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)...
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 was to investigate the antimicrobial activity of inulin nanoparticle (IN)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) for future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in the PINs 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. The antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by coculture 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 with inulin-loaded ACA MCs and PA encapsulated into ACA MCs.
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 was to investigate the antimicrobial activity of inulin nanoparticle (IN)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) for future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in the PINs 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. The antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by coculture 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 with inulin-loaded ACA MCs and PA encapsulated into ACA MCs.
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문제 정의
In this study, we aimed to enhance the antibacterial properties of microencapsulated probiotics internalized with inulin nanoparticles (INs) as prebiotics into alginate/ chitosan/alginate (ACA) microcapsules (MCs) for future in vivo application. To the best of our knowledge, this is the first report to show the improved antimicrobial activity of probiotics against pathogens using INs as prebiotics.
PIN-internalized PA in ACA MCs enhanced the antibacterial activity of the probiotics owing to the production of pediocin, probably through the stimulation of PA by PINs. To the best of our knowledge, this is the first report of the improved antimicrobial activity of probiotic bacteria against enteric pathogens by prebiotic nanoparticles compared with the prebiotic itself. Our results indicate that the encapsulation of PIN-internalized PA into ACA MCs will be a new, promising method for the development of biological antibacterial polypeptides for application in the animal industry.
In this study, we aimed to enhance the antibacterial properties of microencapsulated probiotics internalized with inulin nanoparticles (INs) as prebiotics into alginate/ chitosan/alginate (ACA) microcapsules (MCs) for future in vivo application. To the best of our knowledge, this is the first report to show the improved antimicrobial activity of probiotics against pathogens using INs as prebiotics.
제안 방법
Analysis of the SCFAs (acetate, propionate, and butyrate) was performed by gas chromatography. Briefly, 1 ml of cocultured supernatant was obtained by centrifugation (10,000 ×g, 30 min, 4°C).
In this study, we developed ACA MCs encapsulated with IN-internalized PA to enhance the antimicrobial properties of microencapsulated probiotics. INs can be prepared by the self-assembly of phthalyl inulin conjugated with phthalic acid as a hydrophobic group.
대상 데이터
PA KCTC 21088 and SG KCTC 2931 strains were used in this study. PA was maintained in MRS broth supplemented with 15% (v/v) glycerol and stored at -70°C.
데이터처리
The data are presented as the mean plus standard deviation (SD) from all the replicates. The treatment effects were examined by one-way of variance for multiple comparisons using SPSS 11.5. (SPSS for Windows).
성능/효과
In conclusion, PINs were prepared by the self-assembly of the phthalyl groups of PI in the core as the hydrophobic and hydroxyl moieties of PI were exposed to water in the outer shell, as well as the hydrophilic moieties, when transferred from DMSO to aqueous medium during the dialysis of PI. The sizes of PINs measured by DLS were approximately 203 nm, with a polydispersity index of 0.
3 shows the morphologies of ACA MCs loaded with PA, with PIN-internalized PA, and inulin itself, observed by a stereo digital microscope. It was found that the sizes of ACA MCs after the internalization of PINs were not much changed compared with those of the PAencapsulated ACA MCs or inulin-loaded ACA MCs, having an encapsulation efficiency of almost 100% without a change in the morphologies of the ACA MCs after encapsulation of PA, inulin, and PIN-internalized PA, although the sizes of ACA MCs were increased almost 2 times after encapsulation of PA, inulin, and PINinternalized PA (Table 2). Additionally, we confirmed the encapsulation of FITC-labeled PIN-internalized PA into ACA MCs by CLSM observation (Fig.
Moreover, it was found that pediocin production was the highest for the ACA (PA-PIN) group and the produced polypeptide was confirmed to be pediocin by SDS-PAGE. It can be said that the highest antibacterial activity of ACA (PA-PIN) is closely related with the highest pediocin production, because pediocin produced by various strains of PA inhibited the growth of Listeria and Leuconostoc species associated with food products [30-32].
Inulin acts as a “prebiotic,” promoting the selective development of beneficial microbial “probiotics” [26]. Our results demonstrated that the synergistic inhibitory effect of synbiotic ACA(PA-PIN) MCs (PA as a probiotic and PINs as a prebiotic) and synbiotic ACA (PA-I) MCs on pathogenic bacteria was higher than the inhibitory effect of ACA MCs (PA) alone. Additionally, the antibacterial activity of ACA (PA-PIN) MCs against SG was 7.
98 µg/ml in ACA (PA), ACA (PA-I), and ACA (PA-PIN), respectively. The apparent molecular mass of pediocin was confirmed to be approximately 3.5 kDa by the SDS-PAGE method, and the ACA (PA-PIN) group showed the highest production of pediocin.
The pediocin concentration was measured by Bradford assay and the molecular mass determination of pediocin produced by PA was confirmed with SDS-PAGE (mean ± SD, n = 3, *p ≤ 0.05).
8A-8D show the concentration of acetate, propionate, butyrate, and total SCFAs in the culture medium after treatment with PA, ACA (PA), ACA (PA-I), and ACA (PAPIN) against SG for 9 h. The results indicated that ACA (PA-I) showed the highest production of individual SCFAs, as well as the total ones among the groups, which is different from the antibacterial activity. Figs.
2 and 37°C over time. The results indicated that almost 90% of PA was released within 1 h without differences in release among the groups owing to the swelling of the hydrophilic ACA MC in SIF after the loading of inulin into PA or the internalization of PINs into PA. This suggests that the loading of inulin or the internalization of PINs does not affect the release of PA from ACA MCs owing to the very small amounts of inulin or PINs.
The results indicated that the survivability of PAs themselves was drastically decreased and was almost zero within 1 h at 37°C regardless of pepsin, whereas the other groups slowly decreased over time.
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