Enzymatic Yeast β-Glucan Assay Kit

Vaccine adjuvants are important for enhancing vaccine efficacy, and although aluminium salts (Alum) are the most used, their limited ability to induce specific immune responses has spurred the search for new adjuvants. However, many adjuvants fail during product development due to manufacturability, supply, stability, or safety concerns. This work hypothesizes that protein-free yeast glucans can be used as vaccine adjuvants due to their known immunostimulatory activity and high abundancy. Thus, high molecular weight glucans with over 99% purity, comprising 64-70% β-glucans and 29-35% α-glucans, were extracted from a wild-type yeast and an engineered yeast to produce a steviol glycoside. These glucans underwent carboxymethylation to enhance solubility. Both water-dispersible and particulate glucans were evaluated as adjuvants, either alone or in combination with Alum or squalene stable emulsion (SE), for a SARS-CoV-2 vaccine. The study demonstrated that glucans triggered a robust immune response and enhanced the effects of Alum and SE when used in combination, both in vitro and in vivo. Water-dispersible glucans combined with Alum, and particulate glucans combined with SE, increased the production of specific antibodies against SARS-CoV-2 spike protein and enhanced serum neutralization titers against SARS-CoV-2 pseudovirus. Furthermore, the results indicated that larger molecular weight glucans from engineered yeast exhibited stronger immunogenic activity in comparison to wild-type yeast glucans. In conclusion, appropriately formulated glucans have the potential to be scalable, low-cost vaccine adjuvants, potentially overcoming the limitations of current adjuvants.

Considering the differences in molecular structure and function, the effects of β-1,3-glucans from Euglena gracilis and β-1,3/1,6-glucans from Saccharomyces cerevisiae on immune and inflammatory activities in dogs were compared. Four diets were compared: control without β-glucans (CON), 0.15 mg/kg BW/day of β-1,3/1,6-glucans (Β-Y15), 0.15 mg/kg BW/day of β-1,3-glucans (Β-S15), and 0.30 mg/kg BW/day of β-1,3-glucans (Β-S30). Thirty-two healthy dogs (eight per diet) were organized in a block design. All animals were fed CON for a 42-day washout period and then sorted into one of four diets for 42 days. Blood and faeces were collected at the beginning and end of the food intake period and analysed for serum and faecal cytokines, ex vivo production of hydrogen peroxide (H₂O₂) and nitric oxide (NO), phagocytic activity of neutrophils and monocytes, C-reactive protein (CRP), ex vivo production of IgG, and faecal concentrations of IgA and calprotectin. Data were evaluated using analysis of covariance and compared using Tukey’s test (P<0.05). Dogs fed Β-Y15 showed higher serum IL-2 than dogs fed Β-S30 (P<0.05). A higher phagocytic index of monocytes was observed in dogs fed the B-S15 diet than in those fed the other diets, and a higher neutrophil phagocytic index was observed for B-S15 and B-Y15 than in dogs fed the CON diet (P<0.05). Monocytes from dogs fed B-S15 and B-S30 produced more NO and less H₂O₂ than those from the CON and B-Y15 groups (P<0.05). Despite in the reference value, CRP levels were higher in dogs fed B-S15 and B-S30 diets (P<0.05). β-1,3/1,6-glucan showed cell-mediated activation of the immune system, with increased serum IL-2 and neutrophil phagocytic index, whereas β-1,3-glucan acted on the immune system by increasing the ex vivo production of NO by monocytes, neutrophil phagocytic index, and serum CRP. Calprotectin and CRP levels did not support inflammation or other health issues related to β-glucan intake. In conclusion, both β-glucan sources modulated some immune and inflammatory parameters in dogs, however, different pathways have been suggested for the recognition and action of these molecules, reinforcing the necessity for further mechanistic studies, especially for E. gracilis β-1,3-glucan.

