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1.
J Gastroenterol Hepatol ; 39(3): 480-488, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38149305

RESUMO

BACKGROUND AND AIM: Potassium-competitive acid blockers more strongly suppress the gastric acid barrier than proton pump inhibitors and cause dysbiosis. However, preventive measures in this regard have not been established. We aimed to evaluate whether 1-kestose, a known prebiotic, was effective at alleviating dysbiosis caused by potassium-competitive acid blockers. METHODS: Patients scheduled to undergo endoscopic resection for superficial gastroduodenal tumors were enrolled and randomized 1:1 to receive either 1-kestose or placebo. All patients were started on potassium-competitive acid blocker (vonoprazan 20 mg/day) and took 1-kestose 10 g/day or placebo (maltose) 5 g/day for 8 weeks. The primary outcome was the effect of 1-kestose on potassium-competitive acid blocker-induced alterations in the microbiome. The fecal microbiome was analyzed before and after potassium-competitive acid blocker treatment via MiSeq (16S rRNA gene V3-V4 region). RESULTS: Forty patients were enrolled, and 16 in each group were analyzed. In the placebo group, the Simpson index, an alpha diversity, was significantly decreased and relative abundance of Streptococcus was significantly increased by 1.9-fold. In the kestose group, the Simpson index did not change significantly and relative abundance of Streptococcus increased 1.3-fold, but this was not a significant change. In both groups, no adverse events occurred, ulcers were well healed, and pretreatment and posttreatment short-chain fatty acid levels did not differ. CONCLUSIONS: The potassium-competitive acid blocker caused dysbiosis in the placebo group; this effect was prevented by 1-kestose. Thus, 1-kestose may be useful in dysbiosis treatment.


Assuntos
Disbiose , Microbiota , Pirróis , Sulfonamidas , Trissacarídeos , Humanos , Disbiose/etiologia , RNA Ribossômico 16S , Projetos Piloto , Inibidores da Bomba de Prótons/efeitos adversos , Potássio
2.
Bioprocess Biosyst Eng ; 47(9): 1499-1514, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38904715

RESUMO

The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h-1. The methanol-sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h-1 was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.


Assuntos
Hexosiltransferases , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/enzimologia , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hexosiltransferases/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Carbono/metabolismo , Sacarose/metabolismo , Reatores Biológicos , Metanol/metabolismo , Proteínas de Bactérias
3.
Biosci Biotechnol Biochem ; 87(9): 981-990, 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37280168

RESUMO

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a ß-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180 g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation.


Assuntos
Proteínas de Bactérias , Hexosiltransferases , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Sacarose/metabolismo
4.
Br J Nutr ; 127(4): 513-525, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33849681

RESUMO

SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon in vitro and ex vivo, but little is known in vivo. We tested whether dietary indigestible saccharides, utilised as a substrate to produce SCFA by gut microbiota, would increase colonic 5-HT content in mice. Male C57BL/6J mice were fed a purified diet and water supplemented with 4 % (w/v) 1-kestose (KES) for 2 weeks. Colonic 5-HT content and enterochromaffin (EC) cell numbers were lower in mice supplemented with KES than those without supplementation, while monoamine oxidase A activity and mRNA levels of tryptophan hydroxylase 1 (Tph1), chromogranin A (Chga), Slc6a4 and monoamine oxidase A (Maoa) genes in the colonic mucosa, serum 5-HT concentration and total 5-HT content in the colonic contents did not differ between groups. Caecal acetate concentration and Bifidobacterium pseudolongum population were higher in KES-supplemented mice. Similar trends were observed in mice supplemented with other indigestible saccharides, that is, fructo-oligosaccharides, inulin and raffinose. Intragastric administration of live B. pseudolongum (108 colony-forming units/d) for 2 weeks reduced colonic 5-HT content and EC cell numbers. These results suggest that changes in synthesis, reuptake, catabolism and overflow of 5-HT in the colonic mucosa are not involved in the reduction of colonic 5-HT content by dietary indigestible saccharides in mice. We propose that gut microbes including B. pseudolongum could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.


