ABSTRACT
This review explores the role and health impacts of probiotics, focusing specifically on Bifidobacterium spp. It highlights the functionalities that Bifidobacteria can provide, underscored by the historical evolution of definitions and technological advancements related to probiotics. By examining the association between Bifidobacteria and longevity, this review suggests new avenues for health enhancement. Highlighting case studies of centenarians, it presents examples related to human aging, illuminating the potential links to longevity through research on Bifidobacterium strains found in centenarians. This review not only emphasizes the importance of current research but also advocates for further investigation into the health benefits of Bifidobacteria, underlining the necessity for continuous study in the nutraceutical field.
ABSTRACT
Since the discovery of antibiotics, penicillin has remained the top choice in clinical medicine. With continuous advancements in biotechnology, penicillin production has become cost-effective and efficient. Genetic engineering techniques have been employed to enhance biosynthetic pathways, leading to the production of new penicillin derivatives with improved properties and increased efficacy against antibiotic-resistant pathogens. Advances in bioreactor design, media formulation, and process optimization have contributed to higher yields, reduced production costs, and increased penicillin accessibility. While biotechnological advances have clearly benefited the global production of this life-saving drug, they have also created challenges in terms of waste management. Production fermentation broths from industries contain residual antibiotics, by-products, and other contaminants that pose direct environmental threats, while increased global consumption intensifies the risk of antimicrobial resistance in both the environment and living organisms. The current geographical and spatial distribution of antibiotic and penicillin consumption dramatically reveals a worldwide threat. These challenges are being addressed through the development of novel waste management techniques. Efforts are aimed at both upstream and downstream processing of antibiotic and penicillin production to minimize costs and improve yield efficiency while lowering the overall environmental impact. Yield optimization using artificial intelligence (AI), along with biological and chemical treatment of waste, is also being explored to reduce adverse impacts. The implementation of strict regulatory frameworks and guidelines is also essential to ensure proper management and disposal of penicillin production waste. This review is novel because it explores the key remaining challenges in antibiotic development, the scope of machine learning tools such as Quantitative Structure-Activity Relationship (QSAR) in modern biotechnology-driven production, improved waste management for antibiotics, discovering alternative path to reducing antibiotic use in agriculture through alternative meat production, addressing current practices, and offering effective recommendations.
Subject(s)
Anti-Bacterial Agents , Biotechnology , Penicillins , Biotechnology/methods , Waste Management/methodsABSTRACT
BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.
Subject(s)
Alcohol Dehydrogenase , Ethanol , Probiotics , Humans , Alcohol Dehydrogenase/metabolism , Alcohol Dehydrogenase/genetics , Ethanol/metabolism , Lactobacillus/metabolism , Lactobacillus/genetics , Lactobacillales/genetics , Lactobacillales/metabolism , Lacticaseibacillus rhamnosus/genetics , Lacticaseibacillus rhamnosus/metabolism , Aldehyde Oxidoreductases/metabolism , Aldehyde Oxidoreductases/genetics , Pediococcus acidilactici/metabolismABSTRACT
The use of frozen dough is an intensive food-processing practice that contributes to the development of chain operations in the bakery industry. However, the fermentation activity of yeasts in frozen dough can be severely damaged by freeze-thaw stress, thereby degrading the final bread quality. In this study, chickpea protein hydrolysate significantly improved the quality of steamed bread made from frozen dough while enhancing the yeast survival rate and maintaining yeast cell structural integrity under freeze-thaw stress. The mechanism underlying this protective role of chickpea protein hydrolysate was further investigated by untargeted metabolomics analysis, which suggested that chickpea protein hydrolysate altered the intracellular metabolites associated with central carbon metabolism, amino acid synthesis, and lipid metabolism to improve yeast cell freeze-thaw tolerance. Therefore, chickpea protein hydrolysate is a promising natural antifreeze component for yeast cryopreservation in the frozen dough industry.
Subject(s)
Cicer , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Cicer/metabolism , Protein Hydrolysates/metabolism , Freezing , Saccharomyces cerevisiae Proteins/metabolism , Fermentation , Bread/analysisABSTRACT
Recent developments in tissue clearing methods such as the passive clearing technique (PACT) have allowed three-dimensional analysis of biological structures in whole, intact tissues, thereby providing a greater understanding of spatial relationships and biological circuits. Nonetheless, the issues that remain in maintaining structural integrity and preventing tissue expansion/shrinkage with rapid clearing still inhibit the wide application of these techniques in hard bone tissues, such as femurs and tibias. Here, we present an optimized PACT-based bone-clearing method, Bone-mPACT+, that protects biological structures. Bone-mPACT+ and four different decalcifying procedures were tested for their ability to improve bone tissue clearing efficiency without sacrificing optical transparency; they rendered nearly all types of bone tissues transparent. Both mouse and rat bones were nearly transparent after the clearing process. We also present a further modification, the Bone-mPACT+ Advance protocol, which is specifically optimized for processing the largest and hardest rat bones for easy clearing and imaging using established tissue clearing methods.
Subject(s)
Bone and Bones , Imaging, Three-Dimensional , Rats , Mice , Animals , Imaging, Three-Dimensional/methods , Bone and Bones/diagnostic imagingABSTRACT
In this work, the in vivo functionalities of milk fermented with Weissella confusa VP30 (VP30-EPS) and purified exopolysaccharide (pEPS) from the milk fermented with Weissella confusa VP30 were evaluated for their effect on constipation using an experimental constipated rat model. Rats were randomly divided into four groups: (i) control group (PBS administered normal group), (ii) loperamide treated group (constipation group), (iii) constipation with loperamide plus VP30-EPS (1 g/kg), and (iv) constipation with loperamide plus pEPS (0.6 g/kg) groups. Loperamide treatment induced animal constipation and significantly reduced the frequency of defecation, intestinal transit ratio, and water content of feces. However, all four fecal parameters were improved in both the loperamide plus VP30-EPS and pEPS administered groups as compared to the loperamide group. These results suggest that the addition of VP30-EPS potentially improves the functional laxative effects of commercial products. This study suggests the possibility that VP30-EPS can be applied to fermented and/or functional foods to relieve constipation.
ABSTRACT
BACKGROUND: Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms. METHODS: hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria. RESULTS: The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture. CONCLUSIONS: The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.
Subject(s)
Enterococcus faecium , Osteogenesis , Animals , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Dental Pulp/metabolism , Enterococcus faecium/metabolism , Humans , Infant, Newborn , Mice , Osteogenesis/genetics , Stem Cells/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Inflammatory bowel disease (IBD) is a gastrointestinal disease characterized by diarrhea, rectal bleeding, abdominal pain, and weight loss. Recombinant probiotics producing specific proteins with IBD therapeutic potential are currently considered novel drug substitutes. In this study, a Bifidobacterium bifidum BGN4-SK strain was designed to produce the antioxidant enzymes streptococcal superoxide dismutase (SOD) and lactobacillus catalase (CAT), and a B. bifidum BGN4-pBESIL10 strain was proposed to generate an anti-inflammatory cytokine, human interleukin (IL)-10. In vitro and in vivo efficacy of these genetically modified Bifidobacterium strains were evaluated for colitis amelioration. RESULTS: In a lipopolysaccharide (LPS)-stimulated HT-29 cell model, tumor necrosis factor (TNF)-α and IL-8 production was significantly suppressed in the B. bifidum BGN4-SK treatment, followed by B. bifidum BGN4-pBESIL10 treatment, when compared to the LPS-treated control. Synergistic effects on TNF-α suppression were also observed. In a dextran sodium sulphate (DSS)-induced colitis mouse model, B. bifidum BGN4-SK treatment significantly enhanced levels of antioxidant enzymes SOD, glutathione peroxidase (GSH-Px) and CAT, compared to the DSS-only group. B. bifidum BGN4-SK significantly ameliorated the symptoms of DSS-induced colitis, increased the expression of tight junction genes (claudin and ZO-1), and decreased pro-inflammatory cytokines IL-6, IL-1ß and TNF-α. CONCLUSIONS: These findings suggest that B. bifidum BGN4-SK ameliorated DSS-induced colitis by generating antioxidant enzymes, maintaining the epithelial barrier, and decreasing the production of pro-inflammatory cytokines. Although B. bifidum BGN4-pBESIL10 exerted anti-inflammatory effects in vitro, the enhancement of IL-10 production and alleviation of colitis were very limited.
