Alginate oligosaccharide-mediated butyrate-HIF-1α axis improves skin aging in mice.

The "gut-skin" axis has been proved and is considered as a novel therapy for the prevention of skin aging. The antioxidant efficacy of oligomannonic acid (MAOS) make it an intriguing target for use to improve skin aging. The present study further explored whereby MAOS-mediated gut-skin axis balance prevented skin aging in mice. The data indicated the skin aging phenotypes, oxidative stress, skin mitochondrial dysfunction, and intestinal dysbiosis (especially the butyrate and HIF-1α levels decreased) in aging mice. Similarly, fecal microbiota transplantation (FMT) from aging mice rebuild the aging-like phenotypes. Further, we demonstrated MAOS-mediated colonic butyrate-HIF-1α axis homeostasis promoted the entry of butyrate into the skin, upregulated mitophagy level and ultimately improving skin aging via HDAC3/PHD/HIF-1α/mitophagy loop in skin of mice. Overall, our study offered a better insights of the effectiveness of alginate oligosaccharides (AOS), promised to become a personalized targeted therapeutic agents, on gut-skin axis disorder inducing skin aging.

Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

High-Level Extracellular Production of a Trisaccharide-Producing Alginate Lyase AlyC7 in Escherichia coli and Its Agricultural Application.

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 µg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.

Alginate Oligosaccharide Alleviates Lipopolysaccharide-Induced Apoptosis and Inflammatory Response of Rumen Epithelial Cells through NF-κB Signaling Pathway.

AOS alleviates inflammatory responses; however, whether it exerts an effect on the rumen or regulates rumen inflammatory reaction remains unknown. In this study, firstly, the ovine ruminal epithelial cells (ORECs) were treated with 0, 200, 400, 600, and 800 µg/mL AOS, hoping to explore whether AOS hurt cell health. The results showed that compared with the AOS-0 group, the AOS-400 group could significantly increase (p < 0.05) cell viability, reduce (p < 0.05) reactive oxygen species (ROS) and interleukin (IL)-6 content, and have no adverse effect on cells. Secondly, we used LPS to construct an in vitro inflammatory model of rumen epithelial cells and then explored the protective role of AOS on rumen epithelial cells. The study was divided into three groups: the control group (CON), LPS, and LPS + AOS. The results demonstrated that the LPS + AOS group significantly increased the cell viability and reduced the ROS level in comparison with the LPS group (p < 0.05). Pretreatment with AOS also repressed (p < 0.05) the secretion of IL-1ß, IL-6, IL-8, and immunoglobulin (Ig)A from ORECs in the culture medium following LPS. In terms of tight junction (TJ) proteins, AOS treatment also significantly increased (p < 0.05) the zonula occludens 1 (ZO-1) and Occludin expression. The apoptosis rate, Caspase3, Caspase9, BAD, and BCL-2/BAX were decreased (p < 0.05) after AOS treatment, and the expression of BCL-2 was increased (p < 0.05). In addition, the expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were inhibited (p < 0.05) with the addition of AOS. At the protein level, pretreatment of AOS decreased (p < 0.05) the expression of MyD88 and the phosphorylation level of inhibitor κB α (IκBα) after the LPS challenge. Taken together, our results indicated that AOS could alleviate the LPS-induced apoptosis and inflammatory response of rumen epithelial cells through the NF-κB signaling pathway, which may be a promising strategy for treating apoptosis and inflammation in sheep breeding.

Low-cost and efficient strategy for brown algal hydrolysis: Combination of alginate lyase and cellulase.

