Biotechnological production of ectoine: current status and prospects.

Ectoine is an important natural secondary metabolite in halophilic microorganisms. It protects cells against environmental stressors, such as salinity, freezing, drying, and high temperatures. Ectoine is widely used in medical, cosmetic, and other industries. Due to the commercial market demand of ectoine, halophilic microorganisms are the primary method for producing ectoine, which is produced using the industrial fermentation process "bacterial milking." The method has some limitations, such as the high salt concentration fermentation, which is highly corrosive to the equipment, and this also increases the difficulty of downstream purification and causes high production costs. The ectoine synthesis gene cluster has been successfully heterologously expressed in industrial microorganisms, and the yield of ectoine was significantly increased and the cost was reduced. This review aims to summarize and update microbial production of ectoine using different microorganisms, environments, and metabolic engineering and fermentation strategies and provides important reference for the development and application of ectoine.

Rapid quantification of polysaccharide and the main onosaccharides in Dendrobium huoshanense by near-infrared attenuated total reflectance spectroscopy.

A rapid, green, low cost and nondestructive attenuated total reflection near infrared (ATR NIR) method was developed to quantify the total polysaccharide and the main monosaccharides mannose and glucose in Dendrobium huoshanense. Total 100 D. huoshanense samples from different places were analyzed using ATR NIR method. Potential outlying samples were initially removed from the collected NIR data using the PCA-Mahalanobis distance method. Spectral data preprocessing was studied in the construction of a partial least squares (PLS) model and six different signal pretreatment methods, including multiplicative scattering correction (MSC), standard normal transformation (SNV), first and second derivatives, the combination of MSC with the first derivative, and the combination of SNV with the first derivative, were compared. The results showed that the best signal pretreatment method was the spectral data pretreated by SNV combined with the first derivative due to it showed the lowest root-mean-square error of cross-validation (RMSECV), highest R2 for both the polysaccharide and its main monosaccharides. In order to improve the performance of the model, the pretreated full spectrum was calculated by different wavelength selection method. The results showed that the optional wavelength selection model was the one simultaneously selecting the NIR wavelength ranges 7500-5750 cm-1, 5250-4700 cm-1, 4450-4300 cm-1 and 4200-4100 cm-1 because of the lowest RMSECV and the highest R2 among the ten wavelength selection models. The external validation and the complete external validation confirmed the robustness and reliability of the developed NIR model. The contents of the total polysaccharide and the main monosaccharides are the essential quality assessment criterion for plant medicines while their traditional quantification methods involved sample destruction, tedious sample processing and non-environmentally friendly pretreatment, therefore, our study might provide an efficient technique tool for the rapid, green and nondestructive quantification of the total polysaccharide and the main monosaccharides for D. huoshanense and other rich-in-polysaccharide plant medicines.

Effects of Hyriopsis cumingii Polysaccharides on Mice Immunologic Receptor, Transcription Factor, and Cytokine.

To discuss the molecular mechanism of immunoenhancing activities of Hyriopsis cumingii polysaccharides (HCPS), effects of HCPS on mice immunologic receptors (toll-like receptors-4 [TLR-4] and mannose receptor-1 [MR-1]), transcription factor (nuclear factor kappa-B [NF-κB]), and cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were evaluated by cell model in vitro and cyclophosphamide-induced immunosuppression animal model in vivo. Results showed that HCPS could promote the mRNA synthesis of TLR-4, MR-1, IL-6, and TNF-α in spleen, and the gene expression of TLR-4, MR-1, NF-κB, IL-6, and TNF-α in spleen and serum in a dose-dependent manner. Crude HCPS and its purified fractions (HCPS-1, HCPS-2, and HCPS-3) could strengthen peritoneal macrophage expressing MR-1 and NF-κB in a dose-dependent manner. In addition, HCPS-3 showed stronger promotions on MR-1 and NF-κB than crude HCPS, HCPS-1, and HCPS-2. It suggested that HCPS-stimulated immunostrengthening was mediated, at least in part, by TLR-4/NF-κB/IL-6 and TLR-4/NF-κB/ TNF-α signaling pathways. MR-1, IL-6, and TNF-α might be 3 of the immune regulators mediating immunity and homeostasis when HCPS performed immunoenhancing activities.