G-protein signaling is important for signal transduction, allowing various stimuli that are external to a cell to affect its internal molecules. In Aspergillus fumigatus, the roles of Gβ-like protein CpcB on growth, asexual development, drug sensitivity, and virulence in a mouse model have been previously reported. To gain a deeper insight into Aspergillus fumigatus sexual development, the ΔAfcpcB strain was generated using the supermater AFB62 strain and crossed with AFIR928. This cross yields a decreased number of cleistothecia, including few ascospores. The sexual reproductive organ-specific transcriptional analysis using RNAs from the cleistothecia (sexual fruiting bodies) indicated that the CpcB is essential for the completion of sexual development by regulating the transcription of sexual genes, such as veA, steA, and vosA. The ΔAfcpcB strain revealed increased resistance to oxidative stress by regulating genes for catalase, peroxiredoxin, and ergosterol biosynthesis. The ΔAfcpcB strain showed decreased uptake by alveolar macrophages in vitro, decreased sensitivity to Congo red, decreased expression of cell wall genes, and increased expression of the hydrophobin genes. Taken together, these findings indicate that AfCpcB plays important roles in sexual development, phagocytosis by alveolar macrophages, biosynthesis of the cell wall, and oxidative stress response.

Beta glucans are complex glucose polymers well known for their immune modulatory properties. Therefore they are used and advertised in dietary supplements. Unfortunately there is no standardized test system for quality control of such health-related foods. This approach combined wet chemical and enzyme-based quantification methods (e.g. aniline blue, Glucatell®) with a cytokine secretion assay as parameter for immune activation to resolve this problem and to establish a quality control system for β-glucan containing products and extracts. Commercially available pure β-glucans with different origin and structure were used in this study. None of the methods allowed an accurate β-glucan quantification. Most promising was the test kit K-EBHLG (Megazyme). However, cytokine secretion from whole blood was detectable under the influence of β-glucans, but there was no correlation with the quantification results using the commercially available kits. Therefore, the quality control of β-glucan containing products needs further efforts.

β-Glucan, isolated from the mushroom Pleurotus ostreatus, at a concentration of 0.4%, was used in the manufacture of reduced-fat white-brined cheese from sheep milk. Control reduced-fat cheese was also produced from the same milk without the addition of β-glucan. The resultant cheeses were examined for their physicochemical characteristics, color and textural properties, and level of proteolysis and lipolysis. Furthermore, cheeses were evaluated organoleptically. In general, there were no statistical differences in the physicochemical characteristics and proteolysis levels found between both cheeses. The addition of β-glucan improved textural properties, and the cheeses received favorable grades for all the organoleptic characteristics. There were no flavor defects (such as a bitter taste) described by the panellists in this study. Generally, the addition of β-glucan did not significantly affect total free fatty acid content; however, at 180 days of ripening and storage, cheeses with the addition of β-glucan had a higher (p < 0.05) content than cheeses without β-glucan. The major fatty acids were acetic acid and capric acid.

Three hulless barley varieties were grown under normal conditions during 2017/2018 and 2018/2019, to improve their agronomic yield, and to assess how the genotype influences β‐glucan contents, and its structural, thermal, rheological, and functional properties, as intended to be used in food applications. The extracted gums with hot water at 55°C and pH 8.0, showed contents from 5.75% to 6.41% (w/w), and concentrations from 68.55% to 79.29% of β‐glucan, with some starch and protein impurities. The results of the agronomic trail indicated the highly significant (P ≤ 0.01) influence of the genotype on all studied characteristics, and on the β‐glucan contents (0.28^**and 0.33^**) at both seasons. The morphology of the three gums was significantly different in the distribution and structure of networks. Peak intensities of the –OH and –CH groups and CH₂ stretching were higher and wider in Giza129 and Giza131. β‐Glucan networks melt from 71.5 to 87.18 °C, and Giza131 exhibited the highest thermal stability. The aqueous dispersions (1%) of β‐glucan gums exhibited a non‐Newtonian behavior, and Giza130 presented the highest significant (P ≤ 0.05) apparent viscosity (η) and foaming stability. Giza129 showed the highest significant water and fat binding capacities, whereas Giza131 showed the highest significant foaming capacity. β‐Glucan gums showed different potentials in food applications as fat replacers, stabilizers, thickeners, and foaming agents in food systems. This study suggests planting the proper barley variety in breeding and genetic improvement programs to supply the food industry with the expected β‐glucan content with consistent structural, thermal, rheological, and functional properties.

