In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from gluco-oligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.     

In vitro three-stage continuous fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora

In vitro fermentations were carried out by using a model of the human colon to stimulate microbial activities of gut bacteria. The model consisted of a three-stage culture system. Bacterial populations were evaluated under the effect of three types of arabinoxylan, a nonstarch polysaccharide derived from wheat, the water-unextractable arabinoxylan fraction (WU-AX), WU-AX pretreated with exogenous xylanase and the soluble water-extractable arabinoxylan fraction (WE-AX). The xylanase pretreated (WU-AX) had a stimulatory effect upon colonic bifidobacteria throughout all three vessels. Counts of Bacteroides spp. and Clostridium spp. were also both significantly reduced. Addition of the WU-AX substrates to the first vessel resulted in induction of bacterial synthesis of extracellular hydrolytic enzymes xylanase and ferulic acid esterase which are both required for bacterial metabolism of WU-AX; this induction was significantly greater with the xylanase treated WU-AX.     

A study of dextran production from maltodextrin by cell suspensions of Gluconobacter oxydans NCIB 4943

This study investigated dextran synthesis from a commercial maltodextrin substrate using cell suspensions of G. oxydans NCIB 4943 as catalysts. Experiments were arranged according to a central composite statistical design. The effects of substrate concentration (10-100 g l-1), cell concentration (0.32-32.0 g wet weight l-1), time of reaction (8-48 h) and pH (3.5-5.5), each at three levels, on dextran yield and dextran molecular weight (MW), were investigated. Response surface methodology was used to assess factor interactions, and empirical models describing the two responses were fitted. Most of the variance in dextran yield could be explained by the fitted model (R2 = 0.96). Dextran yield ranged from 1.21 to 41.69%. The presence of significant negative quadratic effects of cell concentration and time indicated that dextran yield reached a plateau and thus, optimum levels of cell concentration and time could be identified to maximize dextran yield. Dextran MW ranged from 6.6 to 38 kDa and was characterized by the significant interactions of reaction time with substrate concentration and cell concentration. The model, however, could account for only 60% of the variance in dextran MW. Possible reasons for this are discussed.     

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.     

Characterization of a novel dextran produced by Gluconobacter oxydans DSM 2003

A novel water-soluble dextran was synthesized from maltodextrin by cell-free extract of Gluconobacter oxydans DSM 2003. The dextran was purified by size exclusion chromatography, and the structure was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and gas chromatography-mass spectrometer. Based on the spectral data, we found that the dextran contained only D-glucose residues. The ratio of nonreducing end glucopyranosyl (Glcp) to 6-linked Glcp to 4,6-linked Glcp was estimated to be 8.62:78.79:12.59 by methylation analysis. This result indicated the existence of a small proportion of α(1,4) branches in α(1,6) glucosyl linear chains. Here, we reported the first time a novel dextran was synthesized by G. oxydans DSM 2003.     

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

AIMS: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. MATERIALS AND METHODS: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. RESULTS: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. CONCLUSIONS: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.     

Biotransformation of Patulin by Gluconobacter oxydans

A bacterium isolated from patulin-contaminated apples was capable of degrading patulin to a less-toxic compound, ascladiol. The bacterium was identified as Gluconobacter oxydans by 16S rRNA gene sequencing, whereas ascladiol was identified by liquid chromatography-tandem mass spectrometry and proton and carbon nuclear magnetic resonance. Degradation of up to 96% of patulin was observed in apple juices containing up to 800 μg/ml of patulin and incubated with G. oxydans.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.     

基于重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种重要的氧化还原酶辅基,具有多种生理生化功能,在食品、医药卫生及农业等领域具有广泛的应用。文中采用重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌。首先构建丙酮酸脱羧酶基因GOX1081敲除的重组菌G. oxydans T1,减少副产物乙酸的形成。然后利用筛选的内源性组成型启动子P0169融合表达pqqABCDE基因簇及tldD基因,构建重组菌G. oxydans T2。最后对发酵培养基添加物和发酵条件进行优化。结果显示重组菌G. oxydans T1、G. oxydans T2生物量较野生菌分别提高43.02%和38.76%,而PQQ的产量分别是野生菌的4.82倍和20.5倍。进一步优化G. oxydans T2碳源及培养条件,最终PQQ产量达(51.3241±0.8997)mg/L,是野生菌的345.62倍。通过基因工程手段,可以有效提高氧化葡萄糖酸杆菌的生物量和合成PQQ的产量,为改善PQQ生物合成效率奠定基础。     

氧化葡萄糖酸菌转化制备米格列醇关键中间体

米格列醇作为一种新型α－葡糖苷酶抑制剂,能够有效治疗Ⅱ型糖尿病,并已迅速成为首选药物。葡萄糖酸菌细胞膜上含有多种脱氢酶,能够催化一系列重要产物的微生物转化,米格列醇就是其中之一。本文将详细说明不同的微生物转化过程,并进行比较。     

Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.     

A novel 3-dehydroquinate dehydratase catalyzing extracellular formation of 3-dehydroshikimate by oxidative fermentation of Gluconobacter oxydans IFO 3244

In addition to the cytoplasmic soluble form of 3-dehydroquinate dehydratase (sDQD) (EC 4.1.2.10), a novel form of DQD occurring in the periplasmic space was found in Gluconobacter oxydans IFO 3244. The novel DQD, tentatively designated as pDQD, appeared to have a practical function involved in oxidative fermentation extracellularly coupling with membrane-bound quinoprotein quinate dehydrogenase (QDH) yielding 3-dehydroshikimate from quinate via 3-dehydroquinate. pDQD was not detached from the membrane by mechanical disruption or extraction with high salt, but was solubilized only with detergent. pDQD and sDQD were purified to homogeneity and compared as to their enzymatic properties. They showed the same apparent molecular weights and same catalytic properties, but they were distinct each other in subunit molecular mass, 16 kDa for pDQD and 47 kDa for sDQD.     

In vitro maintenance of a human proximal colon microbiota using the continuous fermentation system P-ECSIM

Ethical and technical difficulties for in vivo studies on gut microbiotas argue for the development of alternative in vitro models: here, we describe a system simulating the proximal part of a human colon both nutritionally and physico-chemically with a procedure aimed to limit experimental variations over the time (Proximal Environmental Control System For Intestinal Microbiota—P-ECSIM). The continuous culture system P-ECSIM is first inoculated by a −20°C glycerol stock established from the batch culture of a stool-inoculated medium. The anaerobic atmosphere is self-maintained by the gases produced in the ordinary metabolism of fermentations. The monitoring of metabolic activities and microbial constitutions indicates that different steady states are obtained according to the dilution rate. Finally, the glycerol conservation of the batch culture-derived inoculum gives a similar differential response between the two dilution rates (D = 0.08 h⁻¹ and D = 0.04 h⁻¹) after a 1-year storage time as well for their metabolism and constitution in steady states, but with a lower abundance. Molecular fingerprints of the microbiota reveal however alterations over the time. Further efforts are needed concerning the preservation of standardized inoculums in order to improve the process for intra- and inter-lab comparison. Combined with appropriate analytical techniques, this system provides an efficient alternative means of studying functionally human microbiota in its constitution, metabolism and adaptation to environmental changes, particularly nutritional.     

In vitro anaerobic biofilms of human colonic microbiota

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.     

Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased.