New two-stage pH combined with dissolved oxygen control strategy for cyclic ß-1,2 glucans synthesis.

On the basis of a novel two-stage pH combined with dissolved oxygen (DO) control strategy in fed-batch fermentation, this research addresses the influence of pH on cyclic ß-1,2-glucans (CßGs) biosynthesis and melanin accumulation during the production of CßGs by Rhizobium radiobacter ATCC 13,333. Under these optimal fermentation conditions, the maximum cell concentration and CßGs concentration in a 7-L stirred-tank fermenter were 7.94 g L-1 and 3.12 g L-1, which were the maximum production reported for R. radiobacter. The melanin concentration of the fermentation broth was maintained at a low level, which was beneficial to the subsequent separation and purification of the CßGs. In addition, a neutral extracellular oligosaccharide (COGs-1) purified by the two-stage pH combined with DO control strategy fermentation medium was structurally characterized. Structural analyses indicated that COGs-1 was a family of unbranched cyclic oligosaccharides composed of only ß-1,2-linked D-glucopyranose residues with degree of polymerization between 17 and 23, namely CßGs. This research provides a reliable source of CßGs and structural basis for further studies of biological activity and function. KEY POINTS: • A two-stage pH combined with DO control strategy was proposed for CßGs production and melanin biosynthesis by Rhizobium radiobacter. • The final extracellular CßGs production reached 3.12 g L-1, which was the highest achieved by Rhizobium radiobacter. • The existence of CßGs could be detected by TLC quickly and accurately.

Multi-stage glucose/pachymaran co-feeding enhanced endo-ß-1,3-glucanase production by Trichoderma harzianum via simultaneous increases in cell concentration and inductive effect.

Endo-ß-1,3-glucanase is used to hydrolyze curdlan in a wide range of oligosaccharides production processes. Using pachymaran as the sole carbon source resulted in an endo-ß-1,3-glucanase activity of 86.1 U/mL and an Eendo/Etotal ratio of 0.43, which were 3.2 and 1.65 folds of the values from control (glucose as the sole carbon source), due to the inductive effect of pachymaran as a polysaccharide. However, the cell concentration decreased from 25 to 12 g/L during the late fermentation phase. Therefore, a novel multi-stage feeding strategy was developed wherein glucose was fed twice during the cell logarithmic growth phase (24 and 48 h) and pachymaran once during the early stage of the enzyme accumulation phase (72 h). Consequently, the cell concentration remained around 30 g/L during the late fermentation phase. Endo-ß-1,3-glucanase activity and Eendo/Etotal reached 160.0 U/mL and 0.76, respectively, which were 6.0 and 2.92 folds of the values from control. In addition, three typical polysaccharides with ß-1,3-linked glucose residues were successfully hydrolyzed by endo-ß-1,3-glucanase to produce multifunctional ß-1,3-oligoglucosides.

Enhanced N-acetyl-D-neuraminic production from glycerol and N-acetyl-D-glucosamine by metabolically engineered Escherichia coli with a two-stage pH-shift control strategy.

Typical N-acetyl-D-neuraminic acid (Neu5Ac) production uses N-acetyl-D-glucosamine (GlcNAc) and excess pyruvate as substrates in the enzymatic or whole-cell biocatalysis process. In a previous study, a Neu5Ac-producing biocatalytic process via engineered Escherichia coli SA-05/pDTrc-AB/pCDF-pck-ppsA was constructed without exogenous pyruvate. In this study, glycerol was found to be a good energy source compared with glucose for the catalytic system with resting cells, and Neu5Ac production increased to 13.97 ± 0.27 g L-1. In addition, a two-stage pH shift strategy was carried out, and the Neu5Ac yield was improved to 14.61 ± 0.31 g L-1. The GlcNAc concentration for Neu5Ac production was optimized. Finally, an integrated strategy was developed for Neu5Ac production, and the Neu5Ac yield reached as high as 18.17 ± 0.27 g L-1. These results provide a new biocatalysis technology for Neu5Ac production without exogenous pyruvate.

High production of xanthan gum by a glycerol-tolerant strain Xanthomonas campestris WXLB-006.