Brown algae are rich in biostimulants that not only stimulate the overall development and growth of plants but also have great beneficial effects on the whole soil-plant system. However, alginate, the major component of brown algae, is comparatively difficult to degrade. The cost of preparing alginate oligosaccharides (AOSs) is still too high to produce seaweed fertilizer. In this work, the marine bacterium Vibrio sp. B1Z05 is found to be capable of efficient alginate depolymerization and harbors an extended pathway for alginate metabolism. The B1Z05 extracellular cell-free supernatant exhibited great potential for AOS production at low cost, which, together with cellulase, can efficiently hydrolyze seaweed. The brown algal hydrolysis rates were significantly greater than those of the commercial alginate lyase product CE201, and the obtained seaweed extracts were rich in phytohormones. This work provides a low-cost but efficient strategy for the sustainable production of desirable AOSs and seaweed fertilizer.

Alginate oligosaccharide alleviates vascular aging by upregulating glutathione peroxidase 7.

Alginate oligosaccharide (AOS) may delay aging by decreasing oxidative stress, but the effects on vascular aging remain unclear. Here, we evaluate the effect of AOS on vascular aging and investigate the underlying mechanisms. Twenty-month-old rats acted as the natural aging model in vivo. Senescence of human aortic vascular smooth muscle cells (HA-VSMCs) was induced in vitro using angiotensin II (AngII). The aging rats and senescent cells were treated with AOS, followed by assessment of aging makers, oxidative stress, and aging-induced vascular remodeling. AOS treatment alleviated vascular aging and HA-VSMC senescence and decreased the levels of oxidative stress and vascular remodeling-associated indicators. AOS upregulated the expression of glutathione peroxidase 7 (GPX7) in aging rats and GPX7 depletion disrupted the geroprotective effect of AOS. AOS increased the nuclear translocation of nuclear factor erythroid-2-related factor (Nrf2) protein, which interacts with GPX7 protein to induce its expression. In conclusion, AOS alleviates vascular aging and HA-VSMC senescence and reduces aging-related vascular remodeling via the GPX7 antioxidant pathway, which may provide new avenues for treating aging-associated diseases.

Alginate oligosaccharide extends the service lifespan by improving the sperm metabolome and gut microbiota in an aging Duroc boars model.

Introduction: Alginate oligosaccharide (AOS), as a natural non-toxic plant extract, has been paid more attention in recent years due to its strong antioxidant, anti-inflammatory, and even anti-cancer properties. However, the mechanism by which AOS affects animal reproductive performance is still unclear. Methods: The purpose of this study is to use multi-omics technology to analyze the effects of AOS in extending the service lifespan of aging boars. Results: The results showed that AOS can significantly improve the sperm motility (p < 0.05) and sperm validity rate (p < 0.001) of aging boars and significantly reduce the abnormal sperm rate (p < 0.01) by increasing the protein levels such as CatSper 8 and protein kinase A (PKA) for semen quality. At the same time, AOS significantly improved the testosterone content in the blood of boars (p < 0.01). AOS significantly improved fatty acids such as adrenic acid (p < 0.05) and antioxidants such as succinic acid (p < 0.05) in sperm metabolites, significantly reducing harmful substances such as dibutyl phthalate (p < 0.05), which has a negative effect on spermatogenesis. AOS can improve the composition of intestinal microbes, mainly increasing beneficial bacteria Enterobacter (p = 0.1262) and reducing harmful bacteria such as Streptococcus (p < 0.05), Prevotellaceae_UCG-001 (p < 0.05), and Prevotellaceae_NK3B31_group (p < 0.05). Meanwhile, short-chain fatty acids in feces such as acetic acid (p < 0.05) and butyric acid (p < 0.05) were significantly increased. Spearman correlation analysis showed that there was a close correlation among microorganisms, sperm metabolites, and sperm parameters. Discussion: Therefore, the data indicated that AOS improved the semen quality of older boars by improving the intestinal microbiota and sperm metabolome. AOS can be used as a feed additive to solve the problem of high elimination rate in large-scale boar studs.

Genome Sequencing-Based Mining and Characterization of a Novel Alginate Lyase from Vibrio alginolyticus S10 for Specific Production of Disaccharides.

Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties.