Effects of Hyriopsis cumingii polysaccharides on angiogenesis, macrophage chemotaxis, proliferation and phagocytosis.

Anti-angiogenic activities of crude Hyriopsis cumingii polysaccharides (HCPS) and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in vivo using the chicken embryo chorioallantoic membrane (CAM) assay. The promoting effects of crude HCPS and its purified fractions on the chemotaxis, proliferation and phagocytosis of peritoneal macrophage were tested by cell model in vitro and cyclophosphamide-induced immuno-suppression animal model in vivo. The results showed that HCPS could significantly suppress the neovascularization of chicken embryo CAM and promote peritoneal macrophage migrating to monocyte chemotactic protein-1 (MCP-1), propagating and devouring sheep red blood cell (SRBC) in a dose-dependent manner. In addition, HCPS-3 showed stronger immunostimulatory activities in vitro than crude HCPS, HCPS-1 and HCPS-2. The beneficial effects of HCPS on the immune system might be, at least in part, attributed to the improvement of chemotaxis, proliferation and phagocytosis of peritoneal macrophage. All these results suggest that HCPS is a potential immunoenhancing and anti-tumor agent.

Immunostimulatory and antiangiogenic activities of low molecular weight hyaluronic acid.

The immunostimulatory activities of two low molecular weight hyaluronic acids (LMWHA-1 and LMWHA-2 with MW of 1.45×10(5) and 4.52×10(4) Da, respectively) and HA (MW, 1.05×10(6) Da) were evaluated by using in vitro cell models and in vivo animal models, and their effects on angiogenesis were measured in vivo by using the chick embryo chorioallantoic membrane (CAM) assay. The results demonstrated that LMWHA-1, LMWHA-2 and HA could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro in a dose-dependent manner. Furthermore, LMWHA-1 and LMWHA-2 exhibited much stronger immunostimulatory activity than HA. For assay in vivo, LMWHA-1 and LMWHA-2 significantly increased the indices of spleen and thymus, the activity of lysozyme in serum and the swelling rate of earlap in delayed-type hypersensitivity in a dose-dependent manner. In the CAM model, the results showed that LMWHA-1, LMWHA-2 and HA suppressed angiogenesis in chicken embryos. Moreover, LMWHA-1 exhibited higher antiangiogenesis activity than LMWHA-2 and HA. All these results suggested that LMWHA might be a potential natural immunomodulator and a potential candidate compound for antiangiogenic.

Preliminary characterization of proteoglycan from Hyriopsis cumingii.

OBJECTIVE: To study the preliminary characterizations of Hyriopsis cumingii proteoglycan (HCPG). METHODS: The content of carbohydrate and protein were measured by spectrophotometry. FTIR spectrum was used to analyze the functional groups. Relative molecular mass and amino acid composition were detected by HPLC. GC was utilized to determine the monosaccharide composition. The glycopeptide linkage-bond was detected by using the method of beta-elimination reaction. RESULTS: In HCPG,the content of carbohydrate and protein was 80.06% and 9.42%, respectively. FTIR spectrum showed the characteristic absorptions of polysaccharides and protein. Relative molecular mass of HCPG, determined by size-exclusive HPLC, was 503.1 kDa. GC spectra demonstrated that polysaccharide of HCPG was composed of rhamnose, fucose, mannose, glucose and galactose with a molar ratio of 13.80: 4.51: 7.70: 64.92 : 9.07. Fourteen amino acids (13 known and one unknown) have been detected by pre-column derivation HPLC. From beta-elimination reaction, peptide chain was attached to the carbohydrate chain by O-glycosidic bond. CONCLUSION: Basic characterizations of HCPG have been determined preliminarily.

Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus.

