Reduction in Allergenicity and Induction of Oral Tolerance of Glycated Tropomyosin from Crab.

Tropomyosin (TM) is an important crustacean (Scylla paramamosain) allergen. This study aimed to assess Maillard-reacted TM (TM-G) induction of allergenic responses with cell and mouse models. We analyzed the difference of sensitization and the ability to induce immune tolerance between TM and TM-G by in vitro and in vivo models, then we compared the relationship between glycation sites of TM-G and epitopes of TM. In the in vitro assay, we discovered that the sensitization of TM-G was lower than TM, and the ability to stimulate mast cell degranulation decreased from 55.07 ± 4.23% to 27.86 ± 3.21%. In the serum of sensitized Balb/c mice, the level of specific IgE produced by TM-G sensitized mice was significantly lower than TM, and the levels of interleukins 4 and interleukins 13 produced by Th2 cells in spleen lymphocytes decreased by 82.35 ± 5.88% and 83.64 ± 9.09%, respectively. In the oral tolerance model, the ratio of Th2/Th1 decreased from 4.05 ± 0.38 to 1.69 ± 0.19. Maillard reaction masked the B cell epitopes of TM and retained some T cell epitopes. Potentially, Maillard reaction products (MRPs) can be used as tolerance inducers for allergen-specific immunotherapy.

Degraded polysaccharides from Porphyra haitanensis: purification, physico-chemical properties, antioxidant and immunomodulatory activities.

To explore effect of the structural properties of porphyra haitanensis polysaccharide on its biological activity, degraded porphyra polysaccharides were separated and purified by Cellulose DEAE-52 and Sephadex G-100 chromatography, obtaining three purified components (P1, P2 and P3). All the three components were sulfate polysaccharides containing the repeating units of → 3) ß-D-galactose (1 → 4) 3,6-anhydro-α-L-galactose (1 →, and → 3) ß-D-galactose (1 → 4) α-L-galactose-6-S (1 →, and → 3) 6-O-methyl-ß-D-galactose (1 → 4) 3,6-anhydro-α-L-galactose (1 →. The molecular weight of the three fractions was measured to be 300.3, 130.4 and 115.1 kDa, respectively. Their antioxidant activity was investigated by the determination of the free radical scavenging effect and ferric reducing power. It was found that P1, P2 and P3 possessed marked antioxidant activity. It was also found that they appreciably enhanced the proliferation, phagocytic ability and nitric oxide secretion in RAW264.7 cells. Lower molecular weight and higher sulfate content were beneficial to bioactivities of P. haitanensis polysaccharides. Overall, P2 and P3 possess superior immuno-modulatory activity to that of P1 and PHP. Thus, the current work will provide the basis for the better utilization of P. haitanensis to develop the related functional foods.

Purification, structural characterization, and biological activities of degraded polysaccharides from Porphyra yezoensis.

The degraded polysaccharides from Porphyra yezoensis (DPPY) prepared using the H2 O2 -Vc method under optimized conditions were isolated and purified by DEAE Cellulose-52, and Sephadex G-100, providing four pure components, namely, DPPY-0, DPPY-0.1, DPPY-0.3, and DPPY-0.5. Their relative molecular weights were measured to be 10.8, 10.7, 18.7, and 35.5 kDa, respectively. GC-MS analysis revealed that all the four fractions were mainly composed of galactose, together with a small portion of glucose, mannose, xylose, and rhamnose. Structural analysis revealed that the purified polysaccharides mainly possess a backbone of (1 â†’ 3)-ß-D-galactose (1 â†’ 4)-3,6-anhydro-α-L-galactopyranose (G-A) units and (1 â†’ 3)-ß-D-galactose (1 â†’ 4)-α-L-galactose-6-sulfate (G-L6S) units. They were found to promote the proliferation of RAW264.7 macrophages and enhance phagocytosis of the RAW264.7 cells. Antioxidant assays indicated that DPPY-0.5 possessed the most potent reducing power and free radical scavenging ability among the four purified polysaccharides. High sulfate content and proper molecular weight of these fractions are favorable to their immunomodulatory and antioxidant activities. PRACTICAL APPLICATIONS: Porphyra yezoensis, common economic red algae widely distributed in East Asian countries, contains a high content of polysaccharides with a variety of biological activities. However, P. yezoensis polysaccharide (PPY) has not been well utilized due to the relatively low biological activities and lack of understanding of its structure-activity relationship. Thus, it is necessary to improve the bioactivities and elucidate the structure-activity relationship of this polysaccharide for its practical use. In the present work, four purified fractions (DPPY-0, DPPY-0.1, DPPY-0.3, and DPPY-0.5) were isolated from the degraded P. yezoensis polysaccharide, and were investigated for their antioxidant and immunoregulatory activities. The results of the present work will lay a foundation for the application of the degraded P. yezoensis polysaccharide in the food industry as a functional food ingredient.