Characterization of bifunctional alginate lyase Aly644 and antimicrobial activity of enzymatic hydrolysates.

An alginate lyase gene aly644 encoding a member of polysaccharide lyase family 6 was obtained from a metagenome of Antarctic macroalgae-associated microbes. The gene was expressed heterologously in Escherichia coli, and the recombinant protein was purified using a Ni-NTA His Tag Kit. With sodium alginate as the substrate, recombinant Aly644 exhibited an optimum reaction temperature of 50°C and an optimum reaction pH of 7.0. The Vmax and Km values of Aly644 toward sodium alginate were 112.36 mg/mL·min and 16.75 mg/mL, respectively. Substrate specificity analysis showed that Aly644 was a bifunctional alginate lyase that hydrolyzed both polyguluronic acid and polymannuronic acid. The hydrolysis products of Aly644 with sodium alginate as the substrate were detected by thin-layer chromatography, and were mainly di- and trisaccharides. The oligosaccharides produced by degradation of sodium alginate by Aly644 inhibited the mycelial growth of the plant pathogens Phytophthora capsici and Fulvia fulva; the 50% maximal effective concentration (EC50) values were 297.45 and 452.89 mg/L, and the 90% maximal effective concentration (EC90) values were 1341.45 and 2693.83 mg/L, respectively. This highlights that Aly644 is a potential candidate enzyme for the industrial production of alginate oligosaccharides with low degree of polymerization. Enzyme-hydrolyzed alginate oligosaccharides could support the development of green agriculture as natural antimicrobial agents. KEY POINTS: • An alginate lyase was obtained from a metagenome of Antarctic macroalgae-associated microbes. • Aly644 is a bifunctional alginate lyase with excellent thermostability and pH stability. • The enzymatic hydrolysates of Aly644 directly inhibited Phytophthora capsici and Fulvia fulva.

Structure-Activity Relationships of Low Molecular Weight Alginate Oligosaccharide Therapy against Pseudomonas aeruginosa.

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

The effect of alginate oligosaccharides on intestine barrier function and Vibrio parahaemolyticus infections in the white shrimp Litopenaeus vannamei.

The intestine is a host-pathogen interaction site and improved intestinal barrier function help to prevent disease in shrimp. Alginate oligosaccharides (AOS) are derived from resourceful brown algae. The intestine protection properties of AOS were widely recognized, and their benefits in fish have been reported. Nevertheless, there are no reports on AOS in shrimp and other crustaceans. In the present work, we measured the effects of AOS on growth performance and disease resistance in the white shrimp Litopenaeus vannamei and investigated their effects on intestinal health. Shrimps with an initial weight of about 2 g were fed with diets supplemented with 0 (control), 0.07%, 0.2%, 0.6%, or 1.2% of AOS for 56 days and were sampled and challenged with Vibrio parahaemolyticus. Dietary AOS did not significantly influence weight gain or feed utilization (P > 0.05). However, AOS considerably decreased the seven-day cumulative mortality after the challenge at any dose (P < 0.05). Dietary AOS improved the intestinal structure, significantly boosted the intestinal villus height at 0.6% and 1.2% levels, and increased intestinal wall thickness by 0.2%, 0.6%, and 1.2%. The alkaline phosphatase and maltase activities were also increased, suggesting that AOS improved the intestinal condition. Redox homeostasis in intestinal was improved by AOS, as expressed by the enhanced total antioxidant capacity and decreased malonaldehyde content, partly due to the increased superoxide dismutase and catalase activities. Compared with the antioxidant system, AOS's stimulating effects on immunity were more significant. At any level, AOS significantly activated lysozyme activity, the expression of propo and two antimicrobial peptide genes (pen-3 and crusin). However, the lowest concentration of AOS did not stimulate the gene expression of all three assayed pattern recognition receptors (LGBP, Toll, and IMD), and only the highest concentration of AOS increased the expression of imd. These findings suggest that AOS are highly efficient immunostimulants, and various immune pathways in shrimp are differentially sensitive to AOS. Finally, our findings suggest that AOS significantly alter the gut microbiota and their relative abundance at the phylum, family, and genus levels. In conclusion, AOS significantly enhances disease resistance in L. vannamei, possibly attributed to improved intestinal development, increased intestinal immunity and altered microbiota. These findings could provide a basis for future studies on the practical use of AOS and its mechanisms of action.

