Optimization of welan gum extraction and purification using lysozyme and alkaline protease.

Welan gum, a natural polysaccharide produced by Sphingomonas sp. ATCC 31555, has attracted considerable attention in the scientific community due to its desirable properties. However, challenges, such as high viscosity, residual bacterial cells, carotenoids, and protein complexation, hinder the widespread application of welan gum. In this study, we established a method for the extraction and purification of welan gum using a synergistic approach with lysozyme and alkaline protease. Lysozyme hydrolysis conditions were optimized by applying response surface methodology, and the best results for bacterial cell removal were achieved at 11 000 U/g, 44 °C, and pH 9 after 3 h of treatment. Subsequently, we evaluated protein hydrolysis through computer simulation and identified alkaline protease as the most suitable enzyme. Through experimental investigations, we found that the optimal conditions for alkaline protease hydrolysis were 7500 U/g, 50 °C, pH 10, and 600 rpm. These conditions resulted in a sugar recovery rate of 76.1%, carotenoid removal rate of 89.5%, bacterial removal rate of 95.2%, and protein removal rate of 87.3% after 3 h of hydrolysis. The purified welan gum exhibited high transparency and purity. Structural characterization and antioxidant activity evaluation revealed that enzymatically purified welan gum has potential application prospects. Our study provides valuable insights into the optimal method for the enzymatic extraction and purification of welan gum. Such a method is conducive to the development of the multiple potential applications of welan gum. KEY POINTS: • A novel process for the synergistic purification of welan gum using lysozyme and alkaline protease was established. • In silico virtual digestion was employed to select the purification enzyme. • Welan gum with high transparency and purity was obtained.

Dietary sialic acids: distribution, structure, and functions.

Sialic acids (Sias), a group of over 50 structurally distinct acidic saccharides on the surface of all vertebrate cells, are neuraminic acid derivatives. They serve as glycan chain terminators in extracellular glycolipids and glycoproteins. In particular, Sias have significant implications in cell-to-cell as well as host-to-pathogen interactions and participate in various biological processes, including neurodevelopment, neurodegeneration, fertilization, and tumor migration. However, Sia is also present in some of our daily diets, particularly in conjugated form (sialoglycans), such as those in edible bird's nest, red meats, breast milk, bovine milk, and eggs. Among them, breast milk, especially colostrum, contains a high concentration of sialylated oligosaccharides. Numerous reviews have concentrated on the physiological function of Sia as a cellular component of the body and its relationship with the occurrence of diseases. However, the consumption of Sias through dietary sources exerts significant influence on human health, possibly by modulating the gut microbiota's composition and metabolism. In this review, we summarize the distribution, structure, and biological function of particular Sia-rich diets, including human milk, bovine milk, red meat, and egg.

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.

Malto-oligosaccharides as critical functional ingredient: a review of their properties, preparation, and versatile applications.

Malto-oligosaccharides (MOS) are α-1,4 glycosidic linked linear oligosaccharides of glucose, which have a diverse range of functional applications in the food, pharmaceutical, and other industries. They can be used to modify the physicochemical properties of foods thereby improving their quality attributes, or they can be included as prebiotics to improve their nutritional attributes. The degree of polymerization of MOS can be controlled by using specific enzymes, which means their functionality can be tuned for specific applications. In this article, we review the chemical structure, physicochemical properties, preparation, and functional applications of MOS in the food, health care, and other industries. Besides, we offer an overview for this saccharide from the perspective of prospect functional ingredient, which we feel lacks in the current literature. MOS could be expected to provide a novel promising substitute for functional oligosaccharides.

New high-density fermentation method for producing high molecular weight polysialic acid based on the combination fermentation strategy.