Improved antioxidant and immunomodulatory activities of enzymatically degraded Porphyra haitanensis polysaccharides.

Porphyra haitanensis polysaccharide (CPH) was degraded by pectinase to improve its biological activities. Box-Behnken response surface design was used to optimize the hydrolysis conditions. The molecular weight of CPH and the degraded P. haitanensis polysaccharide (DCPH) were measured to be 524 and 217 kDa, respectively. GC-MS spectrometry results showed that CPH and DCPH were mainly composed of galactose. In vitro antioxidant assays indicated that DCPH possessed improved radical scavenging activity and ferric iron reducing power when compared to those of CPH. In H2 O2 -treated RAW264.7 cells, DCPH was also found to be more effective in reducing the generation of malondialdehyde and reactive oxygen species than CPH. The immunomodulatory assays demonstrated that DCPH possessed superior activities in enhancing the proliferation, phagocytosis, and NO secretion in a RAW264.7 macrophage cell model to those of CPH. PRACTICAL APPLICATIONS: Polysaccharide is the most abundant bioactive component of an edible red algae Porphyra haitanensis. However, the use of CPH is limited due to its relatively low biological activities. Thus, in order to fully utilize P. haitanensis, it is necessary to enhance the biological activities of CPH for its practical use. An efficient and practical method to enhance the bioactivities of P. haitanensis polysaccharide has been developed in the present work. The DCPH prepared in this work could have potential applications in food and medicinal areas.

Intestinal inflammation modulates expression of the iron-regulating hormone hepcidin depending on erythropoietic activity and the commensal microbiota.

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

Quality evaluation of farmed whiteleg shrimp, Litopenaeus vannamei, treated with different slaughter processing by infrared spectroscopy.

The present study was to evaluate two slaughter processing methods (ice water immersion (T-1) and individual beheaded (T-2)) on whiteleg shrimp quality farmed in freshwater culture systems using attenuated total reflection infrared (ATR-IR) technology. In addition, the corresponding physical, chemical and microbial properties of shrimp samples were also determined. No significant differences were observed in pH, total volatile basic nitrogen (TVB-N), thiobarbutiric acid (TBA) and K value as well as the contents of moisture, crude protein, crude fat and ash between groups of T-1 and T-2. However, significantly higher springiness and chewiness (P<0.05) were observed in T-1 as compared to those of T-2. As for the result of total viable counts (TVC), significantly lower value (P<0.05) was shown in T-1 than that of T-2, indicating that the quality and shelf life of whiteleg shrimp killed by ice water immersion could be improved and prolonged. Furthermore, all the samples were successfully divided into two categories regarding the two slaughter methods by principal component analysis (PCA) according to the infrared spectra. By analysis of the regression coefficients of PLS-DA, it can be supposed that the quality differences of whiteleg shrimp with different slaughter processing are largely caused by structural changes of their protein and fat. All together, our results indicated that the springiness and chewiness of whiteleg shrimp with different processing could be correctly distinguished using infrared spectroscopy.

Proteomic identification of responsive proteins of Vibrio parahaemolyticus under high hydrostatic pressure.

BACKGROUND: High hydrostatic pressure (HHP) processing is currently being used as a treatment for certain foods to inhibit spoilage organisms and control the presence of foodborne pathogens. In this study proteome profiles were performed by two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification to determine the effects of HHP (50, 100, 150 and 200 MPa, each for 10 min) on Vibrio parahaemolyticus ATCC 17802 (∼8 log CFU mL⁻¹) in order to understand how it responds to mechanical stress injury. RESULTS: Multiple comparisons of 2-DE revealed that the majority of changes in protein abundance occurred in a pressure-dependent fashion. A total of 18 differentially expressed protein spots were successfully identified by MALDI-TOF/TOF analysis. Moreover, quantitative RT-PCR and immunoblotting also substantiated the changes of transcriptional and translational levels of representative proteins. CONCLUSIONS: Our results suggested that V. parahaemolyticus may respond to HHP treatment through suppression of membrane stability and functionality (PfaC, Alr2, MltA, PLA2 and PatH), depression of biosynthesis and cellular processes (NadB, PyrB and ArgB), decreased levels of transcription (RpoD) and translation (RpsA, RplJ and PheS), and effective activation of protein folding and stress-related elements (GroES, DnaK and GroEL). This study may provide insight into the nature of the cellular targets of high pressure and in high-pressure resistance mechanisms in V. parahaemolyticus.

