Development of molecular immunoassay system for probiotics via toll-like receptors based on food immunology

Recent interest has focused on the importance of intestinal immunity for the host defense, but to date, not much is known about the underlying mechanisms. The toll‐like receptor (TLR) family plays an important role in host defense through recognizing bacterial pathogen‐associated molecular patterns. Our recent research on the physiological function of food products has investigated the immunoregulatory effects of probiotic lactic acid bacteria (LAB) via TLR. Studies of swine, which often substitute for a human model, have demonstrated intestinal immunoregulation by the probiotic LAB mediated by TLR in the gut. On the basis of our study, efforts have also been made to develop a molecular immunoassay system for probiotic LAB and find novel immunostimulatory DNA sequences from probiotics and high potential immunobiotic LAB strains via TLR signaling. These findings may provide important clues at the molecular level on TLR signal transduction pathways and recognition mechanisms for the ligands. They also provide impetus to further delineate the activation mechanism of the innate immune response. In addition to identifying immunoregulatory factor immunogenics from LAB, a better understanding of intestinal immune regulation through cytokine networks holds out promise for basic food immunology research and the development of immunobiotic foods to prevent specific diseases.     

Bacteriocin production of probiotic Lactobacillus gasseri LA39 isolated from human feces in milk-based media

The use of bacteriocins from Lactobacillus gasseri , a probiotic lactic acid bacterium, as bio-preservatives in the food industry and animal formulations has been limited because few strains of Lb. gasseri are cultivated and produce a bacteriocin in natural media such as milk and milk-based media. By the determination of the growth-supplements to milk among the 47 nutrients, Lb. gasseri JCM1131^T, LA39 and LA158 isolated from human feces were successfully cultured in reconstituted skim milk and cheese whey using proteose peptone as a nutrient supplement, where Lb. gasseri LA39 produced a useful bacteriocin, gassericin A, with effective growth-inhibiting activity against Gram-positive food-borne pathogens. The data suggest these developed low-cost safe media supporting enough production of bacteriocins by the probiotic Lb. gasseri LA39 could be used to improve the safe bio-preservation of foods and therapy of bovine mastitis, and extra cheese whey produced by cheese making industry is reused in the cultivation for probiotics effectively.     

Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods

In the present review, a new mass screening system for selecting probiotic strains from Lactobacillus (L) acidophilus group lactic acid bacteria (LAB) with strong adhesion to the human intestinal tract is described. Characteristics of antimicrobial peptides (bacteriocin), lactose‐hydrolyzing enzymes and immunostimulative oligo DNA motifs in L. gasseri strains are also described. Finally, the use of L. acidophilus LAB, selected by our screening method, that have strong adhesion to the human colonic mucosa in functional yogurt products is described. Adhesiveness to the human intestine is one of the most important characteristics of probiotic LAB. A new screening system that involves a combination of three methods is proposed: rat colonic mucin (RCM)‐micro plate assay, Carnoy's histochemical staining method and carbohydrate probe binding assay. By using an RCM‐coated poly‐vinylidene‐diflouride membrane that mimics the human colonic mucous layer, a new lectin was isolated and its structure was clarified by gene cloning. Furthermore, the structures and functions of a new cyclic bacteriocin (gassericin A), new lactose‐hydrolyzing enzymes, and new immunostimulating oligo DNA motifs from Lactobacillus gasseri (B₁ subgroup) were clarified. A new functional yogurt ‘Fit down’ is proposed, that is fermented by an adhesive strain of L. acidophilus LA67 selected by our screening and contains antihypertensive peptides derived from whey proteins by protease digestion. In the future, superior functional foods containing more effective probiotic LAB are expected to be developed by the use of the proposed mass screening system.     

Insect growth regulators with hydrazide moiety inhibit strigolactone biosynthesis in rice

Strigolactones (SLs) are carotenoid-derived plant hormones involved in several growth and developmental processes. Also, SLs are allelochemicals that induce the seed germination of root parasitic plants and the hyphal branching of arbuscular mycorrhizal fungi. In this study, to identify novel lead chemicals that inhibit SL biosynthesis, we evaluated the effect of agrochemicals on SL biosynthesis. We found that the diacylhydrazine insect growth regulator, chromafenozide, reduced the endogenous level of 4-deoxyorobanchol (4DO), a major SL in rice. Furthermore, treatment with the same class of insect growth regulator, methoxyfenozide, also resulted in the reduction of 4DO levels in rice root exudates. These results suggest that chromafenozide and methoxyfenozide are novel lead inhibitors of SL biosynthesis.     

Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-ionization tandem mass spectrometry

Pyrroloquinoline quinone (PQQ) is believed to be an important factor for mammalian growth and development and has, therefore, been declared a vitamin by some researchers. However, this issue remains controversial, and from a nutritional viewpoint, accurate determination of PQQ levels in a variety of foods is very important. Here, we describe a simple, highly sensitive, and highly selective method for quantitative analysis of PQQ. Liquid foods or aqueous extracts of solid foods were analyzed using high-performance liquid chromatography (HPLC) combined with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS). (15)N-labeled PQQ was added to the samples as an internal standard. Quantitative analyses of PQQ were performed by multiple reaction monitoring (MRM) with LC/MS/MS. Free PQQ was detected in almost all food samples in the range 0.19-7.02 ng per g fresh weight (for solid foods) or per mL (liquid foods). This method will enable the rapid and simple determination of PQQ levels in many samples.     

Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

Effect of CO2 enrichment on biomass production,photosynthesis, and sink activity in Soybean cv. Bragg and its supernodulating mutant nts 1007

Soybean (Glycine max L. Merr.) cv. Bragg and its supernodulating mutant nts 1007 were grown in pots containing vermiculite with a N-free nutrient solution in order to examine the effect of elevated CO₂ concentration (100+20 Pa CO₂ ) on biomass production, photosynthesis, and biological nitrogen fixation. The whole plant weight increase in Bragg was higher than in the mutant at a high CO₂ concentration. Apparent photosynthetic activities of the upper leaves in both Bragg and the mutant increased up to 14 d after treatment initiation by the CO₂ enrichment and thereafter decreased to some extent. Both leaf area and leaf thickness of Bragg increased more than in nts 1007. With the elevated CO₂ concentration, biological nitrogen fixation (BNF) also responded in the same manner as biomass production in both Bragg and nts 1007. The increase of BNF in Bragg was largely due to an increase in nodule weight. Starch contents in the leaves of both Bragg and the mutant increased significantly by CO₂ enrichment, with a higher increase in Bragg than in its mutant. Sugar content in leaf differed only slightly in both Bragg and the mutant. N content in leaf decreased in both Bragg and its mutant, with the decrease being more pronounced in Bragg. However, in other plant parts (roots, stem, and petiole + pods), N content increased in the mutant while in Bragg, it decreased in the pod. N accumulation rate was higher in Bragg than in the mutant and increased more in Bragg than in the mutant by CO₂ enrichment. The ureide content in leaf decreased in Bragg but increased in the mutant by elevated CO₂ concentration. In the nodules, ureide content increased in both Bragg and the mutant by CO₂ enrichment. Based on these results, it is suggested that in terms of biomass production and photosynthetic rate, Bragg responded more to elevated CO₂ concentration than its mutant nts 1007. The alleviation of the stunted vegetative growth of the mutant by CO₂ enrichment was limited despite the significant increase in the photosynthetic activity, presumably due to the limitation of sink activity in the growing parts and not to insufficient supply of N through BNF.     

Residual effects of organic acid-treated phosphate rocks on some soil properties and phosphate availability

The effects of the application of organic acid-treated phosphate rocks on the growth and nutrient uptake of Italian rye grass (Lolium multiflorum Lam. cv. Tachiwase) and some properties of the soil were evaluated in a greenhouse pot experiment. Phosphate rocks (PRs) collected from six countries; China, Florida (USA), Jordan, Sri Lanka, Togo, and Tanzania, were treated with 1 M oxalic or tartaric acid at the ratio of 2.5 mL g^-1 PR. The organic acid-treated PRs, containing 12–31% water soluble P, were applied to a granitic regosol (pH 5.8) at 200 mg P pot^-1 (4 kg soil). Untreated PRs and single superphosphate (SSP) were included in the treatments. Italian ryegrass was grown for 175 dafter planting (DAP) with ample supply of other nutrients and water. Shoots were harvested at 56, 119, and 175 DAP and the soils were analyzed for pH and Olsen-P after the experiment. Application of organic acid-treated PRs consistently increased the dry matter yield and P uptake of the plants compared with the application of untreated PRs at each harvest, but they were less effective than SSP. A larger amount of P (calculated per unit water-soluble P applied) was recovered from the organic acid-treated PRs than from SSP. The amount of residual extractable P in the soils with the organic acid-treated PRs was about the same as or significantly larger than that in the soil treated with SSP. Soil pH was also significantly higher than in the control and SSP soils. The results suggest that organic acids could be used to improve the P availability of PRs to plants with favorable residual effects in terms of available P and soil pH, without exerting any adverse effects on plant growth or nutrient acquisition.     

Heterologous expression of gassericin A,a bacteriocin produced by Lactobacillus gasseri LA39

Gassericin A, a bacteriocin produced by Lactobacillus gasseri LA39, has a cyclic structure linking N‐ and C‐terminal amino acids. Gassericin A was expressed in Escherichia coli JM109 as a biotinylated fusion protein on the basis of the DNA sequence of mature bacteriocin. A positive clone accumulated the bacteriocin, with no activity, as a soluble fusion protein in the cytoplasm. After release of an N‐terminal tag with factor Xa protease, gassericin A was converted into an active peptide having N‐ and C‐termini. The total amount of purified bacteriocins (expressed and native) was 480 µg/L and 370 µg/L, respectively. However, the specific activity of expressed gassericin A was 15 AU/mg lower than that of native bacteriocin (2600 AU/mg). Although the actual Mr (molecular weight) of the expressed bacteriocin should be 5666, the peptide showed the same mobility (Mr 3800) in sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as native cyclic gassericin A, suggesting that the expressed peptide retains compact folding of the molecule similar to that of native gassericin A.