Identification, expression, and characterization of a marine-derived chitinase Ce0303 from Chitiniphilus eburneus YS-30 with exo- and endo-hydrolytic properties.

N-acetyl-oligosaccharides exhibit antioxidant and antibacterial activities. However, the low catalytic efficiency of chitinase on crystalline chitin hinders the eco-friendly production of N-acetyl-oligosaccharides. A marine-derived chitinase-producing strain Chitiniphilus eburneus YS-30 was screened in this study. The genome of C. eburneus YS-30 spans 4,522,240 bp, with a G + C content of 63.96 % and 4244 coding genes. Among the chitinases secreted by C. eburneus YS-30, Ce0303 showed the highest content at 19.10 %, with a molecular weight of 73.5 kDa. Recombinant Ce0303 exhibited optimal activity at 50 °C and pH 5.0, maintaining stability across pH 4.0-10.0. Ce0303 demonstrated strict substrate specificity, with a specific activity toward colloidal chitin of 6.41 U mg-1, Km of 2.34 mg mL-1, and kcat of 3.27 s-1. The specific activity of Ce0303 toward α-chitin was 18.87 % of its activity on colloidal chitin. Ce0303 displayed both exo- and endo-hydrolytic properties, primarily producing (GlcNAc)1-3 from colloidal chitin. The structure of Ce0303 includes one catalytic domain and two chitin-binding domains. Docking results revealed that the GlcNAc at -1 subsite formed two hydrogen bonds with conserved Trp380. The hydrolytic properties of Ce0303 will provide technical support for the comprehensive utilization of crustacean raw materials.

A Recombinant Alginate Lyase Algt1 with Potential in Preparing Alginate Oligosaccharides at High-Concentration Substrate.

Alginate lyase has been demonstrated as an efficient tool in the preparation of functional oligosaccharides (AOS) from alginate. The high viscosity resulting from the high concentration of alginate poses a limiting factor affecting enzymatic hydrolysis, particularly in the preparation of the fragments with low degrees of polymerization (DP). Herein, a PL7 family alginate lyase Algt from Microbulbifer thermotolerans DSM 19189 was developed and expressed in Pichia pastoris. The recombinant alginate lyase Algt1 was constructed by adopting the structural domain truncation strategy, and the enzymatic activity towards the alginate was improved from 53.9 U/mg to 212.86 U/mg compared to Algt. Algt1 was stable when incubated at 40 °C for 90 min, remaining with approximately 80.9% of initial activity. The analyses of thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC), and electrospray ionization mass spectrometry (ESI-MS) demonstrated that the DP of the minimum identifiable substrate of Algt1 was five, and the main hydrolysis products were AOS with DP 1-4. Additionally, 1-L the enzymatic hydrolysis system demonstrated that Algt1 exhibited an effective degradation at alginate concentrations of up to 20%, with the resulting products of monosaccharides (14.02%), disaccharides (21.10%), trisaccharides (37.08%), and tetrasaccharides (27.80%). These superior properties of Algt1 make it possible to efficiently generate functional AOS with low DP in industrial processing.

Realization process of microalgal biorefinery: The optional approach toward carbon net-zero emission.

Increasing carbon dioxide (CO2) emission has already become a dire threat to the human race and Earth's ecology. Microalgae are recommended to be engineered as CO2 fixers in biorefinery, which play crucial roles in responding climate change and accelerating the transition to a sustainable future. This review sorted through each segment of microalgal biorefinery to explore the potential for its practical implementation and commercialization, offering valuable insights into research trends and identifies challenges that needed to be addressed in the development process. Firstly, the known mechanisms of microalgal photosynthetic CO2 fixation and the approaches for strain improvement were summarized. The significance of process regulation for strengthening fixation efficiency and augmenting competitiveness was emphasized, with a specific focus on CO2 and light optimization strategies. Thereafter, the massive potential of microalgal refineries for various bioresource production was discussed in detail, and the integration with contaminant reclamation was mentioned for economic and ecological benefits. Subsequently, economic and environmental impacts of microalgal biorefinery were evaluated via life cycle assessment (LCA) and techno-economic analysis (TEA) to lit up commercial feasibility. Finally, the current obstacles and future perspectives were discussed objectively to offer an impartial reference for future researchers and investors.

Potential Application of Marine Fucosyl-Polysaccharides in Regulating Blood Glucose and Hyperglycemic Complications.