Subject(s)
Bifidobacterium bifidum , Colitis , Inflammatory Bowel Diseases , Probiotics , Animals , Anti-Inflammatory Agents/adverse effects , Antioxidants/metabolism , Bifidobacterium bifidum/genetics , Colitis/drug therapy , Colitis/therapy , Cytokines/metabolism , Dextran Sulfate/adverse effects , Dextran Sulfate/metabolism , Disease Models, Animal , Humans , Inflammatory Bowel Diseases/drug therapy , Interleukin-10/metabolism , Lipopolysaccharides , Mice , Probiotics/therapeutic use , Superoxide Dismutase/adverse effects , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Butyl-fructooligosaccharides (B-FOSs) are newly synthesized prebiotics composed of short-chain FOS (GF2, 1-kestose; GF3, nystose; GF4, fructofuranosyl-nystose; GF5, 1-F-(1-b-D-fructofuranosyl)-2-nystose) bound with one or two butyric groups by ester bonds. Previous in vitro studies have shown that B-FOS treatment increases butyrate production and protects the growth of butyrate-producing bacteria during fermentation. The aim of this study was to further test B-FOS as a novel prebiotic compound by evaluating the effect of B-FOS on gut microbiota via 16S rRNA metagenomic analysis in an Institute of Cancer Research (ICR) mouse model and examining its anti-inflammatory efficacy in a mouse model of colitis induced by dextran sodium sulphate (DSS). In the healthy ICR mouse study, linear discriminant analysis effect size results revealed that Bifidobacterium was the representative phylotype in the B-FOS treatment compared to the control group. Furthermore, the cecal butyrate concentration of the B-FOS group was significantly higher than that of the control (P < 0.05). The high concentration of butyrate in the B-FOS treatment was probably associated with the high relative abundance of clusters of orthologous group (COG) 4770 (acetyl/propionyl-CoA carboxylase). In the DSS-induced infection study, B-FOS significantly ameliorated the symptoms of DSS-induced colitis, increased the mRNA expression of occludin, decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL-8) in the colon tissues, and significantly increased cecal butyrate concentrations. These findings suggest that B-FOS ameliorated DSS-induced colitis by maintaining the epithelial barrier and reducing the secretion of inflammation related cytokines.
Subject(s)
Colitis, Ulcerative/metabolism , Gastrointestinal Microbiome/drug effects , Oligosaccharides/pharmacology , Animals , Colitis, Ulcerative/chemically induced , Dextran Sulfate/adverse effects , Disease Models, Animal , MiceABSTRACT
Bifidobacterium bifidum BGN4-SK (BGN4-SK), a recombinant strain which was constructed from B. bifidum BGN4 (BGN4) to produce superoxide dismutase (SOD) and catalase, was analyzed to determine its antioxidant and anti-inflammatory properties in vitro. Culture conditions were determined to maximize the SOD and catalase activities of BGN4-SK. The viability, intracellular radical oxygen species (ROS) levels, intracellular antioxidant enzyme activities, and pro-inflammatory cytokine levels were determined to evaluate the antioxidant and anti-inflammatory activities of BGN4-SK in human intestinal epithelial cells (HT-29) and murine macrophage cells (RAW 264.7). Antioxidant enzymes (SOD and catalase) were produced at the highest levels when BGN4-SK was cultured for 24 h in a medium containing 500 µM MnSO4 and 30 µM hematin, with glucose as the carbon source. The viability and intracellular antioxidant enzyme activities of H2O2-stimulated HT-29 treated with BGN4-SK were significantly higher (p < 0.05) than those of cells treated with BGN4. The intracellular ROS levels of H2O2-stimulated HT-29 cells treated with BGN4-SK were significantly lower (p < 0.05) than those of cells treated with BGN4. BGN4-SK more significantly suppressed the production of interleukin (IL)-6 (p < 0.05), tumor necrosis factor-α (p < 0.01), and IL-8 (p < 0.05) in lipopolysaccharide (LPS)-stimulated HT-29 and LPS-stimulated RAW 264.7 cells compared to BGN4. These results suggest that BGN4-SK may have enhanced antioxidant activities against oxidative stress in H2O2-stimulated HT-29 cells and enhanced anti-inflammatory activities in LPS-stimulated HT-29 and RAW 264.7 cells.