Assuntos
Colo , Serotonina , Animais , Bifidobacterium , Colo/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoaminoxidase/metabolismo , Serotonina/metabolismo
5.
Appl Microbiol Biotechnol ; 106(7): 2455-2470, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35267055

RESUMO

Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF2), tetrasaccharide nystose (GF3), and fructosyl nystose (GF4). Enzymes that hydrolyze GF3 may be useful for preparing GF2 from the fructooligosaccharide mixture. A ß-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60 mM sucrose, GF2, and GF3 by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF2 was lower than those for disaccharide sucrose and tetrasaccharide GF3. The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial ß-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF2 and the amino acid residues interacting with fructose at subsite - 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60 mM GF2/GF3 by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117-127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF2. KEY POINTS: • Frischella ß-fructofuranosidase hydrolyzed nystose more efficiently than 1-kestose. • Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference. • Loop consisting of residues 117-127 appears to contribute to the substrate binding.


Assuntos
Oligossacarídeos , beta-Frutofuranosidase , Animais , Abelhas , Frutose , Gammaproteobacteria , Oligossacarídeos/metabolismo , Sacarose , Trissacarídeos/metabolismo , beta-Frutofuranosidase/metabolismo
6.
Appl Microbiol Biotechnol ; 106(12): 4617-4626, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35739346

RESUMO

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated ß-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L-1 of FOS, with a productivity of 1.53 ± 0.03 g L-1 h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented.


Assuntos
Zymomonas , Etanol , Fermentação , Oligossacarídeos , Sacarose , Zymomonas/genética
7.
Crit Rev Biotechnol ; 41(1): 34-46, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33153319

RESUMO

Kestoses, the smallest fructooligosaccharides, are trisaccharides composed of a fructose molecule and a sucrose molecule linked by either ß-(2,1) or ß-(2,6) linkage. 1-kestose, 6-kestose and neokestose are the three types of kestoses occurring in nature. As the main kind of fructooligosaccharide, kestoses share similar physiological effects with other fructooligosaccharides, and they have recently been determined to show more notable effects in promoting the growth of probiotics including Faecalibacterium prausnitzii and Bifidobacterium than those of other fructooligosaccharides. Kestoses exist in many plants, but the relatively low content and the isolation and purification are the main barriers limiting their industrial application. The production of kestoses by enzymatic biosynthesis and microbial fermentation has the potential to facilitate its production and industrial use. In this article, the recent advances in the research of kestoses were overviewed, including those studying their functions and production. Kestose-producing enzymes were introduced in detail, and microbial production and fermentation optimization techniques for enhancing the yield of kestoses were addressed. ß-Fructofuranosidase is the main one used to produce kestoses because of the extensive range of microbial sources. Therefore, the production of kestoses by microorganisms containing ß-fructofuranosidase has also been reviewed. However, few molecular modification studies have attempted to change the production profile of some enzymes and improve the yield of kestoses, which is a topic that should garner more attention. Additionally, the production of kestoses using food-grade microorganisms may be beneficial to their application in the food industry.


Assuntos
Biotecnologia , Oligossacarídeos , Biotecnologia/tendências , Enzimas/metabolismo , Fermentação , Microbiologia de Alimentos , Oligossacarídeos/metabolismo
8.
Plant Foods Hum Nutr ; 76(4): 487-493, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34668149

RESUMO

Yacon (Smallanthus sonchifolius (Poepp.) H. Robinson) leaves is traditionally consumed as herbal tea in many countries including Indonesia. This plant's antidiabetic properties have been extensively researched, but studies on the responsible active compound identification are scarce. Information on the active compounds is critical for the consistency of Yacon herbal tea quality. The aim of this study was to identify α-glucosidase inhibitors in Indonesian Yacon leaves grown in two different locations using FTIR- and LC-MS/MS-based metabolomics in combination with in silico technique. Yacon leaves ethanol (50 and 95%) and water extracts were tested for α-glucosidase inhibitory activity, with the 95% ethanol extract being the most active. Geographical origins were found to have no major impact on the activity. In parallel, chemical profile of Yacon leaves extract was determined using FTIR and LC-MS/MS. Orthogonal Projection to Latent Structure (OPLS) was used to analyze both sets of data. OPLS analysis of FTIR data showed that compounds associated to α-glucosidase inhibitor activity included those with functional groups -OH, stretched CH, carbonyl, and alkene. It was consistent with the result of OPLS analysis of LC-MS/MS data, which revealed that based on their VIP and Y-related coefficient value, nystose, 1-kestose, luteolin-3'-7-di-O-glucoside, and 1,3-O-dicaffeoilquinic acid isomers, strongly linked to Yacon's α-glucosidase inhibitor activity. In silico study supported these findings, revealing that the four compounds were potent α-glucosidase inhibitors with docking score in the range of - 100.216 to - 115.657 kcal/mol, which are similar to acarbose (- 115.774 kcal/mol) as a reference drug.