Diabetes mellitus (DM) has become the world's third major disease after tumors and cardiovascular disease. With the exploitation of marine biological resources, the efficacy of using polysaccharides isolated from marine organisms in blood glucose regulation has received widespread attention. Some marine polysaccharides can reduce blood glucose by inhibiting digestive enzyme activity, eliminating insulin resistance, and regulating gut microbiota. These polysaccharides are mainly fucose-containing sulphated polysaccharides from algae and sea cucumbers. It follows that the hypoglycemic activity of marine fucosyl-polysaccharides is closely related to their structure, such as their sulfate group, monosaccharide composition, molecular weight and glycosidic bond type. However, the structure of marine fucosyl-polysaccharides and the mechanism of their hypoglycemic activity are not yet clear. Therefore, this review comprehensively covers the effects of marine fucosyl-polysaccharides sources, mechanisms and the structure-activity relationship on hypoglycemic activity. Moreover, the potential regulatory effects of fucosyl-polysaccharides on vascular complications caused by hyperglycemia are also summarized in this review. This review provides rationales for the activity study of marine fucosyl-polysaccharides and new insights into the high-value utilization of marine biological resources.

Response of Salmonella enterica serovar Typhimurium to alginate oligosaccharides fermented with fecal inoculum: integrated transcriptomic and metabolomic analyses.

Alginate oligosaccharides (AOS), extracted from marine brown algae, are a common functional feed additive; however, it remains unclear whether they modulate the gut microbiota and microbial metabolites. The response of Salmonella enterica serovar Typhimurium, a common poultry pathogen, to AOS fermented with chicken fecal inocula was investigated using metabolomic and transcriptomic analyses. Single-strain cultivation tests showed that AOS did not directly inhibit the growth of S. Typhimurium. However, when AOS were fermented by chicken fecal microbiota, the supernatant of fermented AOS (F-AOS) exhibited remarkable antibacterial activity against S. Typhimurium, decreasing the abundance ratio of S. Typhimurium in the fecal microbiota from 18.94 to 2.94%. Transcriptomic analyses showed that the 855 differentially expressed genes induced by F-AOS were mainly enriched in porphyrin and chlorophyll metabolism, oxidative phosphorylation, and Salmonella infection-related pathways. RT-qPCR confirmed that F-AOS downregulated key genes involved in flagellar assembly and the type III secretory system of S. Typhimurium, indicating metabolites in F-AOS can influence the growth and metabolism of S. Typhimurium. Metabolomic analyses showed that 205 microbial metabolites were significantly altered in F-AOS. Among them, the increase in indolelactic acid and 3-indolepropionic acid levels were further confirmed using HPLC. This study provides a new perspective for the application of AOS as a feed additive against pathogenic intestinal bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00176-z.

Structural characterization of a novel fucosylated trisaccharide prepared from bacterial exopolysaccharides and evaluation of its prebiotic activity.

Fucosylated oligosaccharides have promising prospects in various fields. In this study, a fucosylated trisaccharide (GFG) was separated from the acidolysis products of exopolysaccharides from Clavibacter michiganensis M1. Structural characterization demonstrated that GFG consists of glucose, galactose, and fucose, with a molecular weight of 488 Da. Nuclear magnetic resonance analysis showed that it has a different structure than that of 2'-fucosyllactose (2'-FL), even though they have the same monosaccharide composition. In vitro prebiotic experiments were conducted to evaluate the differences in the utilization of three selected carbohydrates by fourteen bacterial strains. In comparison with 2'-FL, GFG could be utilized by more beneficial bacteria, leading to generate more short-chain fatty acids. Moreover, GFG could not promote the proliferation of Escherichia coli. This work describes a novel fucosylated oligosaccharide and its preparation method, and the obtained trisaccharide may serve as a promising candidate for fucosylated human milk oligosaccharides.

Genome analysis and 2'-fucosyllactose utilization characteristics of a new Akkermansia muciniphila strain isolated from mice feces.

Akkermansia muciniphila is considered to be a next-generation probiotic, and closely related to host metabolism and immune response. Compared with other probiotics, little is known about its genomic analysis. Therefore, further researches about isolating more A. muciniphila strains and exploring functional genes are needed. In the present study, a new strain isolated from mice feces was identified as A. muciniphila (MucX). Whole-genome sequencing and annotation revealed that MucX possesses key genes necessary for human milk oligosaccharides (HMO) utilization, including α-L-fucosidases, ß-galactosidases, exo-α-sialidases, and ß-acetylhexosaminidases. The complete metabolic pathways for γ-aminobutyric acid and squalene and genes encoding functional proteins, such as the outer membrane protein Amuc_1100, were annotated in the MucX genome. Comparative genome analysis was used to identify functional genes unique to MucX compared to six other A. muciniphila strains. Results showed MucX genome possesses unique genes, including sugar transporters and transferases. Single-strain incubation revealed faster utilization of 2'-fucosyllactose (2'-FL), galacto-oligosaccharides, and lactose by MucX than by A. muciniphila DSM 22959. This study isolated and identified an A. muciniphila strain that can utilize 2'-FL, and expolored the genes related to HMO utilization and special metabolites, which provided a theoretical basis for the further excavation of A. muciniphila function and the compound application with fucosylated oligosaccharides.

Complete-genome sequence and in vitro probiotic characteristics analysis of Bifidobacterium pseudolongum YY-26.

AIMS: The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential. METHODS AND RESULTS: Bifidobacterium pseudolongum YY-26 (CGMCC 24310) was isolated from faeces of mice treated with low-molecular-weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole-genome sequencing obtained using PacBio's single-molecular and Illumina's paired-end sequencing technology. A genome of 2.1 Mb in length, with 1877 predicted protein-coding sequences was obtained. Carbohydrate-Activity enZyme analysis revealed that YY-26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY-26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short-chain fatty acid production was observed with single YY-26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY-26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging. CONCLUSIONS: B. pseudolongum YY-26 was identified with numerous probiotic-associated genes and its probiotic characteristics were verified in vitro. SIGNIFICANCE AND IMPACT OF STUDY: This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY-26.

Fucose-containing bacterial exopolysaccharides: Sources, biological activities, and food applications.

Bacterial exopolysaccharides are high molecular weight polysaccharides that are secreted by a wide range of bacteria, with diverse structures and easy preparation. Fucose, fucose-containing oligosaccharides (FCOs), and fucose-containing polysaccharides (FCPs) have important applications in the food and medicine fields, including applications in products for removing Helicobacter pylori and infant formula powder. Fucose-containing bacterial exopolysaccharide (FcEPS) is a prospective source of fucose, FCOs, and FCPs. This review systematically summarizes the common sources and applications of FCPs and FCOs and the bacterial strains capable of producing FcEPS reported in recent years. The repeated-unit structures, synthesis pathways, and factors affecting the production of FcEPS are reviewed, as well as the degradation methods of FcEPS for preparing FCOs. Finally, the bioactivities of FcEPS, including anti-oxidant, prebiotic, anti-cancer, anti-inflammatory, anti-viral, and anti-microbial activities, are discussed and may serve as a reference strategy for further applications of FcEPS in the functional food and medicine industries.

A novel glucofucobiose with potential prebiotic activity prepared from the exopolysaccharides of Clavibacter michiganensis M1.

Fucose and fucosylated oligosaccharides have important applications in various industries owing to their prebiotic, anti-inflammatory, anticoagulant, and antiviral activities. Here, we aimed to obtain fucosylated oligosaccharides using the acidolysis method to depolymerize exopolysaccharides extracted from Clavibacter michiganensis M1. Based on structural analysis, the prepared glucofucobiose was found to consist of d-glucose and l-fucose, with a molecular weight of 326 Da and a structure of d-Glcp-ß-(1→4)-l-Fucp. The prebiotic activity of glucofucobiose was compared with that of 2'-fucosyllactose (2'-FL), the most abundant oligosaccharide in human milk. According to the results, glucofucobiose could significantly promote the proliferation of six probiotic strains, and short-chain fatty acid production of five probiotic strains on glucofucobiose was substantially higher than that on 2'-FL at 48 h of fermentation. Overall, this study proposed a new technology for obtaining fucosylated oligosaccharides. The prepared glucofucobiose was found to exhibit potential prebiotic activity and should be further assessed.

Genome sequence analysis of Cronobacter phage PF-CE2 and proposal of a new species in the genus Pseudotevenvirus.

The genome of a Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work, and a new species named "Cronobacter virus PF-CE2", in the genus Pseudotevenvirus of the subfamily Tevenvirinae of the family Myoviridae is proposed. The Gp190 gene of phage PF-CE2 is predicted to encode a bacteriophage-borne glycanase that is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Furthermore, we propose changing the taxonomic status of eight additional phages based on nucleotide sequence comparisons. This work provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.

Structural characterization of fucose-containing disaccharides prepared from exopolysaccharides of Enterobacter sakazakii.