Polysialic acid (PSA) is a long-chain linear amino polysaccharide with broad application prospects; however, its relatively low molecular weight limits its application range. This study aims to explore a new fermentation method of combining the three-phase pH control strategy, three-phase mixing speed control strategy, and exogenous substance to produce high molecular weight PSA. In brief, Escherichia coli K235 6E61 (CCTCC M208088) was used as a fermentation strain. 3 g·L-1 Na5P3O10 was added to the initial medium. At 0-12 h, the mixing speed was controlled to 250 r·min-1, and the pH was maintained at 7.2. At 12-20 h, the mixing speed was increased to 400 r·min-1, the pH was changed to 6.8, and 0.75% n-hexadecane was added at hour 16. After 20 h, the mixing speed was adjusted to 250 r·min-1; the pH was restored to 7.2. Air flow was regulated to 1.2 vvm throughout the experiment. The combination fermentation strategy greatly improved the molecular weight of PSA up to 498 kDa at 32 h, which is currently the maximum molecular weight of PSA produced through microbial fermentation. The yield of PSA reached 6.27 g·L-1 at the end of fermentation (36 h), which is also currently the highest yield of PSA produced by natural bacteria. Therefore, the proposed strategy could simultaneously increase the molecular weight and yield of PSA and is of great importance to the industrial production of high molecular weight PSA. Key points • A new fermentation process was explored to produce high molecular weight PSA. • The yield and molecular weight were improved by the combination fermentation strategy. • The maximum molecular weight and highest yield of PSA were obtained.

Study of growth, metabolism, and morphology of Akkermansia muciniphila with an in vitro advanced bionic intestinal reactor.

BACKGROUND: As a kind of potential probiotic, Akkermansia muciniphila abundance in human body is directly causally related to obesity, diabetes, inflammation and abnormal metabolism. In this study, A. muciniphila dynamic cultures using five different media were implemented in an in vitro bionic intestinal reactor for the first time instead of the traditional static culture using brain heart infusion broth (BHI) or BHI + porcine mucin (BPM). RESULTS: The biomass under dynamic culture using BPM reached 1.92 g/L, which improved 44.36% compared with the value under static culture using BPM. The biomass under dynamic culture using human mucin (HM) further increased to the highest level of 2.89 g/L. Under dynamic culture using porcine mucin (PM) and HM, the main metabolites were short-chain fatty acids (acetic acid and butyric acid), while using other media, a considerable amount of branched-chain fatty acids (isobutyric and isovaleric acids) were produced. Under dynamic culture Using HM, the cell diameters reached 999 nm, and the outer membrane protein concentration reached the highest level of 26.26 µg/mg. CONCLUSIONS: This study provided a preliminary theoretical basis for the development of A. muciniphila as the next generation probiotic.

Synthesis of functional oligosaccharides and their derivatives through cocultivation and cellular NTP regeneration.

Carbohydrates play an important role in the life cycle. Among them, functional oligosaccharides show a complex and diverse structures with unique physiological activities and biological functions. However, different preparation methods directly affect the structure, molecular weight, and other functions of oligosaccharides, as well as their application fields and manufacturing costs. In the preparation of ß-1,3-glucan oligosaccharides (OBGs), water insolubility of ß-1,3-glucans hampers the hydrolysis efficiency. The synthesis of some functional oligosaccharides requires the consumption of energy substrates, such as ATP, CTP, and uridine triphosphate, for sugar nucleotide synthesis, leading to increased capital costs. A more economical solution to solve energy supply is to adopt microbial cocultivation or cellular nucleoside triphosphate regeneration. This review focused on the sources, preparation methods, biological activities of OBG, and the cultivation methods and applications of microbial cocultivation and fermentation. We also reviewed the preparation methods of other functional oligosaccharides, such as sialylated oligosaccharides, ß-nicotinamide mononucleotide, and α-galacto-oligosaccharides.

Metabolic profiles of oligosaccharides derived from four microbial polysaccharides by faecal inocula from type 2 diabetes patients.

In vitro digestion of curdlan oligosaccharides (COSs), pullulan oligosaccharides (POSs), xanthan gum oligosaccharides (XGOSs) and gellan gum oligosaccharides (GGOSs) was investigated. These four oligosaccharides showed resistance to simulated saliva and gastric and small intestinal fluid. In further fermentation with faecal microbiota from healthy subjects and type 2 diabetes (T2D) patients, COS fermentation significantly increased the abundance of Bifidobacterium spp. and Lactobacillus spp. and the production of short-chain fatty acids in healthy and T2D groups. Digestion of XGOS enhanced the growth of the Clostridium leptum subgroup and significantly increased butyric acid production in healthy and T2D groups. Sole fermentation with COS, POS, XGOS and GGOS exhibited different metabolic profiles between healthy and T2D groups, and more small molecule polyols were produced in the T2D group than in the healthy group. This study provides a novel perspective on the reconstruction of gut microbiota and metabolism by POS, COS, GGOS and XGOS intervention.

An Oligomannuronic Acid-Sialic Acid Conjugate Capable of Inhibiting Aß42 Aggregation and Alleviating the Inflammatory Response of BV-2 Microglia.