Assuntos
Asteraceae , Inibidores de Glicosídeo Hidrolases , Cromatografia Líquida , Inibidores de Glicosídeo Hidrolases/farmacologia , Metabolômica , Extratos Vegetais/farmacologia , Folhas de Planta , Espectrometria de Massas em Tandem
9.
Biochem Biophys Res Commun ; 529(1): 64-69, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32560820

RESUMO

RegIIIß and RegIIIγ are antimicrobial peptides expressed in intestinal epithelial cells. Expression of these peptides is reportedly decreased by high-fat diet (HFD) and increased by indigestible oligosaccharides in mice. Clearly, these dietary regimens change the structure of intestinal microbiota. We employed an intestinal microbiota transplantation (IMT) to test whether diet-induced changes in the expression of these peptides are mediated by gut microbiota. C57BL/6J mice were fed either a normal-fat diet (NFD), a HFD, or a NFD supplemented with or without 1-kestose (KES), an indigestible oligosaccharide. Ileal RegIIIß and RegIIIγ mRNA levels were lower in mice receiving IMT from HFD-fed mice than in those receiving NFD-fed mice and higher in mice receiving IMT from KES-supplemented mice than in those receiving the mice without KES supplementation. Western blot analysis showed that serum RegIIIß levels changed in parallel with the ileal mRNA levels. We propose that HFD- and KES-induced changes in the ileal RegIIIß and RegIIIγ expression and in the circulating RegIIIß levels are mediated, at least in part, by intestinal microbiota.


Assuntos
Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Proteínas Associadas a Pancreatite/sangue , Proteínas Associadas a Pancreatite/genética , Animais , Dieta , Dieta Hiperlipídica , Íleo/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trissacarídeos/administração & dosagem , Interleucina 22
10.
Anaerobe ; 61: 102076, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31326442

RESUMO

Prebiotics are widely used to shape a balanced microbiota in humans and animals. 1-Kestose (kestose) is one of the major components in commercialized short-chain fructooligosaccharide and is a promising prebiotic for infants. We herein studied the impact of kestose on the healthy adult microbiota in an in vitro fecal batch culture model. Stool samples obtained from seven healthy adults were diluted, inoculated into broth supplemented with or without 0.5% (w/v) kestose (kestose group and control group, respectively), and cultured under anaerobic conditions. Microbiota in the groups and stool samples were analyzed using 16S rRNA gene sequencing. At the phylum level, the kestose group showed increases in Bacteroidetes, whereas the control group showed increases in Proteobacteria. At the species level, Bifidobacterium longum was the only species showing significantly higher levels in the kestose group than in the control group and stool samples. On the other hand, levels of Escherichia coli were significantly higher in the control group than in stool samples, while the levels were not significantly different between the kestose group and stool samples. Quantitative PCR assays also revealed significantly higher levels of B. longum and lower tendency of E. coli in the kestose group than in the control group. These results suggest that supplementation with kestose increased the levels of beneficial microorganism and prevented the growth of risk-associated microorganisms related to disease development. Further interventional studies are needed to understand the health benefits of kestose in adult humans.


Assuntos
Suplementos Nutricionais , Fezes/microbiologia , Microbioma Gastrointestinal , Trissacarídeos/administração & dosagem , Adulto , Fatores Etários , Feminino , Fermentação , Voluntários Saudáveis , Humanos , Masculino , Metabolômica/métodos , Metagenoma , Metagenômica/métodos , Adulto Jovem
11.
Molecules ; 25(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867210

