Supplemental Clostridium butyricum MIYAIRI 588 Affects Intestinal Bacterial Composition of Finishing Pigs.

Animal gastrointestinal tracts are populated by highly diverse and complex microbiotas. The gut microbiota influences the bioavailability of dietary components and is closely associated with physiological processes in the host. Clostridium butyricum reportedly improves growth performance and affects the gut microbiota and immune functions in post-weaning piglets. However, the effects of C. butyricum on finishing pigs remain unclear. Therefore, we herein investigated the effects of C. butyricum MIYAIRI 588 (CBM588) on the gut microbiota of finishing pigs. 16S rRNA gene sequencing was performed using fecal samples and ileal, cecal, and colonic contents collected after slaughtering. The α-diversity of the small intestinal microbiota was lower than that of the large intestinal microbiota, whereas ß-diversity showed different patterns depending on sample collection sites. The administration of CBM588 did not significantly affect the α- or ß-diversity of the microbiotas of fecal and intestinal content samples regardless of the collection site. However, a linear discriminant ana-lysis Effect Size revealed that the relative abundance of Lactobacillaceae at the family level, Bifidobacterium at the order level, and Lactobacillus ruminis and Bifidobacterium pseudolongum at the species level were higher in the fecal samples and cecal and colonic contents of the treatment group than in those of the control group. Therefore, the administration of CBM588 to finishing pigs affected the composition of the gut microbiota and increased the abundance of bacteria that are beneficial to the host. These results provide important insights into the effects of probiotic administration on relatively stable gut microbial ecosystems.

Detection of Cholera Toxin by an Immunochromatographic Test Strip.

As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139 infection, detection of CT is an important biomarker for diagnosis of the disease. The procedure for pathogenicity analysis of V. cholerae isolates must be carefully developed for the reason that the amount of CT produced by V. cholerae varies according to the medium used and culture conditions (i.e. temperature and aeration status) applied. Here we describe a reproducible rapid method for analysis of CT production by toxigenic V. cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of purified recombinant CT.

Diet-Induced Thermogenesis and Expression Levels of Thyroid Hormone Target Genes and Their Products in Rats Differ between Meat Proteins.

We compared the effects of purified meat proteins on postprandial thermogenesis and on the secretion of and responsiveness to thyroid hormones (THs) in rats. Body temperatures at 2 h after feeding were significantly higher in the chicken and mutton protein groups than in the other groups, and these proteins seem to have a strong thermogenic effect. There were no significant differences in plasma TH concentrations among the groups, but levels of TH-responsive Spot 14 protein in the liver and brown adipose tissue were significantly higher in the chicken and mutton protein groups than in the other groups. Levels of malic enzyme 1 protein in the liver and brown adipose tissue were significantly higher in the chicken protein group than in the other groups except for the mutton protein group. Furthermore, levels of uncoupling protein 1 were higher in the chicken and mutton protein groups than in the other groups. The results suggest that the difference in postprandial thermogenesis of meat is strongly dependent on meat proteins; chicken and mutton proteins are strong promoters of postprandial thermogenesis, and THs may contribute to this effect. Since strong postprandial thermogenesis and high expression levels of TH target genes and their products were not observed in the amino acid group, chicken and mutton proteins or their digested peptides might contribute to these effects.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Fibronectin modified expression of Sonic hedgehog in ATRA-mediated neuronal differentiation.

In this study, the effect of fibronectin on the neurite outgrowth from embryoid bodies (EBs) in neurodifferentiated embryonal carcinoma P19 cells was examined. The neurite outgrowth on fibronectin was maintained for a longer time in comparison with those on collagen or laminin. Quantitative RT-PCR revealed that mRNA level corresponding to sonic hedgehog (Shh) in neurodifferentiated P19 cells was upregulated on fibronectin, whereas collagen or laminin did not affect. Further knockdown of integrin αv subunit in P19 cells demonstrated that expression of Shh was mediated through interaction between fibronectin and integrin. Additionally, exogenous Shh agonist accelerated neurite outgrowth from embryonic stem cell-derived EBs without large change of neuronal phenotype expression. Taken together, fibronectin could maintain neurite outgrowth via increased Shh expression.

The validation of the NH Immunochromato O157 test kit.

NH Immunochromato O157 is an immunochromatographic test for detection of Escherichia coli O157:H7 and O157:NM in food. It enables simple and rapid testing for the target organism after 18-24 h enrichment. In inclusivity and exclusivity testing, all 50 O157:H7 strains and 15 O157:NM strains tested positive, while all 33 exclusivity strains yielded negative results. Taken together, all 98 strains tested in inclusivity/exclusivity testing were identified correctly. NH Immunochromato O157 method was compared to U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, Chapter 5.08, reference method for detection of E. coli O157:H7 in 25 g of raw ground beef. The performance of both methods was revealed to be statistically equivalent. Autoclaved and non-autoclaved sample enrichments yielded the same results, showing sterilization is not mandatory for testing with NH Immunochromato O157. The results of the robustness study were not statistically different in all conditions, suggesting that NH Immunochromato produce reliable results under various conditions. However, the users are recommended to follow the instruction when applying sample to the test strip, because a smaller sample volume may produce invalid result.