Fucose-containing oligosaccharides (FCOs) have important applications in the food, medicine, and cosmetics industries owing to their unique biological activities. The degradation of microbial fucose-containing exopolysaccharide (FcEPS) is a promising strategy for obtaining FCOs, and bacteriophage-borne glycanase is a useful tool for degrading FcEPS. Here, we aimed to obtain FCOs using bacteriophage-borne glycanase to depolymerize FcEPS from Enterobacter sakazakii. The FcEPS was mainly composed of l-fucose (42.72 %), d-galactose (20.59 %), and d-glucose (21.81 %). Based on the results of nuclear magnetic resonance and mass spectrometry, the obtained FCOs were disaccharide fragments with backbones of ß-d-Glcp-(1→4)-ß-l-Fucp and α-d-Galp-(1→3)-ß-l-Fucp, respectively. So far, few studies of disaccharides prepared from FcEPS have been reported. This study demonstrated that the FcEPS of E. sakazakii was a reliable fucose-containing disaccharide source and that bacteriophage-borne glycanase was an effective degradation tool for obtaining FCOs fragments from FcEPS.

Dietary galactosyl and mannosyl carbohydrates: In-vitro assessment of prebiotic effects.

The prebiotic activities of hydrolyzed guar gum (GMOS, <1 kDa; GMPS, 1-10 kDa), manno-oligosaccharide (MOS, <1 kDa), and galacto-oligosaccharide (GOS, <1 kDa) were evaluated by in vitro fermentation. The tested carbohydrates showed selective prebiotic effects on bacterial growth, short-chain fatty acid (SCFA)-production, and substrate consumption. GOS and GMOS markedly promoted the growth of bifidobacteria and Clostridium butyricum, respectively, whereas MOS showed the strongest butyrogenic effect. Moreover, SCFA production in the hydrolyzed guar gum groups was closely related to the varied molecular weight (Mw) of the hydrolysate. During in vitro fermentation with human fecal inocula, GMOS gave the highest yields of lactate, propionate, and butyrate after 48 h fermentation. Combined application of MOS and C. butyricum increased the abundance of Clostridiaceae_1. Overall, our results indicate that galactosyl and mannosyl carbohydrates have individualized prebiotic effects which are associated with their chemical structures including their glycoside composition and Mw.

High-efficiency expression of a superior ß-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides.

ß-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg-1) and MEIR (9003.1 U mg-1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg-1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL-1). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.

Properties of hydrolyzed guar gum fermented in vitro with pig fecal inocula and its favorable impacts on microbiota.

In order to identify an appropriate substitute for antibiotic use in livestock production, this study investigates the fermentation of guar gum and its low molecular weight hydrolyzed derivatives (GMLP-1, 1-10 kDa; GMLP-2, < 1 kDa) in pig fecal cultures and the associated effects on the intestinal microbiota. Both the non-hydrolyzed guar gum and GMLP were quickly utilized by fecal microbiota. GMLP-2 showed the most rapid SCFA-producing activity and produced higher concentrations of lactate, acetate, and propionate. However, GMLP-1 showed the highest yield of total SCFAs and butyrate. Both the guar gum and GMLP groups improved the abundance of Clostridium sensu stricto 1 and Bifidobacterium, but the most significant enhancement was observed with GMLP-1. This study showed that by associating with its chemical structure, GMLP-1 can be utilized to direct a targeted promotion of the intestinal microbiota and may offer the most favorable effects in livestock production.

Enzymatic preparation of a low-molecular-weight polysaccharide rich in uronic acid from the seaweed Laminaria japonica and evaluation of its hypolipidemic effect in mice.

Here, we describe a method combining thermo-acid pretreatment and alginate lyase hydrolysis to prepare a low-molecular-weight polysaccharide from the seaweed Laminaria japonica (SP). The in vitro results showed that SP displayed obvious absorption of oil (2.95 g g-1) and cholesterol (21.87 g g-1 at pH 2.0). In addition, the in vivo assessment of SP-related anti-obesity effects in C57BL/6J mice fed a high-fat diet and treated with SP for 8 weeks revealed that SP significantly reduced weight gain and lipid accumulation in white adipose and liver tissues, improved serum lipid profiles, and ameliorated intestinal damage. Moreover, SP activated the AMP-activated protein kinase pathway in liver tissues, downregulated sterol regulatory element-binding protein and fatty acid synthase, and suppressed lipid synthesis. These findings indicated that SP extracted from L. japonica might represent a potent functional food exhibiting anti-obesity effects.

High-level expression of a thermophilic and acidophilic ß-mannanase from Aspergillus kawachii IFO 4308 with significant potential in mannooligosaccharide preparation.

An engineered thermophilic and acidophilic ß-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme ß-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80 °C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.

The research progress in mechanism and influence of biosorption between lactic acid bacteria and Pb(II): A review.

Currently, due to high surface to volume ratio; large availability, rapid kinetics of adsorption and desorption and low cost, the exploitation of microbial biosorption of heavy metals is regarded as a reliable alternative compared to the conventional bioremediation approaches. In parallel with the increasing attractiveness of biosorption research, its pace of advance is also boosted. The barrier that prevent biosorption as an effective method from being applied into wastewater purification is listed, (1) There is not enough data on multi-component biosorption, (2) It remains to be seen that physical-chemical characteristics of different biomasses. (3) Studies on surface modification of strains for enhancement of heavy metals removal efficiency is lack. And extensive literatures involving the mechanism and model of biosorption for particular metal and microbial strains are not available. The present literatures lack systematization, the theory on interaction between lactic acid bacteria and Pb is far from complete. Therefore, the review tries to give a comprehensive explanation about the mechanism of Pb removal from Lactic acid bacteria and provide a brief overview of distinction between biosorption and bioaccumulation, biosorption technology, highlight the underlying features of biosorption and the various affecting factors such as pH, dose required, initial concentration, temperature, and treatment performance as a reference. Biosorption mechanisms can be briefly generalized into several pathways, which are ion exchange, complexation, precipitation, reduction and chelation. Many views holds that complexation is the major absorption mechanisms of Pb. Biosorption mechanisms can be roughly classified as biosorption and bioaccumulation, which have great differences between each other. Biosorption is metabolism-independent but fast, while bioaccumulation is metabolism-dependent but slow. The slight advantages of the bioaccumulation are the metabolite (lactic acid), lactobacillus surface-layers, enzymes and so on. Many factors can greatly affect adsorption process, different factors have different influence and the effects of pretreatment, pH and temperature are relatively greater. Desorption is not a fully reversible process of biosorption, but could not only achieve the goal of the recycle of microorganism, but also contribute to release of trace metal elements. Also the technologies for observation of biosorbents characterics and effect on the metal binding process are reviewed.

Microbial Degradation of Pesticide Residues and an Emphasis on the Degradation of Cypermethrin and 3-phenoxy Benzoic Acid: A Review.

Nowadays, pesticides are widely used in preventing and controlling the diseases and pests of crop, but at the same time pesticide residues have brought serious harm to human's health and the environment. It is an important subject to study microbial degradation of pesticides in soil environment in the field of internationally environmental restoration science and technology. This paper summarized the microbial species in the environment, the study of herbicide and pesticides degrading bacteria and the mechanism and application of pesticide microbial degrading bacteria. Cypermethrin and other pyrethroid pesticides were used widely currently, while they were difficult to be degraded in the natural conditions, and an intermediate metabolite, 3-phenoxy benzoic acid would be produced in the degradation process, causing the secondary pollution of agricultural products and a series of problems. Taking it above as an example, the paper paid attention to the degradation process of microorganism under natural conditions and factors affecting the microbial degradation of pesticide. In addition, the developed trend of the research on microbial degradation of pesticide and some obvious problems that need further solution were put forward.

Study on the synthesis and physicochemical properties of starch acetate with low substitution under microwave assistance.

In this study, synthesis and physicochemical properties of starch acetate with low substitution under microwave were studied. A three-level-three-factorial Central Composite Design using Response Surface Methodology (RSM) was employed to optimize the reaction conditions. The optimal parameters are as follows: amount of acetic anhydride of 12%, radiation time of 11min, and microwave power of 100W. These optimal conditions predicted by RSM were confirmed that the degree of substitution (DS) of acetate starch is 0.0691mg/g and the physical and chemical properties of natural corn starch (NCS) and corn starch acetate (ACS) were further studied.The transparency, water separation, water absorption, expansion force, and solubility of ACS low substitution are better than NCS, while the NCS's hydrolysis percentage is higher than ACS, which indicate that the modified corn starch has better performance than native corn starch. The surface morphology of the corn starch acetate was examined by scanning electron microscope (SEM), which showed that it had a smooth surface and a spherical and polygonal shape. However, samples' shape is irregular. Crystal structure was observed by X-ray diffraction, and the ACS can determine the level of microwave technology that can destroy the extent of the crystal and amorphous regions. Fourier transform infrared (FTIR) spectroscopy shows that around 1750cm-1 carbonyl signal determines acetylation bonding successfully.