Synbiotics Containing Nanoprebiotics: A Novel Therapeutic Strategy to Restore Gut Dysbiosis.

A new formulation, nanoprebiotics [e.g., phthalyl pullulan nanoparticles (PPNs)], was demonstrated to enhance the antimicrobial activity of probiotics [e.g., Lactobacillus plantarum (LP)] in vitro through intracellular stimulation better than that by backbone prebiotics, which are commonly used. In this study, we aimed to investigate whether this combination would exert distinct effects as synbiotics in vivo. Synbiotics combinations of LP, pullulan, and PPNs were used as experimental treatments in a dysbiosis-induced murine model, and their restorative effect was assessed using pathogen Escherichia coli K99 challenge. Our results showed that the E. coli infection was suppressed markedly in the experimental group fed with synbiotics containing PPNs. In addition, the decrease in serum endotoxin level after synbiotics treatment suggested the reinforcement of the gut barrier. Comparison of treatment groups, including a normal control group, showed that synbiotics containing PPNs increased microbial diversity, which is a representative parameter of healthy status. Furthermore, distinct from probiotics treatment alone, synbiotics showed additive effects of enrichment of several well-known beneficial bacteria such as Lactobacillus, Bifidobacterium, and other butyrate-producing bacteria including Faecalibacterium. Collectively, our results indicate that synbiotics containing PPNs are effective at restoring gut dysbiosis, suppressing pathogenic infection, and increasing microbial diversity, suggesting that synbiotics with nanoprebiotics have the potential to be a novel strategy for ameliorating gut dysbiosis and infectious diseases.

Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route.

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.

Additive anti-inflammation by a combination of conjugated linoleic acid and α-lipoic acid through molecular interaction between both compounds.

Alpha lipoic acid (LA) and conjugated linoleic acid (CLA) have been well-documented on a variety of functional effects in health foods. The main purpose of this study was focused on the additive anti-inflammatory activity of the combination of LA and CLA in vitro. Raw 264.7 cells induced by lipopolysaccharide were treated with LA and CLA individually or in combination at a variety of concentration ranges. Co-treating 25 µM of LA and 25 µM of CLA significantly inhibited pro-inflammatory cytokines compared to the same concentration of single LA- or CLA-treated group. The molecular mechanism of anti-inflammation by a combination of these compounds was attributed to extracellular signal-regulated kinase-1 (ERK1) and peroxisome proliferator-activated receptor gamma (PPARγ). Also, the molecular interaction between both compounds was confirmed by NMR. Our findings suggested that the combination of CLA and LA showed potential additive effect on anti-inflammation through the molecular interaction of both compounds.

Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.

Enhanced Efficacy of Immunization with a Foot-and-Mouth Disease Multi-Epitope Subunit Vaccine Using Mannan-Decorated Inulin Microparticles.

BACKGROUND: Despite the many advantages of recombinant subunit vaccines, they have critical weaknesses that include a low efficacy for promoting cellular and humoral immune responses against antigens because of their poor immunogenicity, and a rapidly cleared properties as a result of proteolytic enzymes in the body. To circumvent these problems, we developed mannan-decorated inulin acetate microparticles (M-IA MPs) that functioned as carriers and adjuvants for immunization with the recombinant foot-and-mouth disease multi-epitope subunit vaccine (M5BT). METHODS: The M5BT-loaded M-IA MPs were obtained by a double-emulsion solvent-evaporation method. Their properties including morphology, size and release ability were determined by field emission scanning electron microscope, dynamic light-scattering spectrophotometer and spectrophotometer. To assess the immunization efficacy of the MPs, mice were immunized with MPs and their sera were analyzed by ELISA. RESULTS: The M-IA MPs obtained by a double-emulsion solvent-evaporation method were spherical and approximately 2-3 µm, and M5BT was encapsulated in the M-IA MPs. The M5BT-loaded M-IA MPs showed higher antigen-specific IgG, IgG1, IgG2a and anti-FMDV antibodies than the M5BT-loaded IA MPs and the Freund's adjuvant as a control. CONCLUSION: The M-IA MPs showed a powerful and multifunctional polymeric system that combined two toll-like receptor agonists compared to the conventional adjuvant.

