Enhancement of γ-aminobutyric acid (GABA) in Nham (Thai fermented pork sausage) using starter cultures of Lactobacillus namurensis NH2 and Pediococcus pentosaceus HN8.

The aim was to produce Nham that was enriched with γ-aminobutyric acid (GABA); therefore two GABA producing lactic acid bacteria (Pediococcus pentosaceus HN8 and Lactobacillus namurensis NH2) were used as starter cultures. By using the central composite design (CCD) we showed that addition of 0.5% monosodium glutamate (MSG) together with an inoculum size of roughly 6logCFU/g of each of the two strains produced a maximal amounts of GABA (4051 mg/kg) in the 'GABA Nham' product. This was higher than any current popular commercial Nham product by roughly 8 times. 'GABA Nham' with the additions of both starters and MSG (TSM) supported maximum populations of lactic acid bacteria (LAB) with a minimum of yeasts and no staphylococci or molds when compared to the controls that had no addition of any starters or MSG (TNN), or only the addition of MSG (TNM), or with only the starter (TSN). Based on proximate analysis among the Nham sets, 'GABA Nham' was low in fat, carbohydrate and energy although its texture and color were slightly different from the control (TNN). However, sensory evaluations of 'GABA Nham' were more acceptable than the controls and commercial Nham products for all tested parameters. Hence, a unique novel 'GABA Nham' fermented pork sausage was successfully developed.

Enhancement of gamma-aminobutyric acid in a fermented red seaweed beverage by starter culture Lactobacillus plantarum DW12

Lactobacillus plantarum DW12, a gamma-aminobutyric acid (GABA) producing strain, was used as a starter culture to produce a functional fermented red seaweed beverage (FSB). Optimal conditions for producing FSB were established using Central Composite Design by varying the amounts of monosodium glutamate (MSG), sucrose and the initial pH in MRS medium. After a verification test, 1 percent MSG, 6 percent sucrose and an initial pH of 6 were selected. Four treatments were tested: traditional formula (A), red seaweed-cane sugar-potable water = 3:1:10, w/w/v, initial pH 6; the traditional formula with a 5 percent starter culture consisting of 4.1 x 10(9) CFU of DW 12/ml (B); formula A modified by changing the amounts of cane sugar and MSG to 6 percent and 1 percent, respectively (C); formula C with a 5 percent starter culture added (D). Comparison among the 4 treatments showed that the treatment D produced the highest amount of GABA (4000 mg/L) during days 45-60 while the GABA content of A, B and C treatments was 340, 730 and 1690 mg/L, respectively. However, the results of the sensory test for the treatments C and D showed that the presence of MSG produced an unsatisfactory salty taste. All finished products from the 4 treatments met Thai standard guidelines for chemical and microbiological qualities after 120 days. The results indicated that enrichment of the GABA content in FSB is possible by adding MSG and the GABA producing strain DW12; however, the appropriate amount of MSG addition should be further studied.

Control of Bacillus cereus in foods by Rhodomyrtus tomentosa (Ait.) Hassk. Leaf extract and its purified compound.

Edible natural products, either standardized plant extracts or pure compounds, provide unlimited opportunities as safe new antimicrobial agents. This study investigated the antibacterial properties of ethanolic extract from Rhodomyrtus tomentosa (Ait.) Hassk. leaves against Bacillus cereus. Preliminary screening to evaluate the activities of the extract used a paper disc diffusion method against 65 food isolates. The extract produced large inhibition zones in all isolates, ranging from 10 to 18 mm. The results were confirmed by MIC and MBC (16 to 64 and 32 to 256 µg/ml, respectively). Rhodomyrtone, a purified compound, exhibited MIC and MBC at 0.5 and at 2 to 8 µg/ml, respectively. The antimicrobial activity of the extract on vegetative cells and endospores of a representative B. cereus isolate (MIC=32 µg/ml) was assessed by enumerating viable cells at different time intervals up to 24 h. At 2 MICs and 4 MICs, a reduction in the viability of the bacterial cells and endospores was at least 3 log within 6 to 8 h and 2 h after incubation, respectively. Application of the extract in precooked rice and tuna steak demonstrated that after exposure to 16 MICs and 32 MICs, the numbers of viable cells and endospores in both model systems were reduced by at least 2 log within 12 and 6 h, respectively. Since the extract consistently produced remarkable activity against both cells and endospores, it could be used as an alternative food additive for controlling B. cereus without compromising food safety.