In industrial winery, the use of mixed starter cultures composed of Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers to enhance the sensory and complexity of the wine without compromising the quality. In this work, the oenological properties and enzymatic activities of 196 non-Saccharomyces yeasts, belonging to Hanseniaspora guilliermondii and Hanseniaspora uvarum species, were investigated. This screening has allowed the selection of the best non-Saccharomyces yeast strain and the use of vinification for white grape of Campania Vitis vinifera cv Fiano, in an industrial scale. The experimental fermentations were performed in four different batches: batch A (H. guilliermondii BF1 and S. cerevisiae 404 in sequential inoculum); batch B only inoculum of S. cerevisiae 404; batch C without inoculation (spontaneous fermentation); batch D with the inoculum of H. guilliermondii BF1 strain. The results of chemical and sensorial analyses have showed that the best wine comes from batch A. In conclusion, the use of H. guilliermondii BF1 with good oenological properties and a strong β-glucosidase activity allowed to improve the sensorial complexity of the wine. Selected non-Saccharomyces yeast could be applied profitably to winemaking to enhance the quality of the wine using new fermentation technologies, and could be used in industrial winery.

Colorectal cancer (CRC) is one of leading causes of mortality in western countries and novel treatment strategies are required. The medicinal application of mushrooms has been used in traditional medicine in many oriental countries. Polysaccharide-rich extracts obtained from certain medicinal mushroom species have shown antitumor effects in different experimental models. In the present study, we have developed polysaccharide-rich extracts from Trametes versicolor (TV) and Grifola frondosa (GF) fruit bodies. We aim to evaluate the anticancer effects of these polysaccharide-rich extracts in LoVo and HT-29 human colon cancer cells. The in vitro effects were determined by cytotoxicity assay, proliferation assay, wound healing assay and invasion assay. Moreover, the effect on anchorage independent-cell growth was also determined. Our results showed that TV and GF extracts did inhibit human colon cell proliferation and induce cytotoxicity. Furthermore, both fungal extracts significantly inhibited oncogenic potential, cell migration and invasion in colon cancer cells. In addition, extracts induce a more epithelial phenotype, observed by phase contrast images, together with an increase expression of the E-cadherin epithelial marker, detected by western-blotting analyses. Moreover, by using gelatin zymography assays, it was detected a decrease of MMP-2 enzyme activity, a crucial metalloproteinase important for the degradation of the extracellular matrix. Finally, the combination of the extracts with one the most clinical used agents for colorectal cancer, 5-fluorouracil, increases cell cytotoxicity. Taken together our results underscore a potential antitumor effect of polysaccharide-rich extracts obtained from TV and GF in human colon cancer cells lines. These finding may contribute to the reported health effects of fungal extracts.

Changes in cell wall structure of four strains of Sacccharomyces cerevisiae species (brewer’s, baker’s and probiotic yeast) after culturing on deproteinated potato juice water (DPJW) with diverse addition of glycerol and different pH were investigated. It allowed to select conditions intensifying biosynthesis of β(1,3)/(1,6)-glucan and mannoproteins of cell walls of tested strains. Yeast cell wall structural polysaccharides show biological activity and technological usability in food industry but also decide about therapeutic properties of yeast biomass. The highest increase in the thickness of walls (by about 100%) and β-glucan layer (by about 120%) was stated after cultivation of S. cerevisiae R9 brewer’s yeast in DPJW supplemented with 5 and 10% (w/v) of glycerol and pH 7.0 while S. cerevisiae var. boulardi PAN yeast synthesized by ab. 70% thicker β-glucan layer when the pH of growth medium was equal to 5.0. The cells of brewer’s yeast (S. cerevisiae R9), probiotic (S. cerevisiae CNCM 1-745) and baker’s (S. cerevisiae 102) intensified the ratio of mannoproteins in the structure of cell walls cultivated in mediums supplemented with above 15% of glycerol what point out the protective action of glycoprotein’s under osmotic stress conditions. The study confirms at the first time the possibility of using agro-industrial waste in biosynthesis of functional polysaccharides of S. cerevisiae cell wall. It could be an new advantage in production of yeast biomass with therapeutic properties or β-glucan preparation as a novel food ingredient.