Erratum to: Study on the fatty acid profile of phospholipid and neutral lipid in Hanwoo beef and their relationship to genetic variation.

[This corrects the article DOI: 10.5187/jast.2019.61.2.69.].

Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0.

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.

Novel production of natural bacteriocin via internalization of dextran nanoparticles into probiotics.

Recently, probiotics has drawn much attention as an alternative of antibiotics because overuse use of antibiotics has caused widespread bacterial resistance. Given that prebiotics enhance the activity of probiotics, we prepared phthalyl dextran nanoparticles (PDNs) by conjugation of phthalic anhydride with dextran to form a prebiotic and checked its effects on the cellular and antimicrobial properties of the probiotics. First, we found that the internalization of PDNs by probiotics was dependent on temperature, time, and glucose transporters. Internalization of PDNs enhanced the production of antimicrobial peptides by probiotics through self-defense mechanism and resulted in higher antimicrobial activities against Gram-positive and -negative pathogens compared to probiotics themselves. Moreover, pediocin produced by PDN-internalized probiotics was able to suppress pathogenic gut infections and alter the population of gut microbiome in vivo. The enhanced antimicrobial property of Pediococcus acidilactidi internalized with PDNs could decrease the number of pathogens and increase beneficial bacteria species in mice. Furthermore, the composition of gut microbiome was changed, and resulted in preventing reduction of the diversity of the microflora. Our results indicate that PDNs as a new type of prebiotic can regulate probiotic bacterial metabolism, suggesting a new avenue for probiotic modulation and their use in addressing the challenge of bacterial resistance.

Study on the fatty acid profile of phospholipid and neutral lipid in Hanwoo beef and their relationship to genetic variation.

Maize which has very high omega-6 fatty acid content has been used as a main feed grain for Hanwoo beef production to increase marbling, and thus omega-6 to omega-3 fatty acids ratio in Hanwoo beef is expected to be biased. To elucidate the current status of omega fatty acids ratio in Hanwoo beef, fatty acid profiles of neutral lipid and phospholipid fraction were analyzed separately using 55 Hanwoo steers' longissimus dorsi muscle slaughtered at Pyeongchang, Korea from Oct. to Nov. 2015. In addition, an association study was conducted to evaluate associations between single nucleotide polymorphism (SNP) markers from references and omega fatty acid profiles in phospholipid of Hanwoo beef samples using analysis of variance (ANOVA). In neutral lipid fraction, composition of saturated and monounsaturated fatty acids was higher and polyunsaturated fatty acids was lower compared to those in phospholipid fraction. The mean n-6/n-3 ratios of Hanwoo were 56.059 ± 16.180 and 26.811 ± 6.668 in phospholipid and neutral lipid, respectively. There were three SNPs showing statistically significant associations with omega fatty acid content. GA type of rs41919985 in fatty acid synthase (FASN) was significantly associated with the highest amount of C20:5 n-3 (p = 0.031). CC type of rs41729173 in fatty acid-binding protein 4 (FABP4) was significantly associated with the lowest amount of C22:2n-6 (p = 0.047). AG type of rs42187261 in FADS1 was significantly linked to the lowest concentration of C20:4 n-6 (p = 0.044). The total n-6/n-3 ratio of the steer which has all four SNP types in above loci (27.905) was much lower than the mean value of the total n-6/n-3 ratio in phospholipid of the 55 Hanwoo steers (56.059 ± 16.180). It was found that phospholipid and neutral lipid of Hanwoo have very high n-6/n-3 ratios compared to the reported data from different cow breeds. Four SNPs in genes related with fatty acid metabolism showed significant associations with the fatty acid profile of phospholipid and may have potential as SNP markers to select Hanwoo steers in terms of n-6/n-3 balance in the future.

Pullulan Nanoparticles as Prebiotics Enhance the Antibacterial Properties of Lactobacillus plantarum Through the Induction of Mild Stress in Probiotics.