The superior properties of xanthan gum make it an industrial aginomoto used in many industries, especially in oil recovery. In the present work, xanthan production from glycerol by a mutant strain Xanthomonas campestris WXLB-006 reached as high as 17.8 g/L in flask culture. With the adoption of pH control, varied aeration and agitation, and varied glycerol feeding strategy, xanthan production reached 33.9 g/L in a 7-L fermenter and fermentation time decreased to 60 hr. Instead of difficultly and costly purifying glycerol, this research provides a very good case for glycerol utilization. At the same time, this is the first report on a high glycerol-tolerant strain for microbial polysaccharide production and 33.9 g/L is the highest production of xanthan gum produced from glycerol so far.

Optimization of a low-cost hyperosmotic medium and establishing the fermentation kinetics of erythritol production by Yarrowia lipolytica from crude glycerol.

The production of erythritol by Yarrowia lipolytica from low-cost substitutable substrates for high yield was investigated. Crude glycerol, urea, and NaCl related to osmotic pressure were the most significant factors affecting erythritol production. An artificial neural network model and genetic algorithm were used to search the optimal composition of the significant factors and locate the resulting erythritol yield. Medium with 232.39 g/L crude glycerol, 1.57 g/L urea, and 31.03 g/L NaCl led to predictive maximum erythritol concentration of 110.7 g/L. The erythritol concentration improved from 50.4 g/L to 109.2 g/L with the optimized medium, which was reproducible. Erythritol fermentation kinetics were investigated in a batch system. Multistep fermentation kinetic models with hyperosmotic inhibitory effects were developed. The resulting mathematical equations provided a good description of temporal variations such as microbial growth (X), substrate consumption (S), and product formation (P) in erythritol fermentation. The accordingly derived model is the first reported model for fermentative erythritol production from glycerol, providing useful information to optimize the growth of Y. lipolytica and contributing visual description for the erythritol fermentation process under high osmotic pressure, as well as improvement of productivity and efficiency.

Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor.

Effective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6-1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13-28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.

A new polysialic acid production process based on dual-stage pH control and fed-batch fermentation for higher yield and resulting high molecular weight product.

To determine the factors influencing the resulting molecular weight of polysialic acid (PSA), batch fermentations by using Escherichia coli were conducted. It was found that temperature and pH were significant factors affecting the PSA production and its resulting molecular weight. When pH was set at 6.4, temperature of 37 °C was suitable for cell growth and PSA production while 33 °C facilitated production of higher molecular weight of PSA. pH 6.4 was favorable for PSA production while pH 7.4 was good for higher molecular weight of PSA at 37 °C. Intramolecular self-cleavage of PSA might lead to relatively low molecular weight under mild acidic condition. Our data suggest that the PSA molecular weight is significantly affected by the pH condition rather than the temperature. It is concluded that the resulting PSA molecular weight not only depends on fermentation conditions but also relates to cell growth rate and PSA production rate. Higher PSA molecular weight was made when its production rate was faster than degradation rate. A novel two-stage pH control fermentation process for production of high molecular weight PSA was developed. At the first stage, pH was set at 6.4 to encourage cell growth and PSA production, whereas pH was set at 7.4 at the second stage to promote the formation of higher molecular weight PSA. PSA yield up to 5.65 g/L and its resulting molecular weight of 260 kDa was attained, the highest level ever reported.

A new effective process for production of curdlan oligosaccharides based on alkali-neutralization treatment and acid hydrolysis of curdlan particles in water suspension.

Biologically active ß-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual ß-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes.

In Vitro Digestion and Fecal Fermentation of Low-Gluten Rice and Its Effect on the Gut Microbiota.

Low-gluten rice is part of a special diet for chronic kidney disease patients, but its digestive mechanism in the gastrointestinal tract is unclear. In this study, low-gluten rice (LGR), common rice (CR), and rice starch (RS) were used as experimental samples, and their digestion and bacterial fermentation were simulated using an in vitro gastrointestinal reactor to investigate the mechanism of the effect of LGR on human health. The starch digestibility of CR was higher than that of LGR, with statistically significant differences. LGR has growth-promoting and metabolic effects on Akkermansia muciniphila. Among the beneficial metabolites, the concentration of short-chain fatty acids (SCFAs) from LGR reached 104.85 mmol/L, an increase of 44.94% (versus RS) and 25.33% (versus CR). Moreover, the concentration of lactic acid reached 18.19 mmol/L, an increase of 60.55% (versus RS) and 25.28% (versus CR). Among the harmful metabolites, the concentration of branched-chain fatty acids (BCFAs) in LGR was 0.29 mmol/L and the concentration of ammonia was 2.60 mmol/L, which was 79.31% and 16.15% lower than CR, respectively. A significant increase in the concentration of the beneficial intestinal bacteria Bacteroides and Bifidobacterium occurred from LGR. The 16s rDNA sequencing showed that the abundance of the Bacteroidetes and Firmicutes increased and the abundance of the Proteobacteria and Fusobacteria decreased. Thus, LGR has positive effects on digestion and gut microbiota structure and metabolism in humans.

