobacterium strains was observed and the species was confirmed by molecular method targeting 16S rRNA gene. In pure culture batch fermentation of tryptone peptone yeast (TPY) medium supplemented with 0.5% inulin, B. pseudocatenulatum G4 grew at the growth rate of 0.53 ± 0.06 log10 h-1 as compared to other Bifidobacterium strains namely B. breve ATCC 15700, B. longum BB536, and B. infantis ATCC 15697 which grew at 0.45 ± 0.04 log10 h-1, 0.31 ± 0.08 log10 h-1, and 0.72 ± 0.03 log10 h-1, respectively. The same amount of inulin was then introduced into darkand milk chocolate and caused B. pseudocatenulatum G4, B. breve ATCC 15700, B. longum BB536, and B. infantis ATCC 15697 to grow at 0.54 ± 0.06, 0.44 ± 0.04, 0.36 ± 0.05, 0.73 ± 0.02 log10 h-1 for dark chocolate and 0.57 ± 0.05, 0.46 ± 0.03, 0.41 ± 0.04, 0.75 ± 0.01 log10 h-1 for milk chocolate respectively. Some of the chocolate ingredients had also influenced the growth of B. pseudocatenulatum G4. The addition of 0.5% of cocoa liquor in TPY medium caused B. pseudocatenulatum G4 to grow at 0.29 ± 0.03 log10 h-1, and isomalt at 0.59 ± 0.05 log10 h-1 compared to TPY medium without any additional carbon source which grew at 0.19 ± 0.02 log10 h-1, while the addition of cocoa butter did not support the growth of B. pseudocatenulatum G4. In 24 hours batch fermentation of human faecal bacteria, B. pseudocatenulatum G4 (Probiotic) showed its probiotic effects by inhibiting the growth of Salmonella and Enterococcus faecalis. The addition of inulin (Prebiotic) selectively supported the growth of Bifidobacterium and Lactobacillus as well as inhibits the growth of Bacteroides, Salmonella, and E. faecalis. The synbiotic combination of B. pseudocatenulatum G4 and inulin (Synbiotic) showed a synergestic effect as they reduced the number of Bacteroides, Salmonella, and E. faecalis better than Probiotic or Prebiotic alone. Synbiotic chocolate preparations (DCsynbiotic and MCsynbiotic) showed better synergestic effect with B. pseudocatenulatum G4 compared to Synbiotic when Bifidobacterium increased at 1.64 log10 (DCsynbiotic) and 1.67 log10 cells/ml (MCsynbiotic) from the initial counts. Lactobacillus also increased its cell number higher than Synbiotic treatment. Nevertheless, synbiotic chocolate preparations also gave a positive result towards the growth of potential pathogenic bacteria when compared to Synbiotic. However, the inhibition pattern still can be observed on Salmonella and E. faecalis when compared to glucose (control). The antimicrobial action was largely due to the pattern of lactic and acetic acid production in fermentation. Here, the synbiotic approach was more efficient than prebiotic or probiotic alone to modulate the human GIT microbial composition and B. pseudocatenulatum G4 with inulin is a compatible synbiotic pair to perform the function

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice

Aims:  To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. Methods and Results:  Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 μl of three different concentrations of B. pseudocatenulatum G4 (2 × 104, 1 × 108 and 1 × 1011 CFU day−1) for 4 weeks. One group was orally administered with 50 μl of 1 × 108 CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 μl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. Conclusion:  Administration of high dose of up to 1 × 1011 CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. Significance and Impact of the Study: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption

Mo (VI) reduction to molybdenum blue by Serratia marcescens strain Dr. Y9.

In this work we report on the isolation of a local molybdenum-reducing bacterium. The bacterium reduced molybdate or Mo(6+) to molybdenum blue (oxidation states between 5+ to 6+). Electron donors that supported cellular growth were sucrose, maltose, mannitol,fructose, glucose and starch (in decreasing order) with sucrose supporting formation of the highest amount of molybdenum blue at 10 g/l after 24 hours of static incubation. The optimum molybdate and phosphate concentrations that supported molybdate reduction were 20 and 5 mM, respectively. Molybdate reduction was optimal at 37∞C. The molybdenum blue produced from cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as S. marcescens strain Dr.Y9 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. No inhibition of molybdenum-reducing activity was seen using electron transport system (ETS) inhibitors such as antimycin A,1 HQNO (Hydro-xyquinoline-N-Oxide), sodium azide and cyanide suggesting that the ETS of this bacterium is not the site of molybdate reduction

Effect of Ganoderma lucidum polysaccharides on the growth of Bifidobacterium spp. as assessed using real-time PCR

The use of component from Ganoderma lucidum as prebiotic source is interesting as the G. lucidum itself was known for more than a decade in the traditional Chinese medicine. In this work, Ganoderma lucidum crude polysaccharides (GLCP) and Polysaccharide-fraction number 2 (PF-2) were used as carbon sources in the fermentation with Bifidobacterium sp. The results showed the potential of prebiotic effect of the G. lucidum extract in batch-culture fermentation based on increment in the growth of bacteria used (0.4-1.5 log10CFU/mL) after 18h fermentation. Fermentation was further done using faecal materials as bacterial inocula and bacterial growth changes were examined using real-time PCR. The results showed the ability of GLCP and PF-2 to support the growth of Bifidobacterium genus with 0.3 and 0.7 log10cells/ml increased, respectively. Interestingly, Lactobacillus which is known as beneficial bacterial genus also showed growth increment with 0.7 and 1 log10cells/ml increased. The competition for carbon sources thus inhibits the growth of potentially harmful genus, Salmonella (0.3 and 0.5 log10cells/ml) in comparison to the control

Selected microbial groups and short-chain fatty acids profile in a simulated chicken cecum supplemented with two strains of Lactobacillus.