Detection of soybean proteins in fermented soybean products by using heating extraction.

UNLABELLED: Soybean is used in processed foods worldwide. Because soybean can cause adverse reactions in some atopic patients, appropriate labeling regarding its content in processed foods is needed to better protect consumers. In the previous study, we developed a reliable sandwich Enzyme Linked Immunosorbent Assay (ELISA) method with high sensitivity and specificity for detecting soybean proteins by using antibody to Gly m Bd 30K, which was originally characterized as a vacuolar protein with a molecular mass of 34 kDa in soybean. The ELISA displayed satisfactory repeatability and reproducibility in an interlaboratory evaluation. However, it could not detect soybean protein in fermented soybean products. We therefore developed an extraction method combined with a heating process to inhibit soybean protein degradation by microbial proteolytic enzymes in fermented soybean products. This extraction method enables the sensitive detection of soybean protein in fermented soybean products such as natto and miso. It was able to detect with high-sensitivity soybean protein present at 10 µg/g levels in model processed foods. This method is suitable for quantifying soybean protein in processed foods without the degrading effects of microbial proteolytic enzymes. The present extraction method can be used sensitively to monitor labeling systems in a reliable manner and should be useful for the mandatory inspections required under Japanese regulations. PRACTICAL APPLICATION: The extraction and ELISA methods that we developed enable sensitive detection of soybean protein in soybean products, including fermented foods. These methods should be useful for reliable and sensitive monitoring of product labeling systems and should help to solve the problem of insensitive in soybean labeling of processed foods.

Effects of oral administration of heat-killed Enterococcus faecium strain NHRD IHARA in post-weaning piglets.

Probiotic bacteria such as lactic acid bacteria (LAB) have recently received attention as candidates for alternative anti-microbial feed additives. We previously isolated Enterococcus faecium strain NHRD IHARA (FERM BP-11090, NHRD IHARA strain) and reported its probiotic efficacy. However, we have not determined the effect of oral administration of heat-killed cells of this strain. Here, we performed two experiments to investigate the effect of oral administration of the heat-killed NHRD IHARA strain on post-weaning piglets. In Experiment 1, there was a significant improvement in growth performance (P = 0.04) and increase in serum immunoglobulin A (IgA) production (P = 0.03) in the group fed heat-killed cells. These results were similar to previous results we obtained with live cells. We also found changes in serum and fecal IgA production that were unrelated to the patterns of microbiotal change. In Experiment 2, we detected a significant improvement in villus growth in the jejunum (P = 0.0002). In conclusion, oral administration of the heat-killed NHRD IHARA strain in post-weaning piglets had the same efficacy as administration of the live strain. The heat-killed NHRD IHARA strain can be used as feed additives to improve pig growth and health on commercial farms.

Multiple marker effects of single nucleotide polymorphisms in three genes, AKIRIN2, EDG1 and RPL27A, for marbling development in Japanese Black cattle.

Marbling in beef, measured by Beef Marbling Standard (BMS) number, is an economically important trait for beef cattle breeding and markets in Japan. We previously detected three single nucleotide polymorphisms (SNPs) associated with BMS number of Japanese Black in Oita prefecture: c.*188G>A in AKIRIN2, g.1471620G>T in EDG1 and g.3109537C>T in RPL27A. Here, we carried out single and multiple marker association analyses for the three SNPs in a different commercial Japanese Black population of 892 genotyped animals. The single marker analyses with the model including a single SNP showed significant associations of all SNPs with BMS number. The multiple marker analysis with the model including the main effects of the three SNPs and their interactions detected only significant main effects of g.1471620G>T and g.3109537C>T and a significant interaction between c.*188G>A and g.1471620G>T. These findings suggest the presence of inter-allelic interactions among genes affecting the development of beef marbling. For effective marker-assisted selection for BMS number, interactions among these markers need to be considered.

Development of an immunochromatographic test strip for detection of cholera toxin.

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Altitudinal variation of antioxidant components and capability in Indocalamus latifolius (Keng) McClure leaf.