Oligosaccharides increased both leaf biomass and steviol glycosides content of Stevia rebaudiana.

Steviol glycosides (SGs) are a variety of important natural sweeteners. They are 200-350 times sweeter than sucrose without calories. Currently, their production is still mainly dependent on extraction from Stevia rebaudiana Bertoni (stevia). Oligosaccharides are environmentally friendly elicitors that promote plant growth and accumulation of secondary metabolites. In the present study, different concentrations of chitosan oligosaccharides (COS) and alginate oligosaccharides (AOS) were applied to stevia to explore their effect on growth and SGs biosynthesis. It was found that both COS and AOS promoted biomass production by increasing the leaf number and photosynthetic efficiency, which may be related to the decreased content of abscisic acid. The content of SGs was significantly increased after 50 mg/L AOS treatment, which not only increased the contents of stevioside (STV) and rebaudioside A (Reb A) significantly, but some important minority glucosides, like Reb E, Reb D, and Reb M. The increased SGs contents were the combined effect of the higher expression of SGs biosynthesis related genes, including KAH, UGT74G1, UGT85C2, and UGT91D2. The geometry changes of stem induced by COS and AOS may help to increase the lodging resistance of stevia. Thus, COS and AOS can be used in the field planting of stevia to increase the yield of SGs for industrial purposes.

How Three Self-Secreted Biofilm Exopolysaccharides of Pseudomonas aeruginosa, Psl, Pel, and Alginate, Can Each Be Exploited for Antibiotic Adjuvant Effects in Cystic Fibrosis Lung Infection.

In cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a cause of increased morbidity and mortality, especially in patients for whom infection becomes chronic and there is reliance on long-term suppressive therapies. Current antimicrobials, though varied mechanistically and by mode of delivery, are inadequate not only due to their failure to eradicate infection but also because they do not halt the progression of lung function decline over time. One of the reasons for this failure is thought to be the biofilm mode of growth of P. aeruginosa, wherein self-secreted exopolysaccharides (EPSs) provide physical protection against antibiotics and an array of niches with resulting metabolic and phenotypic heterogeneity. The three biofilm-associated EPSs secreted by P. aeruginosa (alginate, Psl, and Pel) are each under investigation and are being exploited in ways that potentiate antibiotics. In this review, we describe the development and structure of P. aeruginosa biofilms before examining each EPS as a potential therapeutic target for combating pulmonary infection with P. aeruginosa in CF, with a particular focus on the current evidence for these emerging therapies and barriers to bringing these therapies into clinic.

Alginate oligosaccharide alleviates aging-related intestinal mucosal barrier dysfunction by blocking FGF1-mediated TLR4/NF-κB p65 pathway.

BACKGROUND: Alginate oligosaccharide (AOS) has been reported to exert a crucial role in maintaining the intestinal mucosal barrier (IMB) function. The current study aimed at ascertaining the protective effects of AOS on aging-induced IMB dysfunction and to elucidate the underlying molecular mechanisms. METHODS: An aging mouse model and a senescent NCM460 cell model were established using d-galactose. AOS was administered to aging mice and senescent cells, and IMB permeability, inflammatory response and tight junction proteins were assessed. In silico analysis was conducted to identify factors regulated by AOS. Using gain- and loss-of-function approaches, we evaluated the roles of FGF1, TLR4 and NF-κB p65 in the aging-induced IMB dysfunction and NCM460 cell senescence. RESULTS: AOS protected the IMB function of aging mice and NCM460 cells by reducing permeability and increasing tight junction proteins. In addition, AOS up-regulated FGF1, which blocked the TLR4/NF-κB p65 pathway, and identified as the mechanism responsible for the protective effect of AOS. CONCLUSION: AOS blocks the TLR4/NF-κB p65 pathway via inducing FGF1, ultimately reducing the risk of IMB dysfunction in aging mice. This study highlights the potential of AOS as a protective agent against aging-induced IMB disorder and provides insight into the underlying molecular mechanisms.

