Enzymatic Yeast β-Glucan Assay Kit

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.

Oat bran was fermented by filamentous fungi to improve the extractability of β-glucan. Box-Behnken experimental design and response surface methodology were used to obtain the maximum extractability of β-glucan. Inoculum volume, fermentation temperature, and time were evaluated as three variables. Under optimal fermentation conditions, the extractability of β-glucan in oat bran increased to 45.57 ± 1.82% and 51.10 ± 2.32% by Aspergillus niger and Rhizopus oryzae, respectively. The extractability was about 3-fold of that before fermentation (16.86 ± 0.76%). Fermentation of oat bran by Aspergillus niger and Rhizopus oryzae reduced the molecular weight of the extracted β-glucan from 6.74 × 10⁵ to 2.84 × 10⁵ and 2.20 × 10⁵ Da, respectively. Correspondingly, the apparent viscosity of the extracted β-glucan decreased after fermentation. But the increased extractability compensated for the reduced molecular weight in the formation of viscous solution. The molecular structure and cellotriosyl/cellotetraosyl ratio were not affected by filamentous fungi fermentation.

In this study, β-glucan was extracted from wholegrain oat and barley flours by a novel extraction and purification method employing natural flocculants (chitosan, guar gum and gelatin). The use of flocculants decreased the total amount of extracted gum, which was highest in control samples (9.07 and 7.9% for oat and barley, respectively). The β-glucan specific yield, however, increased with the use of chitosan and guar gum, which were able to remove protein and ash impurities resulting in gums with a higher purity.The highest concentration of chitosan (0.6 %) resulted in gums with the highest β-glucan content (82.0 ± 0.23 and 79.0 ± 0.19 for barley and oat, respectively) and highest β-glucan specific yield (96.9 and 93.3 % for oat and barley, respectively). Explanation is in R&D section. The use of gelatin was not successful. All gum samples had a high content of total dietary fiber (>74%) and a high water holding capacity (4.6–7.4 g/g), but differed in apparent viscosity, which was highest for the oat sample extracted with 0.6% chitosan. This sample also showed the highest β-glucan molecular weight among the oat samples, which were in general 10-fold higher than for the barley samples. Among the barley samples, β-glucan molecular weight was highest for the control.

Oral keratinocytes provide the first line of host defense against oral candidiasis. We speculated that interactions of fungal cell wall components with oral keratinocytes regulate stress response against Candida infection, and examined the expression of genes induced by heat-killed Candida albicans in oral immortalized keratinocytes using a cDNA microarray technique. Of 24,000 genes revealed by that analysis, we focused on HO-1, a stress-inducible gene, as its expression was increased by both heat-killed as well as live C. albicans. In histological findings, HO-1 expression in the superficial layers of oral epithelium following Candida infection was elevated as compared to healthy epithelium. We then investigated fungal cell wall components involved in induction of HO-1 expression and found that β-glucan-containing particles (β-GPs) increased tha expression. Furthermore, β-glucan was observed on the surface of both heat-killed C. albicans and Candida that has invaded oral epithelium. Fungal β-GPs also promoted induction of intracellular reactive oxygen species (ROS), NADPH oxidase activation, and p38 MAPK phosphorylation, while those specific inhibitors inhibited HO-1expression induced by fungal β-GPs. Moreover, fungal β-GPs induced Nrf2 translocation into nuclei via p38MAPK signaling, while HO-1 expression induced by fungal β-GPs was inhibited by Nrf2 siRNA. Finally, knockdown of cells by HO-1 and Nrf2 siRNAs resulted in increased β-GPs-mediated ROS production as compared to the control cells.

β-Glucan is a fibrous polysaccharide and good source of soluble dietary fiber having multiple functional and medicinal properties. Functionality of β-glucan extracts was investigated followed by studying the characteristics of cakes containing 2.5-7.5% of this extracts. Yield, purity, foaming ability, DPPH activity, WBC and SP β-glucan extracts of germinated millets improved due to germination. The in vitro antioxidant activity of cake increased from 18.23 to 22.58% with increase in β-glucan level from 2.5 to 7.5%. The volume index of cake was improved with β-glucan addition up to 5.0% and decreased with further addition to 7.5%. TPA characteristics of the cake improved significantly with β-glucan addition where as firmness decreased with increase in β-glucan content. Panelists rated highly cake fortified with β-glucan levels up to 5.0%. The decrease in sensory score at higher β-glucan level could be attributed to the increase in gumminess, cohesiveness and decrease in resilience score. The L and b values of cake crust and crumbs decreased significantly whereas ‘a’ value increased significantly with increase in β-glucan levels. Overall, the addition of up to 5.0% β-glucan extract in wheat flour cake improved its physicochemical, textural, sensory and in vitro antioxidant characteristics.