Indocalamus latifolius (Keng) McClure leaf is a popular food material in East Asia due to its antioxidant and anticorrosive activities. To utilize it more effectively, we investigated the discrepancy of antioxidant activities and active compound content in Indocalamus latifolius leaf along with the altitude change. Total flavonoids, phenolics, titerpenoids and eight characteristic active constituents, i.e, orientin, isoorientin, vitexin, homovitexin, p-coumaric acid, chlorogenic acid, caffeic acid, and ferulic acid, were determined by UV-spectrophotometer and synchronous RP-HPLC, respectively. Antioxidant activity was measured using DPPH and FRAP methods. Our data showed that the content of TP and TF, DPPH radical scavenging ability and ferric reduction power of Indocalamus latifolius leaf changed as altitude altered, with the trends of decreasing gradually when lower than 700 m and then increasing to 1,000 m. Chlorogenic acid and orientin were the main characteristic compounds in Indocalamus latifolius leaf and were also affected by altitude. Our result indicated that higher altitude with an adverse environment is conducive to secondary metabolite accumulation for Indocalamus latifolius. It would provide a theoretical basis to regulate the leaf collection conditions in the industrial use of Indocalamus latifolius leaf.

Characterization of probiotic properties of Enterococcus faecium NHRD IHARA isolated from porcine feces.

We examined in vitro the adhesion of Enterococcus faecium NHRD IHARA (NHRD IHARA) to porcine small intestinal mucin (PSIM) and inhibition of the adherence of enteropathogenic bacteria due to pre-incubation of PSIM with NHRD IHARA. NHRD IHARA exhibited an effective barrier function in porcine small intestinal mucus layer.

Simple, rapid, and reliable detection of Escherichia coli O26 using immunochromatography.

Shiga toxin-producing Escherichia coli (STEC) O26 has been increasingly associated with diarrheal disease all over the world. We developed an immunochromatographic (ic) strip for the rapid detection of E. coli O26 in food samples. To determine the specificity of the IC strip, pure cultures of 67 E. coli and 22 non-E. coli strains were tested with the IC strip. The IC strip could detect all (18 of 18) E. coli O26 strains tested and did not react with strains of any other E. coli serogroup or non-E. coli strains tested (0 of 71). The minimum detection limits for E. coli O26 were 2.2 × 10(3) to 1.0 × 10(5) cfu/ml. To evaluate the ability of the IC strip to detect E. coli O26 in food, 25-g food samples (ground beef, beef liver, ground chicken, alfalfa sprout, radish sprout, spinach, natural cheese, and apple juice) were spiked with E. coli O26. The IC strip was able to detect E. coli O26 at very low levels (approximately 1 cfu/25 g of food samples) after an 18-h enrichment, and the IC strip results were in 100% agreement with the results of the culture method and pcr assay. When 115 meat samples purchased from supermarkets were tested, 5 were positive for E. coli O26 with the IC strip; these results were confirmed with a pcr assay. These results suggest that the IC strip is a useful tool for detecting E. coli O26 in food samples.

Potential rapid and simple lateral flow assay for Escherichia coli O111.

We developed and evaluated a lateral flow assay (LFA) as a simple and rapid method for direct detection of Escherichia coli O111 in food after enrichment. When cell suspensions of 8 E. coli O111 strains and 77 non-E. coli O111 strains were tested with the LFA, the former all yielded positive results and the latter all yielded negative results. The minimum detection limits for the E. coli O111 strains were 1.8 × 10(3) to 5.6 × 10(5) CFU/ml of cell suspension, and the LFA was able to detect live cultures or those killed by autoclaving at nearly the same level of sensitivity. To evaluate the ability of LFA to detect its target in food, enrichment cultures of meat samples inoculated with 10-fold serial dilutions of E. coli O111 were tested with the LFA and PCR. Even when there were very few E. coli O111 cells in the meat samples (1.6 × 10(0) to 1.6 × 10(1) CFU/25 g of food), when they were cultured in modified E. coli broth with novobiocin for 22 h at 42°C, the LFA yielded positive results that corresponded to the PCR results. Although the LFA requires further evaluation and field study, these results suggest that this assay has sufficient sensitivity and specificity. This procedure can be completed with a one-step incubation after the test strip has been inserted into the sample after 22 h of culture, whereas the standard culture method requires multiple cultures, skilled personnel, a well-equipped laboratory, and 4 or 5 days. The speed and simplicity of this LFA make it suitable for use as part of routine screening assays in the food industry.

Effects of a chicken collagen hydrolysate on the circulation system in subjects with mild hypertension or high-normal blood pressure.

We investigated the effects of a chicken collagen hydrolysate (CCH) on the circulation system in humans. A total of 58 subjects with either mild hypertension (systolic blood pressure (SBP) of 140-159 mmHg or diastolic blood pressure (DBP) 90-99 mmHg) or high-normal blood pressure (SBP 130-139 mmHg or DBP 85-89 mmHg) were assigned to two groups, one involving a placebo and the other, the test food (including CCH of 2.9 g/d). The parameters related to each subject's circulation system were monitored over the study period of 18 weeks. The Δbrachial-ankle pulse wave velocity (baPWV), an indicator of arterial stiffness and marker of vascular damage, was significantly lower in the test food group than in the placebo group during the treatment period. The blood pressure in the test food group was also significantly lower than that in the placebo group, while the serum nitrogen oxide was higher in the test food group after the treatment. These results suggest that CCH exerted modulatory effects on the human circulation system.