ABSTRACT
It has been reported that certain probiotic bacteria have inhibitory effects against oral pathogens. Lactobacillus spp. have been studied and used as probiotics globally, but due to difficulties with laboratory cultivation and experimentation with oral microorganisms, there are few studies on Lactobacillus spp. isolated from the oral cavity being used against oral pathogens. The purpose of this study was to evaluate the biosafety and inhibitory effects of Lactobacillus fermentum OK as a potential oral biotherapeutic probiotic against oral pathogens. L. fermentum OK was evaluated based on microbial and genetic characteristics. A 5% dilution of L. fermentum OK culture supernatant showed that 60% inhibition against the growth of S. mutans and L. fermentum OK displayed significant inhibitory effects against the growth of Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus gordonii, and Streptococcus sanguinis. However, proliferation of L. fermentum OK, when co-cultured with harmful oral bacteria, was retarded. L. fermentum OK was shown to produce 1130 µmol/L hydrogen peroxide, aggregate efficiently with Streptococcus sobrinus, S. gordonii, S. mutans, S. sanguinis, and P. gingivalis, and reduce S. mutans that produced artificial dental plaque by 97.9%. The in vitro cell adhesion capacity of L. fermentum OK to an oral epithelial cell line was 3.1 cells per cell and the cell adhesion of F. nucleatum and S. mutans decreased strongly in protection and displacement assays. L. fermentum OK was evaluated for safety using ammonia production, biogenic amine production, hemolytic property, mucin degradation testing, antibiotic susceptibility, and whole genome sequencing (WGS). Based on this study, L. fermentum OK appears to be a safe and bioactive lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.
Subject(s)
Limosilactobacillus fermentum , Probiotics , HumansABSTRACT
BACKGROUND: Lactobacillus spp. have been researched worldwide and are used in probiotics, but due to difficulties with laboratory cultivation of and experimentation on oral microorganisms, there are few reports of Lactobacillus spp. being isolated from the oral cavity and tested against oral pathogens. This research sought to isolate and determine the safety and inhibitory capabilities of a Lactobacillus culture taken from the human body. RESULTS: One organism was isolated, named "L. gasseri HHuMIN D", and evaluated for safety. A 5% dilution of L. gasseri HHuMIN D culture supernatant exhibited 88.8% inhibition against halitosis-producing anaerobic microorganisms and the organism itself exhibited powerful inhibitory effects on the growth of 11 oral bacteria. Hydrogen peroxide production reached 802 µmol/L after 12 h and gradually diminished until 24 h, it efficiently aggregated with P. catoniae and S. sanguinis, and it completely suppressed S. mutans-manufactured artificial dental plaque. L. gasseri HHuMIN D's KB cell adhesion capacity was 4.41 cells per cell, and the cell adhesion of F. nucleatum and S. mutans diminished strongly in protection and displacement assays. CONCLUSION: These results suggest that L. gasseri HHuMIN D is a safe, bioactive, lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.
Subject(s)
Antibiosis , Lactobacillus gasseri/isolation & purification , Lactobacillus gasseri/metabolism , Lactobacillus/metabolism , Mouth/microbiology , Probiotics/metabolism , Bacteria, Anaerobic/growth & development , Bacteria, Anaerobic/metabolism , Humans , Hydrogen Peroxide/metabolism , Lactobacillus/classification , Lactobacillus/pathogenicity , Lactobacillus gasseri/growth & development , Probiotics/administration & dosageABSTRACT
BACKGROUND: Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines. RESULTS: The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001). CONCLUSION: B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.