Microbial source tracking: a tool for identifying sources of microbial contamination in the food chain.

The ability to trace fecal indicators and food-borne pathogens to the point of origin has major ramifications for food industry, food regulatory agencies, and public health. Such information would enable food producers and processors to better understand sources of contamination and thereby take corrective actions to prevent transmission. Microbial source tracking (MST), which currently is largely focused on determining sources of fecal contamination in waterways, is also providing the scientific community tools for tracking both fecal bacteria and food-borne pathogens contamination in the food chain. Approaches to MST are commonly classified as library-dependent methods (LDMs) or library-independent methods (LIMs). These tools will have widespread applications, including the use for regulatory compliance, pollution remediation, and risk assessment. These tools will reduce the incidence of illness associated with food and water. Our aim in this review is to highlight the use of molecular MST methods in application to understanding the source and transmission of food-borne pathogens. Moreover, the future directions of MST research are also discussed.

Temporal genetic variability and host sources of Escherichia coli associated with fecal pollution from domesticated animals in the shellfish culture environment of Xiangshan Bay, East China Sea.

This study was conducted to analyze the genetic variability of Escherichia coli from domesticated animal wastes for microbial source tracking (MST) application in fecal contaminated shellfish growing waters of Xiangshan Bay, East China Sea. (GTG)(5) primer was used to generate 1363 fingerprints from E. coli isolated from feces of known 9 domesticated animal sources around this shellfish culture area. Jackknife analysis of the complete (GTG)(5)-PCR DNA fingerprint library indicated that isolates were assigned to the correct source groups with an 84.28% average rate of correct classification. Based on one-year source tracking data, the dominant sources of E. coli were swine, chickens, ducks and cows in this water area. Moreover, annual and spatial changes of E. coli concentrations and host sources may affect the level and distribution of zoonotic pathogen species in waters. Our findings will further contribute to preventing fecal pollution in aquatic environments and quality control of shellfish.

Differentiation of fecal Escherichia coli from human, livestock, and poultry sources by rep-PCR DNA fingerprinting on the shellfish culture area of East China Sea.

The rep-PCR DNA fingerprinting performed with REP, BOX A1R, and (GTG)(5) primers was investigated as a way to differentiate between human, livestock, and poultry sources of fecal pollution on the area of Xiangshan Bay, East China Sea. Of the three methods, the BOX-PCR DNA fingerprints analyzed by jack-knife algorithm were revealed high rate of correct classification (RCC) with 91.30, 80.39, 89.39, 86.14, 93.24, 87.72, and 89.28% of human, cattle, swine, chicken, duck, sheep, and goose E. coli isolates classified into the correct host source, respectively. The average rate of correct classification (ARCC) of REP-, BOX-, and (GTG)(5)-PCR patterns was 79.88, 88.21, and 86.39%, respectively. Although the highest amount of bands in (GTG)(5)-PCR fingerprints could be observed, the discriminatory efficacy of BOX-PCR was superior to both REP- and (GTG)(5)-PCR. Moreover, the similarity of 459 isolates originated from shellfish and growing water was compared with fecal-obtained strains. The results showed that 92.4 and 96.2% E. coli strains isolated from midstream and downstream shellfish samples, respectively, had a ≥ 80% similarity with corresponding strains isolated from fecal samples. It was indicated that E. coli in feces could spread from human sewage or domestic farms to the surrounding shellfish culture water, and potentially affect the quality of shellfish. This work suggests that rep-PCR fingerprinting can be a promising genotypic tool applied in the shellfish growing water management on East China Sea for source identification of fecal pollution.

Tea polyphenols inhibit Pseudomonas aeruginosa through damage to the cell membrane.

This paper aims to delineate the inhibition mechanism of tea polyphenols (TP) toward Pseudomonas aeruginosa by cell membrane damage. Morphological changes in bacteria treated with TP were investigated by transmission electron microscopy, with results indicating that the primary inhibitory action of TP is to damage bacterial cell membranes. TP also increased the permeability of the outer and inner membranes of P. aeruginosa and disrupted the cell membrane with the release of small cellular molecules. A proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis was used to study the differences in the membrane proteins of TP-treated P. aeruginosa and those of control samples. Twenty-seven differentially expressed proteins were observed in the treated and the control groups. Most of the proteins identified by MALDI-TOF/TOF MS were enzymes (dihdrollpoamide dehydrogenase 50s ribosomal protein, and so on), which may have induced the metabolic disorder of the bacteria and resulted in their death.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice.

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations.

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.