Isolation, characterization, and effect of administration in vivo, a novel probiotic strain from pig feces.

From porcine rectal swabs or feces, we isolated lactic acid bacteria and used porcine Peyer's patch cells to select them for inducibility of IgA production as an indicator of probiotic effects. The strain selected as a new probiotic was named 'Enterococcus faecium NHRD IHARA'. To verify the probiotic effects of this strain in vivo, 536 piglets at age 25 days were assigned to either the trial group, which administrated the strain, or the control group. An increase in IgA in the feces was observed at age 45 days (P < 0.05 compared with the control group); a significant increase in serum IgA was also observed at the end of the study (P < 0.01) in the trial group. In addition, significant differences between the groups in terms of body weight (P < 0.05) and average daily gain (P < 0.01) were observed. The rate of detection of swine-pathogenic Escherichia coli gene in the feces tended to be lower in the trial group than in the controls. The novel probiotic strain; E. faecium NHRD IHARA may have beneficial effects on swine growth by inducing IgA production and reducing rates of colonization by pathogens in the body.

Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli.

The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 10(4)CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6 CFU per 25 g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.

Postprandial thermic effect of chicken involves thyroid hormones and hepatic energy metabolism in rats.

We investigated the postprandial thermic effect of chicken and its mechanisms in rats. A chicken diet showed a strong thermic effect after consumption, and the removal of fat induced more rapid and stronger thermogenesis. Although thermogenesis induced by a purified chicken protein diet was also strong, the thermic reaction was not so rapid and a remarkable rise of peripheral temperatures was not observed. Defatted chicken and purified chicken protein activated the thyroid hormone system and up-regulated rate-limiting enzyme genes of glucose metabolism and the tricarboxylic acid (TCA) cycle in the liver. Moreover, chicken protein up-regulated the mRNA expression of a rate-limiting enzyme of hepatic lipid metabolism. It is possible that the mechanisms by which body temperature is raised are different between chicken protein and defatted chicken. On the other hand, it is possible that chicken fat suppressed the expression of energy metabolism-related genes that was induced by the consumption of lean chicken. As a result, a rise of postprandial body temperature might not have been induced after consumption of chicken fat. These results suggest that the consumption of lean chicken activates the thyroid hormone system and hepatic energy metabolism and consequently induces the postprandial thermic effect of chicken.

Impacts of acute imipramine treatment on plasma and brain amino acid metabolism in mice given graded levels of dietary chicken protein.

Several studies have shown a relationship between depression and animal protein intake. To evaluate whether the difference of dietary chicken protein levels induces an antidepressant-like effect and potentiates acute antidepressant effects, three levels of dietary chicken protein were used as the representative animal protein with imipramine used as the antidepressant. In addition, the effects of dietary chicken protein on brain metabolism were evaluated. Open field test (OFT) and forced swimming test (FST) were conducted on the 27th and 28th days, respectively. OFT and FST were not influenced by both imipramine and dietary protein levels. However, characteristic effects of imipramine treatment on brain monoamine metabolism were observed in the cerebral cortex and hypothalamus. In addition, dietary protein significantly increased taurine and L-ornithine levels even though these amino acids were not contained in the diets. In conclusion, the metabolism of several amino acids in the plasma and brain were altered by dietary chicken protein.

Dietary animal proteins alter monoamine metabolism in the brain.

Several amino acids have effects on mental function, including sedative, antidepressant-like and anxiolytic-like effects. However, the influence of integrated amino acid nutrition as protein constituents on mental function remains unclear. Therefore, the purpose of the present study was to compare the influence of chicken, pork and beef protein extracts on brain monoamine metabolism in mice. Changes in monoamine levels and their turnover rates in the brain were induced by different protein sources. In particular, chicken protein group showed the highest norepinephrine levels in the hippocampus and hypothalamus, and beef protein extract caused an activation of the serotonergic system in the hypothalamus, although there were no significant differences in amino acid compositions of these protein extracts. Therefore, it was revealed that amino acid compositions in dietary protein did not induce alteration in monoamine metabolism. However, there were differences in small molecular peptides, such as creatine, carnosine and anserine levels in animal protein extracts. In conclusion, monoamine metabolism was altered by dietary protein sources. However, it was indicated that the alteration in monoamine metabolism may be independent from amino acid compositions in dietary protein. In addition, alteration in monoamine metabolism depending on the dietary protein sources may be induced by small molecular peptides.