RESUMO

The effects of supercritical CO2 processing on the chemical stability of fructooligosaccharides (FOS) and other functional and nutritional compounds were evaluated employing non-thermal and thermal approaches. Apple juice was enriched with Pfaffia glomerata roots aqueous extract due to its high content of short-chain FOS and then subjected to different levels of temperature (40 and 60 °C), pressure (8 and 21 MPa), and CO2 volume ratio (20 and 50%). The percentage of CO2 volume was evaluated concerning the total volume of the high-pressure reactor. Also, the functional beverage was thermally treated at 105 °C for 10 min. Physicochemical properties (pH and soluble solid content), beta-ecdysone, sugars (glucose, fructose, and sucrose), and FOS (1-kestose, nystose, and fructofuranosylnystose) content were determined. The pH and soluble solid content did not modify after all treatments. The pressure and CO2 volume ratio did not influence the FOS content and their chemical profile, however, the temperature increase from 40 to 60 °C increased the nystose and fructofuranosylnystose content. High-temperature thermal processing favored the hydrolysis of 1-kestose and reduced the sucrose content. Regarding beta-ecdysone, its content remained constant after all stabilization treatments demonstrating thus its high chemical stability. Our results demonstrated that supercritical CO2 technology is a promising technique for the stabilization of FOS-rich beverages since the molecular structures of these fructans were preserved, thus maintaining their prebiotic functionality.


Assuntos
Amaranthaceae/química , Dióxido de Carbono/química , Sucos de Frutas e Vegetais , Alimento Funcional , Oligossacarídeos/química , Pasteurização , Extratos Vegetais/química , Malus/química , Raízes de Plantas/química
12.
Biochem Biophys Res Commun ; 518(2): 294-298, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31420164

RESUMO

Kestose and nystose are short chain fructooligosaccharides (scFOSs) with degrees of polymerization of 3 and 4, respectively. A previous study revealed that these scFOSs have different growth stimulation properties against two human commensals, i.e. Bifidobacterium longum subsp. longum and butyrogenic Anaerostipes caccae. The present study characterized genes involved in FOS metabolism in these organisms. A. caccae possesses a single gene cluster consisting of four genes, including a gene encoding the putative FOS degradation enzyme sucrose-6-phosphate hydrolase (S6PH). B. longum possesses two gene clusters consisting of three genes each, including genes encoding ß-fructofuranosidase (CscA) and sucrose phosphorylase (ScrP). In A. caccae, the genes were highly transcribed in cells cultured with sucrose or kestose but poorly in cells cultured with glucose or nystose. Heterologously expressed S6PH degraded sucrose and kestose but not nystose. In B. longum, transcription of the genes was high in cells cultured with sucrose or kestose but was poor or not detected in cells cultured with glucose or nystose. Heterologously expressed CscA degraded sucrose, kestose and nystose, but ScrP degraded only sucrose. These data suggested that the different growth stimulation activities of kestose and nystose are due to different substrate specificities of FOS degradation enzymes in the organisms and/or induction activity of the genes in the two scFOSs. This is the first study characterizing the FOS metabolism at the transcriptional level and substrate-specificity of the degradation enzyme in butyrogenic human gut anaerobes.


Assuntos
Bifidobacterium longum/enzimologia , Clostridiales/enzimologia , Oligossacarídeos/metabolismo , Bifidobacterium longum/genética , Bifidobacterium longum/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Genes Bacterianos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Família Multigênica , Especificidade por Substrato , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
13.
Appl Microbiol Biotechnol ; 103(1): 279-289, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30357454

RESUMO

The ß-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.


Assuntos
Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , beta-Frutofuranosidase/metabolismo , Domínio Catalítico , Dissacaridases/metabolismo , Microrganismos Geneticamente Modificados , Modelos Moleculares , Oligossacarídeos/química , Prebióticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/genética , Especificidade por Substrato , Sacarose/metabolismo , Trissacarídeos/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética
14.
Biosci Biotechnol Biochem ; 82(9): 1599-1605, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29873621

RESUMO

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some ß-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production. ABBREVIATIONS: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: ß-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1F-ß-fructofuranosylnystose.


Assuntos
Aspergillus/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Trissacarídeos/metabolismo , beta-Frutofuranosidase/biossíntese , Aspergillus/enzimologia , Escherichia coli/genética , Mutagênese , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
15.
Biosci Biotechnol Biochem ; 81(9): 1786-1795, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28715279

RESUMO

ß-Fructofuranosidases belonging to glycoside hydrolase family (GH) 32 are enzymes that hydrolyze sucrose. Some GH32 enzymes also catalyze transfructosylation to produce fructooligosaccharides. We found that Aspergillus kawachii IFO 4308 ß-fructofuranosidase (AkFFase) produces fructooligosaccharides, mainly 1-kestose, from sucrose. We determined the crystal structure of AkFFase. AkFFase is composed of an N-terminal small component, a ß-propeller catalytic domain, an α-helical linker, and a C-terminal ß-sandwich, similar to other GH32 enzymes. AkFFase forms a dimer, and the dimerization pattern is different from those of other oligomeric GH32 enzymes. The complex structure of AkFFase with fructose unexpectedly showed that fructose binds both subsites -1 and +1, despite the fact that the catalytic residues were not mutated. Fructose at subsite +1 interacts with Ile146 and Glu296 of AkFFase via direct hydrogen bonds.