Recent advances in curdlan biosynthesis, biotechnological production, and applications.

Curdlan is a water-insoluble ß-(1,3)-glucan produced by Agrobacterium species under nitrogen-limited condition. Its heat-induced gelling properties render curdlan to be very useful in the food industry initially. Recent advances in the understanding of the role curdlan plays in both innate and adaptive immunity lead to its growing applications in biomedicine. Our review focuses on the recent advances on curdlan biosynthesis and the improvements of curdlan fermentation production both from our laboratory and many others as well as the latest advances on the new applications of curdlan and its derivatives particularly in their immunological functions in biomedicine.

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of Agrobacterium sp. ATCC 31749.

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45-60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD(+), NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20-50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.

Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy.

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03-0.08 h(-1) during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h(-1). With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.

Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift.

The production of porcine interferon-α (pIFN-α) by Pichia pastoris was largely enhanced when adopting sorbitol/methanol co-feeding induction strategy at 30 °C in a 10-L fermentor. Analysis of energy regeneration pattern and carbon metabolism revealed that major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle under the methanol/sorbitol co-feeding induction strategy. The sorbitol/methanol co-feeding induction strategy weakened formaldehyde dissimilatory pathway and repressed the accumulation of toxic metabolite-formaldehyde, reduced theoretical oxygen consumption rate and oxygen supply requirement, and increased energy/methanol utilization efficiency so that more methanol could be effectively used for pIFN-α synthesis. As a result, pIFN-α antiviral activity reached a highest level of 1.8 × 10(7) IU/mL which was about 10- to 200-folds of those obtained under pure methanol induction at 20 and 30 °C, respectively.

Identification and analysis of the metabolic functions of a high-salt-tolerant halophilic aromatic yeast Candida etchellsii for soy sauce production.

Salt-tolerant yeasts are very important for the flavor formation in soy sauce fermentation production. A halophilic aromatic yeast was isolated on the basis of the molecular biological and metabolic functions from soy sauce. The ITS nucleotide sequence alignment method was used to identify the strain as Candida etchellsii by subjecting the sequence to NCBI-BLAST in comparison with that of the C. etchellsii strain Miso 0208 (a typical high-salt-tolerant halophilic aromatic yeast strain). Organic acids, amino acids and volatile flavor compounds were produced by the yeast strain which were analyzed by HPLC and SPME-GC/MS methods. Tartaric acid (0.979 ± 0.040 g/l), formic acid (0.636 ± 0.030 g/l), lactic acid (2.80 ± 0.10 g/l), α-alkone glutaric acid (0.132 ± 0.015 g/l), citric acid (2.59 ± 0.10 g/l) and succinic acid (3.03 ± 0.20 g/l) were detected at 72 h of fermentation, respectively. Free and acid hydrolyzed amino acids at levels of 3.7355 ± 0.0027 and 11.5604 ± 0.0037 g/l, respectively, 4-ethyl guaiacols as well as other volatile flavor compounds were also detected.

Enhanced solubility of curcumin by complexation with fermented cyclic ß-1,2-glucans.

Curcumin (CUR) is a low-solubility polyphenolic compound with many physiological functions. Cyclic ß-1,2-glucans (cyclosophoraoses [Cys]), which contain rings of different sizes with degrees of polymerization ranging from 17 to 23, were obtained from Rhizobium radiobacter ATCC 1333, a soil microorganism. The complexation ability and solubility enhancement of cyclic ß-1,2-glucans with insoluble curcumin were investigated. Phase-solubility analysis revealed that the stoichiometric ratio of the inclusion complexes was 1:1. The stability constant of Cys was 930 M-1, which was 7.68 times that of α-cyclodextrin (α-CD) and 2.09 times that of ß-cyclodextrin (ß-CD). The characteristics of the curcumin/Cys inclusion complexes were successfully determined by using Fourier transform infrared (FTIR) spectrometry, differential scanning calorimetry (DSC), nuclear magnetic resonance (1H NMR) spectroscopy, and scanning electron microscopy (SEM). Moreover, a 1:1 molecular model of the curcumin/Cys inclusion complexes was established through molecular docking analysis. These findings indicated that cyclic ß-1,2-glucans successfully formed complexes with curcumin, which suggested that they could be used as solubility-increasing agents. To the best of our knowledge, this is the first report in which curcumin has been embedded into cyclic ß-1,2-glucans resulting in an increase in its aqueous solubility.