Subject(s)
Bifidobacterium bifidum/metabolism , Escherichia coli/metabolism , Interleukin-10/metabolism , Recombinant Proteins/metabolism , Animals , Base Sequence , Bifidobacterium bifidum/genetics , Blotting, Western , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , HT29 Cells , Humans , Interleukin-10/genetics , Mice , Plasmids/genetics , Promoter Regions, Genetic/genetics , RAW 264.7 Cells , Sequence Homology, Nucleic AcidABSTRACT
The advent of tissue clearing methods, in conjunction with novel high-resolution imaging techniques, has enabled the visualization of three-dimensional structures with unprecedented depth and detail. Although a variety of clearing protocols have been developed, little has been done to quantify their efficacies in a systematic, reproducible fashion. Here, we present two simple assays, Punching-Assisted Clarity Analysis (PACA)-Light and PACA-Glow, which use easily accessible spectroscopy and gel documentation systems to quantify the transparency of multiple cleared tissues simultaneously. We demonstrate the use of PACA-Light and PACA-Glow to compare twenty-eight tissue clearing protocols on rodent brains. We also show that regional differences exist in tissue transparency in the rodent brain, with cerebellar tissue consistently achieving lower clearing levels compared to the prefrontal or cerebral cortex across all protocols. This represents the largest comparative study of tissue clearing protocols to date, made possible by the high-throughput nature of our PACA platforms.
ABSTRACT
B-FOS (butyl-fructooligosaccharide) is a newly synthesized prebiotic molecule, formed by the combination of FOS and butyrate by ester bonds. B-FOS has been reported to have the potential prebiotic effect of promoting intestinal flora diversity and enhancing butyrate production. The aim of this study was to investigate the potential acute and sub-chronic toxicity of B-FOS in Institute of Cancer Research (ICR) mice and Wistar rats to verify its biosafety. ICR mice were administered a single oral gavage of B-FOS at doses of 0, 500, 1000, and 2000 mg/kg body weight and observed for signs of acute toxicity for 14 days. Sub-chronic toxicity was evaluated by repeated oral administration of B-FOS at 2000 mg/kg for 28 days, in accordance with Organization for Economic Co-operation and Development (OECD) protocol test numbers 420 and 407. No mortality or abnormal clinical signs were observed during the experimental periods after B-FOS administration. Furthermore, no significant changes in body weight, organ weight, serum biochemical parameters, or tissue histology were observed after animal sacrifice. These in vivo results indicate that B-FOS does not exert any acute or sub-chronic toxicity at a dose of 2000 mg/kg, and this novel molecule can be regarded as a safe prebiotic substance for use in the food and nutraceutical industries.
ABSTRACT
The prevention and treatment of chronic inflammation using food-derived compounds are desirable from the perspectives of marketing and safety. Monascus pigments, widely used as food additives, can be used as a chronic inflammation treatment. Orange Monascus pigments were produced by submerged fermentation in a 5 L bioreactor, and multiple orange Monascus pigment derivatives with anti-inflammatory activities were synthesized using aminophilic reaction. A total of 41 types of pigment derivatives were produced by incorporating amines and amino acids into the orange pigments. One derivative candidate that inhibited nitric oxide (NO) production in Raw 264.7 cells and exhibited low cell cytotoxicity was identified via in vitro assay. The 2-amino-4 picoline derivative inhibited NO production of 48.4%, and exhibited cell viability of 90.6%. Expression of inducible NO synthase, an important enzyme in the NO synthesis pathway, was suppressed by such a derivative in a dose-dependent manner. Therefore, this derivative has potential as a functional food colorant with anti-inflammatory effects.
ABSTRACT
Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 µM Ara-27 in both HDF and hDPSC. In contrast, 2 µM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 µM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.
Subject(s)
Cell-Penetrating Peptides , Drug Delivery Systems/methods , Nanoparticles , Amino Acid Sequence , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , Cells, Cultured , Flow Cytometry , Humans , Microscopy, Confocal , Nanoparticles/chemistry , Nanoparticles/metabolism , Zinc/chemistryABSTRACT
Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.
Subject(s)
Cell Lineage/drug effects , Complex Mixtures/pharmacology , Enterococcus faecium/chemistry , Gene Expression Regulation/drug effects , Probiotics/pharmacology , Stem Cells/drug effects , Animals , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Differentiation/drug effects , Cell Lineage/genetics , Cell Proliferation/drug effects , Complex Mixtures/chemistry , Embryo, Mammalian , Enterococcus faecium/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Inbred ICR , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Primary Cell Culture , Probiotics/chemistry , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Biological systems consist of a variety of distinct cell types that form functional networks. Super-resolution imaging of individual cells is required for better understanding of these complex systems. Direct visualization of 3D subcellular and nano-scale structures in cells is helpful for the interpretation of biological interactions and system-level responses. Here we introduce a modified magnified analysis of proteome (MAP) method for cell super-resolution imaging (Cell-MAP) which preserves cell fluorescence. Cell-MAP expands cells more than four-fold while preserving their overall architecture and three-dimensional proteome organization after hydrogel embedding. In addition, Optimized-Cell-MAP completely preserves fluorescence and successfully allows for the observation of tagged small molecular probes containing peptides and microRNAs. Optimized-Cell-MAP further successfully applies to the study of structural characteristics and the identification of small molecules and organelles in mammalian cells. These results may give rise to many other applications related to the structural and molecular analysis of smaller assembled biological systems.
Subject(s)
Fluorescence , Proteome/metabolism , Animals , Cell Line , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/metabolism , Flow Cytometry , HEK293 Cells , Humans , Male , MicroRNAs/metabolism , Microscopy, Fluorescence , Rats , Rats, Sprague-DawleyABSTRACT
Short-chain fatty acids (SCFAs), especially butyrate, produced in mammalian intestinal tracts via fermentation of dietary fiber, are known biofunctional compounds in humans. However, the variability of fermentable fiber consumed on a daily basis and the diversity of gut microbiota within individuals often limits the production of short-chain fatty acids in the human gut. In this study, we attempted to enhance the butyrate levels in human fecal samples by utilizing butyl-fructooligosaccharides (B-FOS) as a novel prebiotic substance. Two major types of B-FOS (GF3-1B and GF3-2B), composed of short-chain fructooligosaccharides (FOS) bound to one or two butyric groups by ester bonds, were synthesized. Qualitative analysis of these B-FOS using Fourier transform infrared (FT-IR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), nuclear magnetic resonance (NMR) and low-resolution fast-atom bombardment mass spectra (LR-FAB-MS), showed that the chemical structure of GF3-1B and GF3-2B were [O-(1-buty-ß-D-fru-(2â1)-O-ß-D-fru-(2â1)-O-ß-D-fru-O-α-D-glu] and [O-(1-buty)-ß-D-fru-(2â1)-O-ß-D-fru-(2â1)-O-(4-buty)-ß-D-fru-O-α-D-glu], respectively. The ratio of these two compounds was approximately 5:3. To verify their biofunctionality as prebiotic oligosaccharides, proliferation and survival patterns of human fecal microbiota were examined in vitro via 16S rRNA metagenomics analysis compared to a positive FOS control and a negative control without a carbon source. B-FOS treatment showed different enrichment patterns on the fecal microbiota community during fermentation, and especially stimulated the growth of major butyrate producing bacterial consortia and modulated specific butyrate producing pathways with significantly enhanced butyrate levels. Furthermore, the relative abundance of Fusobacterium and ammonia production with related metabolic genes were greatly reduced with B-FOS and FOS treatment compared to the control group. These findings indicate that B-FOS differentially promotes butyrate production through the enhancement of butyrate-producing bacteria and their metabolic genes, and can be applied as a novel prebiotic compound in vivo.