Assuntos
Aspergillus/enzimologia , Frutose/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Modelos Moleculares
16.
Biosci Biotechnol Biochem ; 78(5): 861-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25035990

RESUMO

To examine the effect of dietary supplementation with 1-kestose on the IgA levels in milk, BALB/c mice were fed diets with or without 5% 1-kestose during pregnancy and lactation. The total and specific IgA levels in the milk were measured at 7 and 14 days after delivery. A two-way ANOVA with repeated measures resulted in a significant effect of 1-kestose-supplementation on total IgA concentrations (p < 0.05) and the level of anti-Bacteroides IgA (p < 0.05). A significant positive correlation was found between the mean count of Bacteroides spp. in maternal feces and the total IgA concentration in maternal milk (r = 0.55, p < 0.05), suggesting a potential link between the gut and mammary gland immune system. In conclusion, this study demonstrated the effects of dietary prebiotics on milk IgA production.


Assuntos
Imunoglobulina A/metabolismo , Lactação/metabolismo , Leite/efeitos dos fármacos , Leite/metabolismo , Trissacarídeos/farmacologia , Animais , Bacteroides/imunologia , Bacteroides/fisiologia , Suplementos Nutricionais , Fezes/microbiologia , Feminino , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Lactação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Microbiota/efeitos dos fármacos , Gravidez
17.
Biosci Microbiota Food Health ; 43(2): 110-119, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38562544

RESUMO

How bifidobacteria colonize and survive in the intestine is not fully understood. The administration of bifidobacteria to conventional mice can be used to evaluate their ability to colonize the intestine in the presence of endogenous gut microbiota. However, human-derived bifidobacteria do not readily colonize the intestines of conventional mice, and although colonization by Bifidobacterium breve UCC2003 has been achieved, the viability of such populations requires improvement. Therefore, we aimed to establish a colonization system with human-derived bifidobacteria of high viability in conventional mice using Bifidobacterium longum subsp. longum 105-A. Lactose, raffinose, and 1-kestose were identified as the preferred carbohydrate sources for the growth of this strain in culture. The administration of B. longum 105-A to conventional BALB/c mice fed these carbohydrates showed that diets containing 6% (w/w) raffinose or 1-kestose facilitated colonization with >108 colony-forming units/g feces for 2 weeks. The population of this strain was more stable in the raffinose-fed group than in the 1-kestose-fed group. The ingestion of these prebiotics had a greater impact on the composition of the microbiota than the administration of B. longum 105-A. The ingestion of these prebiotics also increased the fecal concentrations of organic acids, which was indicative of greater intestinal fermentation. Collectively, we established a colonization system for B. longum 105-A with high viability in conventional mice by feeding the mice raffinose or 1-kestose. This system should be useful for elucidation of the mechanisms of colonization and survival of bifidobacteria in the intestines in the presence of the endogenous gut microbiota.