Among the bacterial fermentation end products in the chicken cecum, butyrate is of particular importance because of its nutritional properties for the epithelial cell and pathogen inhibitory effects in the gut. An in vitro experiment, operated with batch bioreactor, was conducted to quantify butyric-producing bacteria in a simulated broiler cecum supplemented with Lactobacillus salivarius ssp. salicinius JCM 1230 and Lactobacillus agilis JCM 1048 during 24 h of incubation. Selected bacterial species were determined by real-time PCR and short-chain fatty acids and lactate concentrations were monitored. The results showed that after 24 h of incubation, Lactobacillus supplementation significantly increased the number of lactobacilli, bifidobacteria and Faecalibacterium prausnitzii in medium containing cecal content and lactobacilli supple-mentation (Cc + L) compared with the control (Cc). Addition of lactobacilli did not alter Escherichia coli and Clostridium butyricum, whereas it significantly (P < 0.05) reduced Salmonella in treatment Cc + L compared with the Cc treatment. Propionate and butyrate formation were significantly (P < 0.05) increased in treatment Cc + L as compared with the Cc treatment. Lactate was only detected in treatment containing 2 Lactobacillus strains. After 24 h of incubation, acetate concentration significantly (P < 0.05) decreased in all treatments. It was suggested that lactate produced by Lactobacillus in the cecal content improved the growth of butyric producers such as F. prausnitzii, which significantly increased butyrate accumulation. Additionally, the results showed that butyrate and propionate inhibited Salmonella without influencing the E. coli profile

Kesan penggunaan inulin dan coklat berinulin terhadap pertumbuhan in vitro bifidobakteria (Effects of inulin and inulin containing chocolate on in vitro growth of bifidobacteria)

Coklat adalah salah satu daripada makanan pembekal tenaga segera dan mempunyai kandungan antioksida yang tinggi. Dalam kajian ini, fungsi coklat telah ditingkatkan dengan menjadikannya sebagai pembawa bahan prebiotik melalui penukaran keseluruhan kandungan gula dengan inulin. Ujian awal telah dilakukan untuk memilih kepekatan atau dos inulin yang paling sesuai sebagai sumber karbon untuk merangsang pertumbuhan bifidobakteria dalam fermentasi kultur kelompok selama 24 jam dengan menilai kesannya terhadap perubahan kepekatan sel. Ujian dilakukan terhadap 4 strain bifidobakteria iaitu Bifidobacterium longum BB536, B. breve ATCC 15700, B. infantis ATCC 15697 dan B. pseudocatenulatum G4. Keputusan menunjukkan kesemua strain bifidobakteria mampu menggunakan inulin sebagai sumber karbon pada kepekatan 2, 5, 10 hinggalah 15 g/L inulin. Manakala kepekatan 5 g/L inulin dilihat mampu digunakan secara optimum (tiada perbezaan signifikan pada p>0.05) oleh keempat-empat spesies bifidobakteria tersebut. Inulin pada kepekatan 5 g/L telah digunakan untuk menghasilkan produk coklat susu (MC-1) dan coklat gelap (DC-2) berinulin, yang seterusnya dinilai kemampuan produk merangsang pertumbuhan spesis bifidobakteria berkenaan secara in vitro. Didapati wujud korelasi negatif yang signifikan (p<0.01) antara pertambahan bilangan bifidobakteria dan pH untuk setiap kultur tulen B. pseudocatenulatum G4, B. Infantis, B. Breve (masing-masing r= -0.97) dan B. longum BB536 (r= -0.95). Inulin dengan kepekatan 5 g/L telah digunakan sebagai ramuan produk coklat berinulin atau prebiotik. Ia didapati dapat meningkatkan pertumbuhan keempat-empat spesis bifidobakteria berkenaan dengan lebih berkesan berbanding penggunaan inulin sahaja sebagai sumber karbon

COCONUT WATER VINEGAR: NEW ALTERNATIVE WITH IMPROVED PROCESSING TECHNIQUE

Vinegar is a condiment made from various sugary and starchy materials by alcoholic and subsequent acetic fermentation. Vinegar can be produced via different methods and from various types of raw material. A new alternative substrate for vinegar production namely mature coconut water has been tested and was compared with 2 common substrates which were coconut sap and pineapple juice. Substrates such as sap and juices have been found to have high amount of total soluble solids which corresponding to high sugar content in the substrates which is more than 14oBrix. Therefore, both substrates could be directly used for vinegar production without requirement of other carbon sources. However, coconut water which showed low Brix value need to be adjusted to 14oBrix by adding sucrose prior to the fermentation process. Substrates fermented with Saccharomyces cerevisiae have yielded 7-8% of alcohol within 7-10 days aerobic incubation at room temperature. The alcoholic medium were then used as a seed broth for acetic fermentation with Acetobactor aceti as inoculums and fermented for approximately 2 months to obtain at least 4% of acetic acid. Investigation on the effect of inoculum sizes and implementation of back-slopping technique were performed to improve the processing method for coconut water vinegar production. The results show that 10% of inoculum size was the best for acetic acid fermentation and the back-slopping technique has helped to reduce the process time of coconut water vinegar production