A Novel Cold-Adapted and High-Alkaline Alginate Lyase with Potential for Alginate Oligosaccharides Preparation.

Alginate oligosaccharides (AOs) prepared through enzymatic reaction by diverse alginate lyases under relatively controllable and moderate conditions possess versatile biological activities. But widely used commercial alginate lyases are still rather rare due to their poor properties (e.g., lower activity, worse thermostability, ion tolerance, etc.). In this work, the alginate lyase Alyw208, derived from Vibrio sp. W2, was expressed in Yarrowia lipolytica of food grade and characterized in order to obtain an enzyme with excellent properties adapted to industrial requirements. Alyw208 classified into the polysaccharide lyase (PL) 7 family showed maximum activity at 35 °C and pH 10.0, indicating its cold-adapted and high-alkaline properties. Furthermore, Alyw208 preserved over 70% of the relative activity within the range of 10-55 °C, with a broader temperature range for the activity compared to other alginate-degrading enzymes with cold adaptation. Recombinant Alyw208 was significantly activated with 1.5 M NaCl to around 2.1 times relative activity. In addition, the endolytic Alyw208 was polyG-preferred, but identified as a bifunctional alginate lyase that could degrade both polyM and polyG effectively, releasing AOs with degrees of polymerization (DPs) of 2-6 and alginate monomers as the final products (that is, DPs 1-6). Alyw208 has been suggested with favorable properties to be a potent candidate for biotechnological and industrial applications.

Improvement of functional characteristics of Hypophthalmichthys molitrix protein by modification with chitosan oligosaccharide.

In the food processing field, it is very often that fish proteins are denatured affecting the nutritional value of the product which is vital to be solved. By using appropriate sugar donors for glycosylation with protein, improving the stability and emulsification properties of fish proteins can be achieved. This research looks into the impacts of enzymatic chitosan oligosaccharide (CO) at various concentration (0.15%, 0.30%, 0.45%, 0.60%, w/v) upon the molecular makeup and function of silver carp myofibrillar protein (MP) in an attempt to comprehend the impact of electrostatic binding among MP as well as CO on protein conformation. Analysis was done on the impact of various CO concentrations upon MP's secondary structure, conformational changes, and functional characteristics. Twelve sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assays were implemented to monitor MP; Fourier transform infrared spectroscopy, endogenous fluorescence spectroscopy, and UV absorption spectra were carried out to investigate the influence of CO on MP; Particle size distribution, emulsifying activity index (EAI), solubility, turbidity, sulfhydryl content, carbonyl content, foaming capacity, surface hydrophobicity, emulsifying stability index (ESI), and foam persistence were all investigated. In addition, we used dynamic light scattering, scanning electron microscope, and atomic force microscope to analyze myosin (MO) and 0.60% CO-MO complex. The results demonstrated that CO and MP form complexes through hydrogen bonding and electrostatic interactions. CO modification not only delayed the oxidation of MP but also promoted MP to show better solubility, foaming, and foaming stability. In addition, CO modified myosin particle size decreased, reducing myosin's roughness and making myosin's structure more compact. To sum up, molecular interaction could change functional characteristics, and products with special properties could be developed after modification with chitosan oligosaccharide.

Solid state fermentation of mung beans by Bacillus subtilis subsp. natto on static, shaking flask and soft elastic tubular reactors.

Given that mung beans constitute a significant nutrient source in many cultures, it is worthwhile to investigate ways to improve their nutritional and functional properties. The effect of fermentation of mung beans by Bacillus subtilis subsp. natto was investigated in various reactor designs, including static, shaking flasks, and soft elastic tubular reactors (SETR). The results showed that all three processes might affect the substrate, resulting in changes in the protein and carbohydrate fractions. We noticed an increase in soluble protein and serine levels, which we attribute to the proteases produced during fermentation. Through XRD, FTIR, and DSC analyses, it was also discovered that whereas static and shaking flask fermentation might raise relative crystallinity and peak temperature, fermentation performed on the SETR decreased these values. It was also possible to notice that SETR might induce a change in the particle size distribution of the substrate through a complex impact of mechanical forces, mixing, and microbial activity, which could be helpful to some aspects of the process. To summarize, fermentation of mung beans by Bacillus. subtilis subsp. natto could be an attractive approach for producing a food ingredient with various functional and nutritional properties. Furthermore, the SETR has been shown to be a viable technique for dealing with high solid load substrates, whether as the reactor for the entire process or as a first stage/pre-treatment step, and its applicability in bioprocesses should be explored further.

Changes in Myosin from Silver Carp (Hypophthalmichthys molitrix) under Microwave-Assisted Water Bath Heating on a Multiscale.