18.
J Vet Med Sci ; 86(2): 193-201, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38171739

RESUMO

Despite the well-known potential health benefits of prebiotics and non-viable probiotics (paraprobiotics) in various animal species, research regarding their use in penguins is scarce. Our study aimed to investigate the impact of a combined administration of prebiotics and paraprobiotics (referred to here as "parasynbiotics") on the gut microbiome and overall health of Magellanic penguins (Spheniscus magellanicus). The parasynbiotics consisted of 1-kestose, which is a fructooligosaccharide comprising sucrose and fructose, and heat-killed Lactiplantibacillus plantarum FM8, isolated from pickled vegetables. It was administered to eight penguins aged <3 years (Young-group) and nine penguins aged >17 years (Adult-group) for 8 weeks. Results from 16S rRNA sequencing revealed that compared to baseline, parasynbiotic administration significantly decreased the relative abundance of intestinal Clostridiaceae_222000 in both groups and significantly increased that of Lactobacillaceae in the Young-group. Quantitative real-time polymerase chain reaction revealed a significant decrease in the plc gene levels encoding alpha-toxin of Clostridium perfringens in the Young-group after parasynbiotic administration (P=0.0078). In the Young-group, parasynbiotic administration significantly increased the plasma levels of total alpha-globulin (P=0.0234), which is associated with inflammatory responses. Furthermore, exposure of dendritic cells to heat-killed L. plantarum FM8 promoted the secretion of interleukin 10, a major anti-inflammatory cytokine. Overall, parasynbiotic administration enhanced the activity of gut Lactobacillaceae, decreased the levels of C. perfringens and its toxin encoding plc gene, and reduced inflammatory response in penguins. These results provide novel insights into the potential benefits of parasynbiotics for improving penguin health.


Assuntos
Prebióticos , Spheniscidae , Trissacarídeos , Animais , Clostridium perfringens , RNA Ribossômico 16S
19.
Front Microbiol ; 15: 1383779, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38741747

RESUMO

Introduction: The effects of fructo-oligosaccharides (FOS) on atopic dermatitis (AD) have not been determined. Methods: In a randomized, double-blind, placebo-controlled trial, children with AD aged 24 months to 17 years received either advanced FOS containing 4.25 g of 1-kestose or a placebo (maltose) for 12 weeks. Results: The SCORAD and itching scores were reduced in patients treated with both FOS (all p < 0.01) and maltose (p < 0.05 and p < 0.01). Sleep disturbance was improved only in the FOS group (p < 0.01). The FOS group revealed a decreased proportion of linoleic acid (18:2) esterified omega-hydroxy-ceramides (EOS-CERs) with amide-linked shorter chain fatty acids (C28 and C30, all p < 0.05), along with an increased proportion of EOS-CERs with longer chain fatty acids (C32, p < 0.01). Discussion: FOS may be beneficial in alleviating itching and sleep disturbance, as well as improving skin barrier function in children with AD.

20.
J Nutr Sci Vitaminol (Tokyo) ; 70(4): 311-317, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39218692

RESUMO

Chronic inflammation in adipose tissue is thought to contribute to insulin resistance, which involves the gut microbiota. Our previous studies have demonstrated that ingestion of 1-kestose can alter the gut microbiota composition, increase cecal butyrate levels, and improve insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Additionally, we found that 1-kestose supplementation ameliorated insulin resistance in obese rat models fed a high-fat diet (HFD), although the effects of 1-kestose on the abundance of inflammation-related gene in adipose tissue and gut microbiota composition in these rats were not explored. This study aimed to investigate the impact of 1-kestose on these parameters in HFD-fed rats, compared to OLETF rats. Male Sprague-Dawley rats were divided into two dietary groups, control or HFD, for 19 wk. Each group was further subdivided to receive either tap water or tap water supplemented with 2% (w/v) 1-kestose throughout the study. We evaluated gene expression in adipose tissue, as well as short-chain fatty acids (SCFAs) levels and microbial composition in the cecum contents. 1-Kestose intake restored the increased relative abundance of tumor necrosis factor (Tnf) mRNA in adipose tissue and the reduced level of butyrate in the cecum contents of HFD-fed rats to those observed in control diet-fed rats. Additionally, 1-kestose consumption changed the composition of the gut microbiota, increasing Butyricicoccus spp., decreasing UGC-005 and Streptococcus spp., in the cecum contents of HFD-fed rats. Our findings suggest that 1-kestose supplementation reduces adipose tissue inflammation and increases butyrate levels in the gut of HFD-fed rats, associated with changes in the gut microbiota composition, distinct from those seen in OLETF rats.


Assuntos
Tecido Adiposo , Ceco , Dieta Hiperlipídica , Ácidos Graxos Voláteis , Microbioma Gastrointestinal , Inflamação , RNA Mensageiro , Ratos Sprague-Dawley , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Masculino , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Inflamação/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ácidos Graxos Voláteis/metabolismo , Ceco/microbiologia , Ceco/metabolismo , Resistência à Insulina , Ratos Endogâmicos OLETF , Obesidade/metabolismo , Obesidade/microbiologia , Suplementos Nutricionais , Butiratos/metabolismo
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