Pustulan

Scheme 1-Method for Extracting Pustulan

Fermentation: The strain Byssochlamys spectabilis SWP35 (preserved as CGMCC NO.11602) is cultured from seeds and then fermented in a fermentation tank at 27°C with an aeration rate of 0.8 vvm for 70 hours to obtain the fermentation broth.

Sterilization: Mix sodium alginate, potassium alginate, and ammonium alginate in any proportion to create a mixed alginate. Prepare a 30 g/L (w/v) alginate solution and mix it with the fermentation broth at a 1:9 (v/v) ratio, stirring thoroughly. Then, mix calcium chloride, calcium lactate, and calcium citrate in any proportion to create a mixed calcium salt. Prepare a 300 g/L (w/v) calcium salt solution and mix it with the fermentation broth at a 1:9 (v/v) ratio, stirring again to form flocculent precipitates. Adjust the pH to 5 with a base solution, heat and stir, and then maintain at 60°C for 0.5 hours. The mixture will separate into solid and liquid phases. Remove the biomass and insoluble materials by centrifugation to obtain the first filtrate and recover the biomass.

Protein Removal: Adjust the pH of the first filtrate to 5 with a base solution, then filter it through a filter press equipped with diatomaceous earth (filter aid #10) to obtain the second filtrate.

Ultrafiltration and De-salting: Filter the second filtrate using a 3 kDa molecular weight cut-off ultrafiltration membrane. Circulate purified water to remove salts and other impurities, resulting in a polysaccharide solution with a weight-average molecular weight of 10 kDa.

Evaporation and Concentration: Concentrate the polysaccharide solution (Pustulan) obtained in step 4 by evaporation to achieve a concentration of 10%. The condensate from the evaporation process is recycled as pure water for future use.

In-situ Sterilization: Transfer the concentrated Pustulan solution from step 5 to a sterilization tank for in-situ sterilization at 115°C for 30 minutes.

Filling: Fill the sterilized high-concentration Pustulan solution into sterile containers to obtain the liquid product.

Case Study 1-Pustulan Enhances MHCII Presentation in Avian Vaccines

Infectious bronchitis virus (IBV) is a highly contagious avian coronavirus that causes significant economic losses in the poultry industry worldwide. Consequently, the use of live-attenuated viral vaccines is crucial for vaccination. However, live-attenuated vaccines have the potential for reversion to virulence and recombination with virulent field strains. Therefore, alternative approaches, such as subunit vaccines, are needed, along with suitable adjuvants, as subunit vaccines are less immunogenic than live-attenuated vaccines. In vitro assessments were conducted on several glycan-based adjuvants that directly target mammalian C-type lectin receptors. The β-1-6-glucan, pustulan, induced up-regulation of MHC class II (MHCII) cell surface expression, enhanced a strong proinflammatory cytokine response, and increased endocytosis in a cation-dependent manner. In ex vivo co-cultures of peripheral blood monocytes from IBV-immunized chickens and BM-DCs pulsed with pustulan-adjuvanted recombinant IBV N protein (rN), a strong recall response was induced. Compared to rN, pustulan, or media alone, pustulan-adjuvanted rN induced a significantly higher percentage of CD4+ blast cells. However, the percentages of CD8+ and TCRγδ+ blast cells were significantly lower with pustulan-adjuvanted rN compared to pustulan or media. Thus, pustulan enhanced the efficacy of MHCII antigen presentation but did not appear to enhance cross-presentation on MHCI. In conclusion, the study found an immunopotentiating effect of pustulan in vitro using chicken BM-DCs. Future in vivo studies may demonstrate that pustulan is a promising glycan-based adjuvant for use in the poultry industry to control the spread of coronaviruses and other avian viral pathogens.

Notes: BM-DCs were pulsed overnight with media, recombinant IBV N protein (rN) (20 µg/mL), pustulan (5 µg/mL), or rN (20 µg/mL) + pustulan (5 µg/mL). Pulsed BM-DCs were washed and co-cultured for 72 hours in flat-bottom 96-well plates with PBMCs isolated from the heparin-stabilized blood of IBV-vaccinated MHC-matched chickens (n = 6). The blast transformations of CD4+, CD8+, and TCRγδ+ T cells were analyzed by flow cytometry. Results are presented as violin plots with 95% confidence intervals showing individual blast percentage values from the technical replicates (n = 2/treatment) and biological replicates (n = 6/treatment). The data were log-transformed, and a two-tailed Student’s t-test with Bonferroni multiple comparison correction was employed. A p-value ≤ 0.05 was considered statistically significant, and differences between treatments are indicated by bars. Significance levels are indicated by asterisks: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (Larsen FT., 2020)

Case Study 2-GMSusD: A Pustulan-Binding Protein from Gramella sp. and Its Role in Ocean Glycan Cycling

Marine bacteria degrade carbohydrate polymers from algae, which are synthesized by algae in ocean surface waters. Although algal glycans are a rich carbon and energy source in the ocean, the molecular details of the specific recognition between algal glycans and bacterial degraders remain largely unknown. This study characterizes a surface protein, GMSusD, from the planktonic Bacteroidetes Gramella sp. MAR_2010_102, which is active during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides, such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan-binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We searched global surface water metagenomes for the occurrence of SusD-like proteins and found sequences with these three structurally conserved residues in different oceanic locations. The molecular selectivity of GMSusD highlights the necessity of specific interactions for laminarin recognition. In summary, our findings provide insights into the molecular details of β-glucan binding by GMSusD, and bioinformatics analysis suggests that this molecular interaction may contribute to glucan cycling in the surface ocean.

Notes: Phylogenetic analysis of predicted laminarin binding proteins and conservation of binding site residues of GMSusD among homologs from marine bacteria. (Mystkowska AA.,2018)

Pustulan has a wide range of applications across various fields. Its unique physical and chemical properties provide significant advantages in the food, pharmaceutical, agricultural, and other industrial sectors.

Food industry: Pustulan is used as a coating agent and thickening agent in candies, chocolate coatings, film sheets, composite seasonings, and fruit and vegetable juice beverages. It provides a smooth, refreshing sensation and improves taste. Adding a small amount can significantly enhance food quality, such as boosting the flavor of fish cakes, preserving the aroma of tofu, stabilizing the viscosity of seasonings and pickles, and increasing the thickness and smoothness of sweetened fish and shrimp dishes.

Filler: Used as a filler in the preparation of pharmaceuticals and cosmetics, it helps control the texture, volume, and consistency of the product. It can increase the volume of tablets or capsules, making them easier to handle and consume, while maintaining the stability of the active ingredients.

Binder: In pharmaceutical formulations and cosmetics, it acts as a binder to help combine different components into a stable solid form. It improves the mechanical strength and integrity of the product, such as enhancing the binding force in tablets and ensuring the stability and efficacy of the product during use.

• Show All
• Q&As (0)
• Reviews (0)