To further prove the advantages of microwave-assisted water bath heating (MWH) in low-value fish processing, the effects of different heating methods (two heating stage method, high temperature section respectively using MWH1, MWH2, MWH3, WH-water heating, MH-microwave heating) on secondary and tertiary myosin structures, SDS-PAGE, surface morphology, scanning electron microscopy (SEM), and particle size distribution were compared and analyzed. The findings revealed that MH and MWH aided in the production of gel formations by promoting myosin aggregation. Myosin from silver carps demonstrated enhanced sulfhydryl group and surface hydrophobicity after MWH treatment, as well as a dense network structure. The distribution of micropores becomes more uniform when the microwave time is increased. Actually, the total effect of microwave time on myosin is not substantially different. The correlation between particle size distribution and protein aggregation was also studied, in terms of time savings, the MWH of short microwave action is preferable since it not only promotes myosin aggregation but also avoids the drawbacks of a rapid warming rate. These discoveries give a theoretical foundation for understanding silver carp myosin under microwave modification, which is critical in the food industry.

Secretory Expression of an Alkaline Alginate Lyase With Heat Recovery Property in Yarrowia lipolytica.

Alginate lyase possesses wide application prospects for the degradation of brown algae and preparation of alginate oligosaccharides, and its degradation products display a variety of biological activities. Although many enzymes of this type have been reported, alginate lyases with unique properties are still relatively rare. In the present work, an alginate lyase abbreviated as Alyw203 has been cloned from Vibrio sp. W2 and expressed in food-grade Yarrowia lipolytica. The Alyw203 gene consists of an open reading frame (ORF) of 1,566 bp containing 521 amino acids, of which the first 17 amino acids are considered signal peptides, corresponding to secretory features. The peak activity of the current enzyme appears at 45°C with a molecular weight of approximately 57.0 kDa. Interestingly, Alyw203 exhibits unique heat recovery performance, returning above 90% of its initial activity in the subsequent incubation for 20 min at 10°C, which is conducive to the recovery of current enzymes at low-temperature conditions. Meanwhile, the highest activity is obtained under alkaline conditions of pH 10.0, showing outstanding pH stability. Additionally, as an alginate lyase independent of NaCl and resistant to metal ions, Alyw203 is highly active in various ionic environments. Moreover, the hydrolyzates of present enzymes are mainly concentrated in the oligosaccharides of DP1-DP2, displaying perfect product specificity. The alkali suitability, heat recovery performance, and high oligosaccharide yield of Alyw203 make it a potential candidate for industrial production of the monosaccharide and disaccharide.

The efficient enrichment of marine peptides from the protein hydrolysate of the marine worm Urechis unicinctus by using mesoporous materials MCM-41, SBA-15 and CMK-3.

Peptides found in marine life have various specific activities due to their special growth environment, and there is increasing interest in the isolation and concentration of these biofunctional compounds. In this study, the protein hydrolysate of the marine worm Urechis unicinctus was prepared by enzymolysis and enriched by using mesoporous materials of silica MCM-41 and SBA-15 and carbon CMK-3. The differences in pore structures and elemental composition of these materials lead to differences in surface area and hydrophobicity. The adsorption capacities of peptides were 459.5 mg g-1, 431.3 mg g-1, and 626.3 mg g-1 for MCM-41, SBA-15 and CMK-3, respectively. Adsorption kinetics studies showed that the pseudo-second-order model fit the adsorption process better, where both external mass transfer and intraparticle diffusion affected the adsorption, while the Langmuir model better fit the adsorption of peptides on MCM-41 and SBA-15 and the Freundlich model was more suitable for CMK-3. Aqueous acetonitrile (ACN, 50/50, v/v) yielded the most extracted peptides. MALDI-TOF mass spectrometry of the extracted peptides showed that the three mesoporous materials, especially the CMK-3, gave good enrichment results. This study demonstrates the great potential of mesoporous materials in the enrichment of marine biofunctional peptides.

Gastric protective activities of fucoidan from brown alga Kjellmaniella crassifolia through the NF-κB signaling pathway.

Fucoidan has been reported to have abundant biological activities. The objective of the present study was to detect the protective effects of fucoidan from Kjellmaniella crassifolia (KF) newly cultured in Dalian, North of China on aspirin-induced gastric ulcers of the Wistar rat model. The present study showed that inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 were effectively regulated in rats pretreated with KF. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities increased significantly in the KF pretreated groups, while the levels of maleic dialdehyde (MDA) decreased. The findings obtained by RT-PCR and western blotting indicated that KF could suppress aspirin-induced NF-κB activation via stabilization of IκB-α and thereby induced the downregulation of COX-2 and iNOS. It was demonstrated that KF exerted positive gastric protective effects via suppression of the inflammatory response and oxidative stress, and the mechanism of KF appeared to mediate the NF-κB signaling pathway.