Synbiotics, which are the combination of probiotics and prebiotics, have recently attracted attention because of their synergistic net health benefits. Probiotics have been used as alternatives to antibiotics. Among the probiotics, Lactobacillus plantarum (LP) has shown strong antimicrobial activity against Escherichia coli K99, a major livestock pathogen. In this study, we aimed to investigate the antimicrobial activity of phthalyl pullulan nanoparticle (PPN)-treated LP. Interestingly, when PPNs were added to LP, the PPNs were internalized into the LP through an energy-dependent and galactose transporter-dependent mechanism. Additionally, more plantaricin, a natural antibacterial peptide, was secreted from PPN-treated LP than from untreated or pullulan-treated LP. Furthermore, antimicrobial activity against Gram-negative Escherichia coli K99 and Gram-positive Listeria monocytogenes by PPN-treated LP was higher than those of untreated or pullulan-treated LP. It is thought that the enhanced antimicrobial properties of the PPN-treated LP are due to intracellular stimulation. Overall, this research provides a new method of producing plantaricin in LP through intracellular stimulation by internalized PPNs.

Oral Delivery of Probiotics Using pH-Sensitive Phthalyl Inulin Tablets.

Probiotics show low cell viability after oral administration because they have difficulty surviving in the stomach due to low pH and enzymes. For the oral delivery of probiotics, developing a formula that protects the probiotic bacteria from gastric acidity while providing living cells is mandatory. In this study, we developed tablets using a new pH-sensitive phthalyl inulin (PI) to protect probiotics from gastric conditions and investigated the effects of different compression forces on cell survival. We made three different tablets under different compression forces and measured survivability, disintegration time, and kinetics in simulated gastric-intestinal fluid. During tableting, there were no significant differences in probiotic viability among the different compression forces although disintegration time was affected by the compression force. A higher compression force resulted in higher viability in simulated gastric fluid. The swelling degree of the PI tablets in simulated intestinal fluid was higher than that of the tablets in simulated gastric fluid due to the pH sensitivity of the PI. The probiotic viability formulated in the tablets was also higher in acidic gastric conditions than that for probiotics in solution. Rapid release of the probiotics from the tablet occurred in the simulated intestinal fluid due to the pH sensitivity. After 6 months of refrigeration, the viability of the PI probiotics was kept. Overall, this is the first study to show the pH-sensitive properties of PI and one that may be useful for oral delivery of the probiotics.

A new way of producing pediocin in Pediococcus acidilactici through intracellular stimulation by internalized inulin nanoparticles.

One of the most challenging aspects of probiotics as a replacement for antibiotics is to enhance their antimicrobial activity against pathogens. Given that prebiotics stimulate the growth and/or activity of probiotics, we developed phthalyl inulin nanoparticles (PINs) as prebiotics and observed their effects on the cellular and antimicrobial activities of Pediococcus acidilactici (PA). First, we assessed the internalization of PINs into PA. The internalization of PINs was largely regulated by glucose transporters in PA, and the process was energy-dependent. Once internalized, PINs induced PA to produce substantial amounts of antimicrobial peptide (pediocin), which is effective against both Gram-positive (Salmonella Gallinarum) and Gram-negative (Listeria monocytogenes) pathogens. When treated with small-sized PINs, PA witnessed a nine-fold increase in antimicrobial activity. The rise in pediocin activity in PA treated with PINs was accompanied by enhanced expression of stress response genes (groEL, groES, dnaK) and pediocin biosynthesis genes (pedA, pedD). Although the mechanism is not clear, it appears that the internalization of PINs by PA causes mild stress to activate the PA defense system, leading to increased production of pediocin. Overall, we identified a prebiotic in nanoparticle form for intracellular stimulation of probiotics, demonstrating a new avenue for the biological production of antimicrobial peptides.

Tracing of the fecal microbiota of commercial pigs at five growth stages from birth to shipment.