Oligomannuronic acid (MOS) from seaweed has antioxidant and anti-inflammatory activities. In this study, MOS was activated at the terminal to obtain three different graft complexes modified with sialic acid moiety (MOS-Sia). The results show that MOS-Sia addition can reduce the ß-structure formation of Aß42, and the binding effect of MOS-Sia3 is more obvious. MOS-Sia conjugates also have a better complexing effect with Ca2+ while reducing the formation of Aß42 oligomers in solutions. MOS-Sia3 (25-50 µg/mL) can effectively inhibit the activation state of BV-2 cells stimulated by Aß42, whereas a higher dose of MOS-Sia3 (>50 µg/mL) can inhibit the proliferation of BV-2 cells to a certain extent. A lower dose of MOS-Sia3 can also inhibit the expression of IL-1ß, IL-6, TNF-α, and other proinflammatory factors in BV-2 cells induced by Aß42 activation. In the future, the MOS-Sia3 conjugate can be used to treat Alzheimer's disease.

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.

Recycling of cooking oil fume condensate for the production of rhamnolipids by Pseudomonas aeruginosa WB505.

Rhamnolipids (RLs) are anionic biosurfactants with great application potential. This study explored the possibility of producing RLs from cooking oil fume condensates (COFCs) collected from range hoods. A mutant of Pseudomonas aeruginosa AB93066 was obtained and used to produce RLs from COFCs as a substrate. RL yields in a 7-L fermenter reached 12.3 g/L, and MALDI-TOF MS showed that Rha2-C10-C10 and Rha-C10-C10 are the most abundant (39.6% and 26.4%, respectively) RL components. The critical micellar concentration (CMC) of the RLs was 45.0 mg/L and the surface tension of water decreased from 60.5 to 25.3 mN/m. Using six kinds of common hydrocarbons as indices, the emulsification coefficients of the RLs obtained were found to exceed 60%; in particular, the emulsification coefficient for benzene was 80.3%. COFCs provide an inexpensive alternative as a substrate for RL production, and the synthetic process is relatively harmless and economical.

Bioproduction, purification, and application of polysialic acid.

Polysialic acid (PSA) is a negatively charged linear homopolymer linked by N-acetylneuraminic acid and widely present in vertebrates and some pathogens. PSA, commonly found on cell surfaces as glycoproteins and glycolipids, plays important roles in intercellular adhesion, cell migration, and formation and remodeling of the neural system by regulating the adhesive property of nerve cell adhesion molecules. PSA with a molecular weight that can reach as high as 260 kDa also belongs to the group II capsule polysaccharide of neonatal meningitis-causing Escherichia coli K1. To date, much effort has been devoted to developing the biotechnological production of PSA. As a non-glycosaminoglycan, PSA is a non-immunogenic and biodegradable polysaccharide that can be used as a biomaterial in protein polysialylation, tissue engineering, and drug delivery. PSA can also combine with other macromolecules to form multifunctional composites. In this mini-review, the production, purification, and application of PSA are summarized to provide a basis for further PSA applications.

Effective production of biologically active water-soluble ß-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum.

Water-soluble ß-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10 g L-1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0 g L-1 hr-1 and 600 rpm, respectively, and at the w-glucan production stage, they were 0.5 g L-1 hr-1 and 400 rpm, respectively. W-glucan production reached 17.31 g L-1, with a degree of polymerization of 19-25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.

Potential application of a low-viscosity and high-transparency xanthan gum produced from Xanthomonas campestris CCTCC M2015714 in foods.

Xanthan gum is commonly used as a thickener in food industry, while the usage of xanthan gum as a dietary fiber is restricted for its low additive volume. Herein, the potential use of a low-viscosity and high-transparency xanthan gum as a dietary fiber was evaluated in vitro. This new xanthan shows better transparency and faster dissolution rate than most commercial products, and its viscosity increases along with the treatment of freeze-thaw cycles at -20°C. Moreover, this new xanthan can absorb heavy metals (Pb, Cd, Cu) and retard starch digestion by glucoamylase. In summary, this new xanthan could be potentially used as a dietary fiber or fiber ingredient for preventing and treating diabetes, hyperlipemia, heavy metal poisoning, and cardiovascular diseases effectively.

Different nitrogen sources change the transcriptome of welan gum-producing strain Sphingomonas sp. ATCC 31555.