Effects of a combined fucoidan and traditional Chinese medicine formula on hyperglycaemia and diabetic nephropathy in a type II diabetes mellitus rat model.

In this study, we innovatively propose a fucoidan mixed with traditional Chinese medicine formula (FCM) and evaluate its effects on hyperglycaemia and diabetic nephropathy in a type II diabetes mellitus Wistar rat model. After treatment with FCM for 8 weeks, the blood glucose, insulin resistance, serum lipid and antioxidant stress levels were significantly decreased (P < 0.05 or P < 0.01, vs. negative group). Via gene expression analysis, we found that three genes (InsR, GCK and GLUT-2) in the glucose metabolism pathway were significantly increased (P < 0.01, vs. negative group) in the FCM-treated groups and play important roles in hypoglycaemic activity. Moreover, FCM treatments alleviated (P < 0.01, vs. negative group) the urine protein, urine creatinine and pathological changes in the kidneys, producing significant improvements in renal function and structure. In summary, FCM exerts protective effects in diabetic rats and could be used in medicinal treatment for diabetes mellitus and its complications.

Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides.

Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from Saccharomyces cerevisiae, ScCDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. ScCDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that ScCDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. ScCDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore ScCDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

In vitro and in vivo immunomodulatory effects of fucoidan compound agents.

Fucoidan extracted from brown algae displays diverse biological activities. In the present study, fucoidan was mixed with Chinese herb extracts, and the in vitro and in vivo immunomodulatory effects of two fucoidan compound agents were evaluated. The results showed that fucoidan from Kjellmaniella crassifolia (KF) and Undaria pinnatifida (UF) were sulfated polysaccharides, Astragalus polysaccharide (AP) was composed of α-d-glucose, and Codonopsis pilosula polysaccharide (CPP) was a furanose. Furthermore, fucoidan compound agents stimulated mouse macrophage RAW264.7 cell proliferation and enhanced the secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α) in vitro. In addition, KCA (KF + AP + CPP) and UCA (UF + AP + CPP) could improve the nonspecific immunity and the specific immunity of BALB/c mice. Fucoidan compound agents also increased the secretion of GM-CSF, TNF-α, interleukin (IL)-4 and IL-10 in vivo. Therefore, we confirmed that fucoidan compound agents have promise for development as supplementary immunopotentiators.

Studies on the hepatoprotective effect of fucoidans from brown algae Kjellmaniella crassifolia.

For illustrating the relation of different structural characteristics correlated with the hepatoprotective effect, studies of fucoidan from algae Kjellmaniella crassifolia, a brown alga distributed in north Japan, were carried out. The fucoidan fractions of K. crassifolia were extracted, separated, and purified using a combinatorial procedure consisting of enzymolysis, ethanol precipitation, DEAE and size-exclusion chromatographies. The fundamental characteristics of the four enriched fucoidan fractions (KF1-KF4), including their sulphate content and monosaccharide composition, were investigated. The fucose was the main composition of monosaccharide for KF1-KF4, that of KF4 was up to 91.4%. The Glu-UA was the special composition of monosaccharide. FTIR and NMR spectroscopy were employed to elucidate the structural features of all fractions which further illustrated that fucose was the main monosaccharide. It was found all the four fractions showed antioxidative activity against hydroxyl radical and had the bioactive effects on CCl4-induced liver injury.

Development of shelf-stable, ready-to-eat (RTE) shrimps ( Litopenaeus vannamei ) using water activity lowering agent by response surface methodology.

Three water-activity-lowering agents (composite phosphate, sorbitol and glycerol) were used to develop a kind of shelf-stable, ready-to-eat (RTE) shrimp. Formula of water-activity- lowering agents was optimized by response surface methodology (RSM) using a central composite design. Model equation was proposed with regard to the contents of composite phosphate (X1), sorbitol (X 2) and glycerol (X3) : [Formula: see text]. The model with a very low probability value (P < 0.0003) was highly significant and the value of lack-of-fit was 0.4028, indicating that the model could predict water activity of shrimps using different agents. Composite phosphate of 0.22%, sorbitol of 3.12% and glycerol of 2.51% were found to be the optimal condition to obtain the lowest water activity of 0.884. Compared to the control shrimps, RTE shrimps treated with water-activity-lowering agents had a longer shelflife and higher sensorial acceptability. During storage at temperature of 35 °C, the quality of RTE shrimps in term of appearance, flavor and texture was found to be superior to the untreated ones. Texture profile, TBARS value, contents of astaxanthin and free amino acid of treated samples were found to be decreased slower from origin value compared to that of untreated samples. These RTE shrimps were biologically safe and sensorially acceptable after 30 days of storage at temperature of 35 °C. Briefly, the application of water-activity-lowering agents extent the shelflife of RTE shrimps obviously and would be beneficial for the exploitation of white shrimp.