The intestinal microbiota affect various physiological traits of host animals such as brain development, obesity, age, and the immune system. In the swine industry, understanding the relationship between intestinal microbiota and growth stage is essential because growth stage is directly related to the feeding system of pigs, thus we studied the intestinal microbiota of 32 healthy pigs across five sows at 10, 21, 63, 93, and 147 d of ages. The intestinal microbiota were altered with growth of pigs and were separated into three distinct clusters. The relative abundance of several phyla and genera were significantly different between growth stages. We observed co-occurrence pattern of the intestinal microbiota at each growth stage. In addition, we predicted the functions of the intestinal microbiota and confirmed that several KEGG pathways were significantly different between growth stages. We also explored the relationship between the intestinal microbiota and innate factors such as the maternal effect and gender. When pigs were young, innate factors affected on construction of intestinal microbiota, however this tendency was disappeared with growth. Our findings broaden the understanding of microbial ecology, and the results will be used as a reference for investigating host-microbe interactions in the swine industry.

A New Method of Producing a Natural Antibacterial Peptide by Encapsulated Probiotics Internalized with Inulin Nanoparticles as Prebiotics.

Synbiotics are a combination of probiotics and prebiotics, which lead to synergistic benefits in host welfare. Probiotics have been used as an alternative to antibiotics. Among the probiotics, Pediococcus acidilactici (PA) has shown excellent antimicrobial activity against Salmonella Gallinarum (SG) as a major poultry pathogen and has improved the production performances of animals. Inulin is widely used as a prebiotic for the improvement of animal health and growth. The main aim of this study is to investigate the effect of the antimicrobial activity of inulin nanoparticles (INs)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) in future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in phthalyl inulin were estimated by ¹H-NMR measurement as 25.1 mol.-%. The sizes of the PINs measured by DLS were approximately 203 nm. Internalization into PA was confirmed by confocal microscopy and flow cytometry. Antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by co-culture antimicrobial assays on SG. PIN-internalized probiotics had a higher antimicrobial ability than that of ACA MCs loaded with PA/inulin or PA. Interestingly, when PINs were treated with PA and encapsulated into ACA MCs, as a natural antimicrobial peptide, pediocin was produced much more in the culture medium compared with other groups inulin-loaded ACA MCs and PA-encapsulated into ACA MCs.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles.

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

Pan-Genomic Approaches in Lactobacillus reuteri as a Porcine Probiotic: Investigation of Host Adaptation and Antipathogenic Activity.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, reuterin-producing Lactobacillus reuteri is getting attention as an alternative to AGPs. In this study, we investigated genetic features of L. reuteri associated with host specificity and antipathogenic effect. We isolated 104 L. reuteri strains from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (group HS) and eight strains exhibiting the lower effect (group LS), were selected for genomic comparison. We generated draft genomes of the 16 isolates and investigated their pan-genome together with the 26 National Center for Biotechnology Information-registered genomes. L. reuteri genomes organized six clades with multi-locus sequence analysis, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein-coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host gastrointestinal epithelia and that these host-specific traits were acquired by horizontal gene transfer. We also identified differences between groups HS and LS in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. This study provides a deeper understanding of the relationship between probiotic properties and genomic features of L. reuteri.

Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate.

BACKGROUND: Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. RESULTS AND CONCLUSION: We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

Oral Delivery of Probiotics in Poultry Using pH-Sensitive Tablets.

As alternatives to antibiotics in livestocks, probiotics have been used, although most of them in the form of liquid or semisolid formulations, which show low cell viability after oral administration. Therefore, suitable dry dosage forms should be developed for livestocks to protect probiotics against the low pH in the stomach such that the products have higher probiotics survivability. Here, in order to develop a dry dosage forms of probiotics for poultry, we used hydroxypropyl methylcellulose phthalate 55 (HPMCP 55) as a tablet-forming matrix to develop probiotics in a tablet form for poultry. Here, we made three different kinds of probiotics-loaded tablet under different compression forces and investigated their characteristics based on their survivability, morphology, disintegration time, and kinetics in simulated gastrointestinal fluid. The results indicated that the probiotics formulated in the tablets displayed higher survival rates in acidic gastric conditions than probiotics in solution. Rapid release of the probiotics from the tablets occurred in simulated intestinal fluid because of fast swelling of the tablets in neutral pH. As a matrix of tablet, HPMCP 55 provided good viability of probiotics after 6 months under refrigeration. Moreover, after oral administration of probiotics-loaded tablets to chicken, more viable probiotics were observed, than with solution type, through several digestive areas of chicken by the tablets.