To reveal effects of different nitrogen sources on the expressions and functions of genes in Sphingomonas sp. ATCC 31555, it was cultivated in medium containing inorganic nitrogen (IN), organic nitrogen (ON), or inorganic-organic combined nitrogen (CN). Welan gum production and bacterial biomass were determined, and RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEGs) between the different ATCC 31555 groups were identified, and their functions were analyzed. Welan gum production and bacterial biomass were significantly higher in the ON and CN groups compared with those in the IN group. RNA-seq produced 660 unigenes, among which 488, 731, and 844 DEGs were identified between the IN vs. ON, IN vs. CN, and ON vs. CN groups, respectively. All the DEGs were related significantly to metabolic process and signal transduction. DEGs between the IN vs. CN and ON vs. CN groups were potentially associated with bacterial chemotaxis. Real-time PCR confirmed the expressions of selected DEGs. Organic nitrogen led to higher bacterial biomass and welan gum production than inorganic nitrogen, which might reflect differences in gene expression associated with metabolic process, signal transduction, and bacterial chemotaxis induced by different nitrogen sources.

Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic, degradation and transmembrane pathways.

OBJECTIVE: To develop a strategy for producing N-acetyl-D-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate. RESULT: An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l-1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l-1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l-1. CONCLUSION: A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.

Phosphoenolpyruvate-supply module in Escherichia coli improves N-acetyl-D-neuraminic acid biocatalysis.

OBJECTIVES: N-Acetyl-D-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1-9, 2016). RESULTS: PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l-1, compared with 3.6 ± 0.15 g l-1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l-1 in a 1 l fermenter. CONCLUSIONS: The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

Sol-gel encapsulation of pullulanase in the presence of hybrid magnetic (Fe3O4-chitosan) nanoparticles improves thermal and operational stability.

Pullulanase was sol-gel encapsulated in the presence of magnetic chitosan/Fe3O4 nanoparticles. The resulting immobilized pullulanase was characterized by scanning electron microscopy, vibrating sample magnetometry, Fourier transform infrared spectroscopy and thermogravimetric analysis. The results showed that the addition of pullulanase created a more regular surface on the sol-gel matrix and an enhanced magnetic response to an applied magnetic field. The maximal activity retention (83.9%) and specific activity (291.7 U/mg) of the immobilized pullulanase were observed under optimized conditions including an octyltriethoxysilane:tetraethoxysilane (OTES:TEOS) ratio of 1:2 and enzyme concentration of 0.484 mg/mL sol. The immobilized enzyme exhibited good thermal stability. When the temperature was above 60 °C, the immobilized pullulanase showed significantly higher activity than the free enzyme (p < 0.01); enzyme immobilized by simple sol-gel encapsulation and co-immobilized by crosslinking-encapsulation retained 52 and 69% of their initial activity after 5 h at 62 °C, respectively, compared to 11% for the free enzyme. Moreover, the stability of the pullulanase was improved by crosslinking-encapsulation, as the enzyme retained more than 85 and 81% of its original activity after 5 and 6 consecutive reuses, respectively, compared to 80 and 72% of its original activity for simple sol-gel encapsulated enzymes. This indicated the leakage of enzyme molecules through the pores of the gel was substantially abated by cross-linking. Such immobilized pullulanase provides high stability and ease of enzyme recovery, characteristics that are advantageous for applications in the food industry that involve continuous starch processing.

Production of rhamnolipids by semi-solid-state fermentation with Pseudomonas aeruginosa RG18 for heavy metal desorption.

Foaming problem and cost of substrate limit the commercial application of rhamnolipids, a potential biosurfactant produced by Pseudomonas aeruginosa. We explored the production of rhamnolipids by a semi-solid-state (SSS) fermentation strategy with glycerol as carbon source and rapeseed/wheat bran as matrix, along with the capacity of the produced rhamnolipids to solubilize lead and cadmium in aqueous solution. Structural analysis by MALDI-TOF MS indicated the increased proportion of mono-rhamnolipids from SSS fermentation. E24 results showed the stronger emulsification capacity and reduced water tension of the SSS fermentation product. Rhamnolipids from SSS fermentation can desorb lead/cadmium from contaminated soil effectively and heavy metals in exchangeable and carbonate forms were easily removed. Our findings suggest that SSS fermentation is an alternative for the economical production of rhamnolipids and the product can be used to solubilize heavy metals from soils.