In the present study, crude capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 (CP) were purified by DEAE cellulose-52 column and Sephadex G-100 column chromatography to afford purified fractions of CP-1, CP-2 and CP-3. The relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. CP-1 was composed of mannose, glucose, arabinose and galactose in a percentage ratio of 66.15:28.97:2.43:2.45. CP-2 was composed of mannose, glucose, galactose and glucuronic acid in a percentage ratio of 40.94:27.71:5.96:25.39. In CP-1, there were pyranose rings, α-configuration glycosidic bond residues, one backbone chain (1→6 glycosidic bonds) and two branch chains (1→3 and 1→4 glycosidic bonds) in one repeating unit. In vitro immunostimulatory assay, it was found that CP could promote the splenocyte proliferation and increase the activity of acid phosphatase in peritoneal macrophages. The immunostimulatory activity of CP-1 might be related to its monosaccharide composition, molecular weight and α-configuration glycosidic bond.

Ultrasonic-assisted extraction and in vitro antioxidant activity of polysaccharides from Agaricus bisporus.

OBJECTIVE: To optimize ultrasonic-assisted extraction parameters of polysaccharides from Agaricus bisporus and evaluate antioxidant activities of A. bisporus polysaccharides. METHODS: Polysaccharides from A. bisporus was extracted by using methods of ultrasonic-assisted hot water lixiviation, ethanol precipitation, Sevag's deproteination and ethanol precipitation again. Extraction temperature, extraction time, ratio of water to raw material and ultrasonic power were selected in single-factor tests. Based on the single-factor tests, parameters combination for the ultrasonic-assisted extraction of A. bisporus polysaccharides was optimized by using four-factor-three-level orthogonal test. Antioxidant activities (reductive potential, superoxide anion scavenging activity and H2O2 scavenging activity) of A. bisporus polysaccharides were evaluated in vitro. RESULTS: Optimum conditions for the extracting of A. bisporus polysaccharides were extracting temperature 65 degrees C, extracting time 40 min, ratio of water to raw material 30 mL/g and ultrasonic power 170 w. Practicing this optimal condition, extraction yield of polysaccharides from A. bisporus was 5.6 014%. In crude polysaccharides of A. bisporus, carbohydrates content, determined by applying the phenol-sulfuric acid method, was 75.48%. Polysaccharides of A. bisporus could reduce ferric ion, scavenge superoxide anion and hydrogen peroxide in a dose-dependent manner. CONCLUSION: Utrasonic-assisted extraction could be used in the extracting of A. bisporus polysaccharides. Polysaccharides of A. bisporus, had direct and potent antioxidant activities, might be developed and utilized as natural antioxidant.

Antioxidant acitivity of low molecular weight hyaluronic acid.

Two polysaccharides, low molecular weight hyaluronic acid-1 (LMWHA-1) and LMWHA-2, with their molecular weight of 1.45×10(5) and 4.52×10(4)Da, respectively, were prepared from high molecular weight hyaluronic acid (HA,1.05×10(6)Da). LMWHA-1, LMWHA-2 and HA were studied for their antioxidant activities. In vitro antioxidant assay, LMWHA showed strong inhibition of lipid peroxidation and scavenging activities of hydroxyl radical, moderate 1,1-diphenyl-2-picryldydrazyl radical and superoxide anion scavenging activity. In addition, the LMWHA-1 exhibited much stronger antioxidant activity than LMWHA-2 and HA. For antioxidant testing in vivo, LMWHA-1, LMWHA-2 and HA were orally administrated over a period of 7days in a cyclophosphamide(CY) induced immunosuppressed mice model. As results, administration of LMWHA was able to overcome CY-induced immunosuppression and significantly raised the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (TAOC) in immunosuppressed mice. The results showed that the LMWHA, possessing pronounced free radical scavenging and antioxidant activities.

Immunostimulatory activity of the polysaccharides from Hyriopsis cumingii.

The immunostimulatory activity of Hyriopsis cumingii polysaccharides (HCPS) was evaluated by using in vitro cell models and in vivo animal models. The results demonstrated that crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro in a dose-dependent manner. Furthermore, HCPS-3 exhibited much stronger immunostimulatory activity in vitro than crude HCPS, HCPS-1 and HCPS-2, probably due to the higher sulfate content of HCPS-3. For assay in vivo, crude HCPS significantly increased the indices of spleen and thymus, the activity of lysozyme in serum and the swelling rate of earlap in delayed-type hypersensitivity in a dose-dependent manner. These results suggested that HCPS might be a potential natural immunomodulator.