Releasing Bioactive Compounds from Brown Seaweed with Novel Cold-Adapted Alginate Lyase and Alcalase.

Seaweeds are considered to be third-generation renewable biomasses, the comprehensive utilization of which has drawn increasing attention in recent years. A novel cold-active alginate lyase (VfAly7) was identified from Vibrio fortis and biochemically characterized for brown seaweed utilization. The alginate lyase gene was high-level expressed in Pichia pastoris, with an enzyme yield of 560 U/mL and a protein content of 9.8 mg/mL by high-cell density fermentation. The recombinant enzyme was most active at 30 °C and pH 7.5, respectively. VfAly7 was a bifunctional alginate lyase with both poly-guluronate and poly-mannuronate hydrolysis activities. On the basis of VfAly7, a bioconversion strategy for the utilization of brown seaweed (Undaria pinnatifida) was developed. The obtained AOSs showed stronger prebiotic activity towards tested probiotics when compared to that of commercial fructooligosaccharides (FOSs), while the obtained protein hydrolysates displayed strong xanthine oxidase inhibitory activity with IC50 of 3.3 mg/mL. This study provided a novel alginate lyase tool as well as a biotransformation route for the utilization of seaweeds.

Alginate Oligosaccharides Enhance Autophagy to Rejuvenate H2O2-Induced Senescent IEC-6 Through the AMPK/mTOR Signaling Pathway.

Intestinal epithelial cellular senescence contributes to the physiological decline of intestine and induces age-associated intestinal diseases. Therefore, the intestine is a vital target to delay intestinal epithelial cellular senescence and extend healthy lifespan. Alginate oligosaccharides (AOSs) have a wide range of biological and pharmacological activities. However, there are no related reports of AOSs on intestinal epithelial cellular senescence. Our study aimed to investigate the effect of AOSs on hydrogen peroxide (H2O2)-induced senescent intestinal epithelial cells (IEC-6) and its antiaging mechanism. A senescent model was successfully constructed by H2O2 (200 µmol/L) treatment on IEC-6 for 4 hours. Different concentrations of AOSs (10, 50, 100 µg/mL) were used to intervene in H2O2-induced senescent IEC-6. The number of ß-galactosidase staining-positive cells was significantly reduced by AOS intervention. The expression levels of p21 and p16, known as the senescent biomarkers, were also decreased. In addition, AOSs alleviated oxidative stress by reducing reactive oxygen species and improving antioxidative ability. To understand how AOSs rejuvenate H2O2-induced senescent IEC-6, we detected the expression level of genes in autophagy process. The results indicated that AOSs restored the expression level of Beclin 1, Atg7, and LC3 to enhance autophagy process by activating activated protein kinase (AMPK) and inhibiting mammalian target of rapamycin in H2O2-induced senescent IEC-6. Compound C, an AMPK inhibitor, abolished the effect of AOSs on activating autophagy and rejuvenating senescent IEC-6. Altogether, our study suggests that AOS is a promising drug for delaying intestinal epithelial cellular senescence.

Effects of Alginate Oligosaccharide on Testosterone-Induced Benign Prostatic Hyperplasia in Orchiectomized Rats.