The impact of bread fortification with β-glucans and with proteins/proteolytic enzymes from brewers’ spent yeast on physical characteristics was evaluated. β-Glucans extraction from spent yeast cell wall was optimized and the extract was incorporated on bread to obtain 2.02 g β-glucans/100 g flour, in order to comply with the European Food Safety Authority guidelines. Protein/proteolytic enzymes extract from spent yeast was added to bread at 60 U proteolytic activity/100 g flour. Both β-glucans rich and proteins/proteolytic enzymes extracts favoured browning of bread crust. However, breads with proteins/proteolytic enzymes addition presented lower specific volume, whereas the incorporation of β-glucans in bread lead to uniform pores that was also noticeble in terms of higher specific volume. Overall, the improvement of nutritional/health promoting properties is highlighted with β-glucan rich extract, not only due to bread β-glucan content but also for total dietary fibre content (39% increase). The improvement was less noticeable for proteins/proteolytic enzymes extract. Only a 6% increase in bread protein content was noted with the addition of this extract and higher protein content would most likely accentuate the negative impact on bread specific volume that in turn could impair consumer acceptance. Therefore, only β-glucan rich extract is a promising bread ingredient.

Powdered β-glucan extracted from brewer's yeast (Yestimun, Leiber GmbH, Bramsche, Germany) was incorporated into skimmed-milk yogurt at varying concentrations (0.2-0.8% wt/wt) to investigate its potential application as a thickener. The effect of β-glucan fortification on the nutritional profile, microstructure, physicochemical properties, and texture of freshly prepared yogurts was investigated. Sensory evaluation was also conducted and was correlated with instrumental analysis. The addition of Yestimun significantly reduced the fermentation time of the yogurt mix from 4 h to 3 h. Scanning electron microscopy revealed that β-glucan particles formed small spherical clusters within the yogurt matrix. The majority of the physicochemical properties (syneresis, viscosity, color, and titratable acidity) remained unaffected by the incorporation of Yestimun in the recipe. Textural properties showed a gradual increment with increasing β-glucan concentration. Hardness, total work done, adhesive force, and adhesiveness increased by 19.27, 23.3, 21.53, and 20.76%, respectively, when using the highest amount of Yestimun powder. Sensory analysis (n = 40) indicated that fortifying yogurt with Yestimun at 0.8% (wt/wt) concentration may affect overall acceptance ratings, which was attributed to adverse flavor and aftertaste effects. However, the overall liking score of the yogurt (5.0/9.0) shows potential for commercialization of the product.

Encapsulation of caffeine in different polysaccharide materials (β-glucan, resistant starch, and β-cyclodextrin) by freeze drying technique to form caffeine loaded microparticles and determine their morphological, structural, and steady release behavior in simulated gastrointestinal (GI) conditions was studied. Swelling index of β-glucan and resistant starch was observed highest at pH 3 whereas at pH 6.5 swelling index decreased significantly (p ≤ 0.05). The morphology of the particles was characterized using SEM. The peaks at 1700 cm⁻¹, 950 cm⁻¹ and 1300-1350 cm⁻¹ confirm the presence of caffeine in encapsulated wall materials as shown by FTIR. DSC analysis revealed decrease in peak melting temperature of caffeine loaded microparticles. The particle size distribution revealed largest size of 297.553 μm for resistant starch and smallest mean particle size of 95.17 μm corresponding to that of β-cyclodextrin whereas the highest encapsulation efficiency (96%) was observed in β-glucan. β-glucan showed maximum decline in the release of caffeine followed by resistant starch and β-cyclodextrin under mimicked stomach conditions whereas RS provided more slow release in intestinal conditions.