Studies on Microbial Quality, Protein Yield, and Antioxidant Properties of Some Frozen Edible Insects.

This research investigated the microbial quality and the protein contents of Thai commercial frozen products of silkworm (Bombyx mori), bamboo caterpillar (Omphisa fuscidentalis), and field cricket (Gryllus bimaculatus). Total mesophilic aerobic counts were about 8-8.4 log cfu/g, while lactic acid bacteria numbers were up to 5.2 log cfu/g samples. Yeasts and mold as well as Enterobacteriaceae numbers were found to be no more than 6.3 and 6.6 log cfu/g, respectively, while Clostridium spp. enumeration detected 3.2-3.6 cfu per gram frozen insect samples. The protein content in cases of cricket and bamboo caterpillar after the Soxhlet defatting method and the Folch lipid extraction combined with isoelectric point precipitation were similar when compared at pH 3.5 or pH 4.5. In contrast, the protein yield from silkworm was higher at pH 3.5 in the Soxhlet defatted sample, as opposed to the Folch method treated sample where higher protein yield was obtained at pH 4.5. Furthermore, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP) of field cricket subcritical water (121 or 135°C for 15 or 30 minutes) hydrolysates were also measured on a pilot basis. These tests revealed higher antioxidant activities in treated samples than in the untreated control. The highest DPPH radical scavenging activity and FRAP values were detected in samples treated at 135°C, while the protein content of these samples was lower than that of the untreated control. These results indicate that hydrolytic compounds of proteins and probably other types of cricket materials are possibly involved in the antioxidant activities of the treated defatted cricket samples.

Potential Prebiotic Oligosaccharide Mixtures from Acidic Hydrolysis of Rice Bran and Cassava Pulp.

Two agricultural wastes, rice bran and cassava pulp were subjected to acidic hydrolysis by 2 M sulfuric acid which resulted in hemicellulosic oligosaccharide mixtures. Monosaccharide component analysis of these mixtures revealed that the oligosaccharides of rice bran acid hydrolysate (RAHF) composed of glucose and arabinose while cassava pulp acid hydrolysate (CAHF) was found to be comprised of glucose, galactose and arabinose. Both RAHF and CAHF were able to fuel all of the tested three Lactobacillus, five Bifidobacterium and three Bacteroides strains indicating the prebiotic potential of these oligosaccharide mixtures. Moreover, Lb. gasseri grew significantly better on RAHF than on inulin, a benchmark prebiotic oligo- and polysaccharide mixture. When the digestibility of RAHF and CAHF were tested it was found that these oligosaccharide mixtures were only slightly hydrolyzed upon exposure to simulated human gastric (by less than 8%) and pancreatic juices (by less than 3%). Additionally, most sensory attributes of the above obtained oligosaccharide mixtures supplemented two model cereal drink formulations were generally not different from those of the control, while the overall acceptance was not affected significantly in one cereal drink formulation.

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.

Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production.

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H(+)-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 degrees C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici.

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H(+)-ATPase activities in energized cells by N,N'-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C(18:1)+C(18:3n6))/(C(16:0)+C(18:0))) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.

Mechanism of growth inhibition by free bile acids in lactobacilli and bifidobacteria.

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.

Cholic acid accumulation and its diminution by short-chain fatty acids in bifidobacteria.

Cholic acid (CA) transport was investigated in nine intestinal Bifidobacterium strains. Upon energization with glucose, all of the bifidobacteria accumulated CA. The driving force behind CA accumulation was found to be the transmembrane proton gradient (Delta pH, alkaline interior). The levels of accumulated CA generally coincided with the theoretical values, which were calculated by the Henderson-Hasselbalch equation using the measured internal pH values of the bifidobacteria, and a pK(a) value of 6.4 for CA. These results suggest that the mechanism of CA accumulation is based on the diffusion of a hydrophobic weak acid across the bacterial cell membrane, and its dissociation according to the Delta pH value. A mixture of short-chain fatty acids (acetate, propionate and butyrate) at the appropriate colonic concentration (117 mM in total) reduced CA accumulation in Bifidobacterium breve JCM 1192(T). These short-chain fatty acids, which are weak acids, reduced the Delta pH, thereby decreasing CA accumulation in a dose-dependent manner. The bifidobacteria did not alter or modify the CA molecule. The probiotic potential of CA accumulation in vivo is discussed in relation to human bile acid metabolism.