The effects of high-fat foods on gut microbiota and small molecule intestinal gases: release kinetics and distribution in vitro colon model.

Profiling intestinal gases and their responses to dietary changes can reveal the products and functions of the gut microbiota and their influence on human health. High-fat foods (HFF) can alter the gut microbiota and its metabolites, posing a potential health risk. However, little is known about the effects of HFF on intestinal gas distribution. Therefore, in this study, we used human fecal microorganisms as strains, an in vitro three-chamber colon model and an intestinal gas array sensor as tools. We performed in vitro fermentation using HFF as the fermentation substrate to reveal the effects of HFF on the kinetics of intestinal gas production and changes in the gut microbiota and its metabolites. We found that dietary fatty acids stimulated the production of H2S and volatile organic compounds in the colon, promoted Firmicutes abundance, and decreased Bacteroidetes abundance. These results highlight the potential role of HFF in altering the gut microbiota and intestinal gas, which can lead to health hazards.

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Changes of curdlan biosynthesis and nitrogenous compounds utilization characterized in ntrC mutant of Agrobacterium sp. ATCC 31749.

The regulatory function of global regulator NtrC on curdlan biosynthesis and nitrogen consumption under nitrogen-limited condition in Agrobacterium sp. ATCC 31749 was investigated. The ntrC mutant of Agrobacterium sp. was constructed by homologous recombination. The ability to utilize NH4Cl and KNO3 was impaired in the mutant. Other nitrogenous compounds, such as glutamic acid and glutamine, were utilized normally. Curdlan production capability was impaired severely in the mutant. Curdlan production was 5-fold lower than the wild type strain in batch fermentation with NH4Cl as the sole nitrogen source. However, up to 6.5 g l(-1) of a newly found alkali-insoluble biopolymer was produced by the ntrC mutant when glutamic acid was used as nitrogen source. The new biopolymer had glycosidic bond and hydroxyl group but no ß-configuration absorption peak on IR spectrum was found as different from curdlan. In addition, the mutant exhibited a rapid morphological change from the dot to rod form. These results deduced that the global regulator NtrC was involved in curdlan and other biopolymer biosynthesis in Agrobacterium sp. ATCC 31749 in response to nitrogen-limited condition.

In vitro digestion and fecal fermentation of highly resistant starch rice and its effect on the gut microbiota.

Highly resistant starch rice (HRSR) is of particular interest in terms of its capacity to deliver short-chain fatty acids (SCFAs) to the colon in the prevention of diabetes mellitus and obesity. In this study, HRSR was processed into cooked rice, rice milk, rice cake, and rice popcorn, and the in vitro digestion and fermentation processes were monitored. The results showed that the starch digestibility of the four samples conformed to a first-order two-phase equation, and the resistant starch content of rice cake was the highest (11.98%). Compared with inulin, rice cake had a slower fermentation rate, and the butyrate concentration increased by 67.85%. The abundances of Prevotellaceae, which promotes the synthesis of SCFAs, and anti-inflammatory Faecalibacterium increased. The abundances of Proteobacteria and Megamonas, markers of gut microbiota imbalance, decreased. The results might facilitate the design and production of functional food products for type 2 diabetic and obese patients and improving colonic health.

Improvement of the CuZn-superoxide dismutase enzyme activity and stability as a therapeutic agent by modification with polysialic acids.

The optimal process for the polysialylation reaction was as follows: polysialicacid (PSA) was activated by periodate oxidation, then coupled to CuZn superoxide dismutase (SOD) with a PSA:SOD molar ratio of 40:1 for 24 h. The resulting polysialylated protein contained 3.9 ± 0.3 mol PSA per mol SOD. SDS-PAGE and atomic force microscopy revealed that the molecular weight of polysialylated SOD was about 90-100 kDa. The average size was 10-15 nm, about four-fold of the native enzyme. Compared to the native enzyme, the activity and stability of the polysialylated SOD, as well as resistance to heat, acid, alkali and proteases present in human digestive system such as pepsin and trypsin, were improved significantly as therapeutic agent.