Benign prostatic hyperplasia (BPH) is an age-related disease of the urinary system that affects elderly men. Current treatments for BPH are associated with several adverse effects, thus highlighting the need for alternative agents. Alginate oligosaccharide (AOS), a water-soluble functional oligomer derived from brown algae, inhibits prostate cancer cell proliferation. However, the effects of AOS on BPH and the underlying molecular mechanisms remain unclear. Therefore, here, we aimed to investigate the therapeutic potential of AOS in BPH by using human benign prostatic epithelial cells (BPH-1) and a rat model of testosterone-induced BPH. Treatment with AOS inhibited in vitro and in vivo proliferation of prostatic epithelial cells and the testosterone-induced expression of androgen receptor (AR) and androgen-associated genes, such as those encoding 5α-reductase type 2 and prostate-specific antigen. Oral administration of AOS remarkably reduced the serum levels of dihydrotestosterone (DHT) and testosterone as well as the expression of proliferating cell nuclear antigen, inflammatory cytokines, and enzymes, which showed increased levels in prostatic tissues of rats with testosterone-induced BPH. Taken together, these data demonstrate that AOS suppresses testosterone-induced BPH in rats by downregulating AR and the expression of androgen-associated genes, supporting the hypothesis that AOS might be of potential use for the treatment of BPH.

Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

The preservable effects of ultrasound-assisted alginate oligosaccharide soaking on cooked crayfish subjected to Freeze-Thaw cycles.

To improve the quality of cooked and frozen crayfish after repeated freeze-thaw cycles, the effects of alginate oligosaccharide (1 %, w/v) with ultrasound-assisted (40 W, 3 min) soaking (AUS) on the physicochemical properties were investigated. The AUS samples improved water-holding capacity with 19.47 % higher than the untreated samples. Low-field nuclear magnetic resonance confirmed that mobile water (T22) in the samples after 5 times of freeze-thaw cycles was reduced by 13.02 % and 29.34 % with AUS and without treatment, correspondingly; and with AUS and without treatment, average size of the ice crystals was around 90.26 µm2 and 113.73 µm2, and average diameter of the ice crystals was 5.83 µm and 8.14 µm, respectively; furthermore, it enhanced the solubility and zeta potential, lowered the surface hydrophobicity, reduced the particle size, and maintained the secondary and tertiary structures of myofibrillar protein (MP) after repeated freeze-thawing. Gel electrophoresis revealed that the AUS treatment mitigated the denaturation of MPs. Scanning electron microscopy revealed that the AUS treatment preserved the structure of the tissue. These findings demonstrated that the AUS treatment could enhance the water retention and physicochemical properties of protein within aquatic meat products during temperature fluctuations..

Improving the thermostability of alginate lyase FlAlyA with high expression by computer-aided rational design for industrial preparation of alginate oligosaccharides.

FlAlyA, a PL7 alginate lyase with industrial potential, is widely applied in the preparation the alginate oligosaccharide because of its high activity of degradation the alginate. However, heat inactivation still limits the industrial application of FlAlyA. To further enhance its thermostability, a group of mutants were designed, according to evaluating the B-factor value and free energy change via computer-aided calculation. 25 single-point mutants and one double-points mutant were carried out by site-directed mutagenesis. The optimal two single-point mutants H176D and H71K showed 1.20 and 0.3°C increases in the values of T m, while 7.58 and 1.73 min increases in the values of half-life (t 1/2) at 50°C, respectively, compared with that of the wild-type enzyme. Interestingly, H71K exhibits the comprehensive improvement than WT, including expression level, thermal stability and specific activity. In addition, the mechanism of these two mutants is speculated by multiple sequence alignment, structural basis and molecular dynamics simulation, which is likely to be involved in the formation of new hydrogen bonds and decrease the SASA of the mutants. These results indicate that B-factor is an efficient approach to improves the thermostability of alginate lyase composed of ß-sheet unit. Furthermore, the highest yield of the mutant reached about 650 mg/L, which was nearly 36 times that of previous studies. The high expression, excellent activity and good thermal stability make FlAlyA a potential candidate for the industrial production of alginate oligosaccharides.