Characterization of the antiyeast compound and probiotic properties of a starter lactobacillus plantarum DW3 for possible use in fermented plant beverages

Lactobacillus plantarum DW3 produced antifungal compounds that inhibited the growth of Rhodotorula mucilaginosa DKA, contaminating yeast in fermented plant beverages (FPBs) and various potential human pathogens. Phenyllactic acid (PLA) identified by gas chromatography- mass spectrometry (GC-MS) was produced at 31 mg/L PLA in MRS medium and 5 mg/ml inhibited growth of the target yeast in vitro by 90 percent. Other inhibitors were also present but not specifically identified. Results of in vitro tests showed that DW3 also had probiotic properties as it survived various human biological barriers resistance to pH 3, bile salts, growth without vitamin B12 and the presence and absence of oxygen. Its inhibitory effect against food borne pathogenic bacteria and spoilage organisms was higher than that found for a commercial strain Lactobacillus casei R. An acute oral toxicity test on ICR mice at a high single dose of either 10(9) and 10(12) cells per mouse for 14 days showed that DW3 had no adverse effect on the general health status and there was no evidence of bacteremia. Mice fed DW3 had a reduced weight gain compared to the control. No significant difference (p > 0.05) was found for the spleen weight index (SWI) among the treatment and control groups whereas there was a significant difference (p < 0.05) for the liver weight ratio (LWR) in a group fed with 10(12) cells per mouse when compared with the control group.

Microbial succession in a fermenting of wild forest noni (Morinda coreia Ham) fruit plus molasses and its role in producing a liquid fertilizer

The numbers of lactic acid bacteria (LAB) and yeasts that were present during a wild forest noni (Morinda coreia Ham) fermentation, the changes in its physico-chemical properties and levels of plant nutrients were investigated. LAB increased rapidly during the first 7 days and were the dominant population until after day 21 when the LAB were declining and the yeasts began to dominate. Identification of the LAB and yeasts to species level showed that the dominant LAB throughout was Lactobacillus plantarum while Lactobacillus pentosus was found but only at day 21. Saccharomyces cerevisiae was the most dominant species of yeast throughout but was slowly replaced by Pichia membranifaciens and then Pichia anomala. Rhodotolura mucilaginosa, an aerobic yeast, was only detected at the beginning of the fermentation process. It is suggested that the Pichia spp. were responsible for consuming lactic acid. After 56 days, the values of pH, acetic acid, ethanol and electrical conductivity in the fermented product were 3.66, 3.34 g L-1, 16.98 g L-1 and 14.47 mS cm-1, respectively. Increased amounts of plant nutrients were present at day 56 mostly derived from the degradation of plant material. At day 56 the amounts were as follows (in mg L-1): N 633, P 1210, K 4356, Ca 693, Mg 536, Mn 7, B 51, Zn 169, and total carbon/total nitrogen ratio (C/N ratio) 18. Based on the seed germination index (GI) of cherry tomato (Lycopersicon esculentum Mill), the extract diluted 256-fold gave the best GI of 157 percent.

Effects of initial air removal methods on microorganisms and characteristics of fermented plant beverages.

The effects of 3 different methods for removing the initial air on the properties of fermented plant beverages produced from phom-nang seaweed (Gracilaria fisheri) and wild forest noni (Morinda coreia Ham.) were investigated. Only method M which covered the space above the fermentation liquid with a water filled plastic bag produced no surface film of yeast, had the highest acidity and also antibacterial activity from both plants after 90 days of fermentation. However, the yeast count still exceeded the standard guidelines for plant beverages. The fermented beverage from wild forest noni showed more antibacterial activity against 3 of 4 pathogenic bacteria tested than that from the phomnang seaweed, probably for its higher levels of acidity and ethanol content. Lactic Acid Bacteria (LAB) isolated from the fermentation samples from days 1-5 using the method M from both fermented plant beverages were Leuconostoc mesenteroides supsp. mesenteroides and Leu. mesenteroides subsp. dextranicum while presence of Lactobacilus plantarum was only recorded at days 4-5 in the wild forest noni beverage. From days 6-14 the isolates were Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus brevis from wild forest noni beverage, whereas only L. brevis was not detected in the seaweed beverage. During days 21-45 both beverages had a similar LAB population of L. plantarum and L. brevis while L. coryniformis was only found in the wild forest noni beverage. Between days 60-90 in both plant beverages only L. plantarum and Lactobacillius sp. were detected.

Selection of sulfur oxidizing bacterium for sulfide removal in sulfate rich wastewater to enhance biogas production

Sulfur oxidizing bacteria (SOB) were isolated and tested in order to remove sulfide from high sulfate wastewater to reduce the amount of hydrogen sulfide (H2S) in the produced biogas. A promising SOB isolate, designated as isolate T307, was selected due to its best sulfide removal (86.7 percent) in the effluent of a sulfate reduction reactor (SRR) over a 24 hrs incubation. The bacterium was able to grow better as a mixotroph (yeast extract as a carbon source) than as a chemolithoautotroph. In addition, as a heterotroph, the bacterium grew well with yeast extract and peptone. Based on partial 16S rRNA gene sequence, the isolated T307 was an Alcaligenes sp. and was able to convert most of sulfide species (total sulfide: TS; dissolved sulfide: DS and H2S) into elemental sulfur or sulfate over a 20 hrs period of cultivation by controlling the speed of shaking. In a biogas reactor set, after pre-treating a sulfide medium with Alcaligenes sp. T307 there was a much higher specific yield of CH4 (238 ml CH4 g-1COD removed) and more biogas (154 ml L-1 d-1) was produced with the biogas containing more methane (48.1 percent CH4, 51.5 percent CO2 and 0.41 percent H2S) in comparison to a control with a specific yield of CH4, (72 ml CH4 g-1COD removed) 86 ml L-1 d-1 biogas produced with a composition of 35.5 percent CH4, 63.7 percent CO2 and 0.86 percent H2S.

Selection of lactic acid bacteria from fermented plant beverages to use as inoculants for improving the quality of the finished product.

Lactic Acid Bacteria (LAB) from fermented plant beverages were selected based on their antibacterial actions against potential food borne pathogenic bacteria (Staphylococcus aureus PSSCMI 0004, Escherichia coli PSSCMI 0001, Salmonella typhimurium PSSCMI 0034 and Vibrio parahaemolyticus VP 4). Antibacterial activities were measured using an agar spot method. The Lactobacillus plantarum W90A strain isolated from a wild forest noni (Morinda coreia Ham) beverage was used as an inoculant. Three different inoculation procedures were conducted with the fruit of wild forest noni fermentations to establish which one was the best for controlling the numbers of yeast in the finished product. A 5% inoculum of L. plantarum W90A (LAB set), initial cell density 8.6 log cfu mL(-1), produced a better product and inhibitory properties against the test organisms, particularly E. coli PSSCMI 0001 than one with no inoculum or with a 5% inoculum from a previous natural fermented product. An LAB inoculum resulted in a reduced total bacterial count and no yeast throughout fermentation period (90 days). The lower yeast resulted in a reduction of the ethanol content to 2.9 g L(-1) compared to 12.2 g L(-1) inthe culture with no inoculum. The highest acidity (1.3-1.4%) with the same pH (3.3) was observed in both sets of inoculated fermentations, whereas the uninoculated set gave a pH value of 3.7 (1.2% acidity).