The effect of ultrasound technology to extract the water soluble polysaccharides from dried and milled by-products generated from Agaricus Bisporus production was studied. Amounts of β-glucan 1.01 and 0.98 g/100 g dry mass were obtained in particle sizes of 355-250 µm and 150-125 µm from the mushroom by-products. Three parameters of extraction were studied; extraction time (0-15 min), ultrasonic amplitude (20-100 µm) and precipitation time (1 or 18 h). The application of ultrasounds enhanced the extraction polysaccharide yields compared to the untreated samples. The highest extraction yield of 4.7% was achieved with an extraction time of 15 min, maximum amplitude of 100 µm with 1 h of precipitation in 80% ethanol. The coefficient of determinations for predicted water soluble polysaccharides extraction yields showed good correlation with the experimental data at the 95% confidence level and indicated that the non-exponential Peleg's model could be employed to predict the extraction polysaccharide yields after ultrasound treatment.

Antibacterial and cytotoxic activities of cyclohexane, dichloromethane, methanol, and aqueous extracts of Cantharellus cibarius were tested. Broth microdilution assay was performed against 10 bacterial strains (Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Bacillus subtilis, Enterococcus feacalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella abony), with emphasis on Helicobacter pylori. Methanol extract was the most active against H. pylori strains with minimal inhibitory concentration values between 4 and 32 µg/mL. All extracts were active against antibiotic resistant H. pylori. Methanol and aqueous extracts had no cytotoxicity against tested cell lines, whereas cyclohexane and dichloromethane extracts were active against HeLa and N87 cells, but also against healthy MRC-5 cells (IC⁵⁰ 39.26 ± 1.24-134.79 ± 0.01 µg/mL). The tested aqueous extracts have shown 68% of angiotensin-converting enzyme inhibitory activity in doses of 1.25 mg/mL. Chemical analysis has shown the presence of linoleic, cis-vaccenic, and oleic acids, sterols, β-glucans, and polyphenolic compounds.

In this study, efforts were made to utilize hulless barley (variety BHS352) to enhance the nutritive value of chapatti and biscuit made from wheat flour. Barley flour was added to wheat flour in different ratios (5 to 30%). Antioxidant activity, total phenolic content and β-glucan content were determined both in flour blends and their products. Changes in physical quality and taste of chapatti and biscuits after blending of hulless barley flour with wheat flour were measured. The chapatti quality score decreased by 15% and biscuit spread factor by 33% after 30% barley flour blending. Significant increase in β-glucan content and antioxidant activity of flour blends and their products was observed at 30% blending level. The phenolic content increased from 63 to 135 &microg for biscuits and 237 to 287 &microg GAE/g for chapatti with blending of 30% barley flour.

The use of agroindustry by-products (BP) for fortification of wheat bread can be an alternative to waste disposal because BP are appealing sources of dietary fiber. Moreover, it may also contribute to indirect income generation. In this study, sensory, color, and crumb structure properties of breads fortified with fiber rich fraction recovered from four types of agroindustry BP were tested, namely orange (OE), pomegranate (PE), elderberry (EE), and spent yeast (YE). Statistical models for sensory preference evaluation and correlation with color and crumb structure were developed. External preference mapping indicated consumer preferences and enabled selection of the concentrations of BP fibre-rich fraction with best acceptance, namely 7.0% EE, 2.5% OE, 5.0% PE, and 2.5% YE. Data collected from image analysis complemented sensory profile information, whereas multivariate PLS regression provided information on the relationship between “crust color” and “crumb color” and instrumental data. Regression models developed for both sensory attributes presented good fitting (R²Y > 0.700) and predictive ability (Q² > 0.500), with low RMSE. Crust and crumb a^* parameters had a positive influence on “crust color” and “crumb color” models, while crust L^* and b^* had a negative influence.

In this study, the crude ethanol Rhynchosia nulubilis cultivated with Ganoderma lucidummycelium (RNGM) extract was solvent fractionated with organic solvents such as n-hexane, chloroform, ethyl acetate, and water. The anticancer activities, anti-inflammatory activity total polyphenols, total flavonoids, isoflavones, and β-glucan of the solvent fractions of RNGM were studied. The ethyl acetate fraction showed the highest 2,2-diphenyl-1-picrylhydrazyl scavenging activity of 76.60% at 800 µg/mL. The ethyl acetate fraction also showed higher antioxidant activity in 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and ferric reducing ability of plasma assays compared to the other fractions. In addition, this study confirmed that the ethyl acetate fraction strongly inhibited nitric oxide production. The ethyl acetate fraction had the highest amount of total polyphenol and total flavonoid (65.33 mg gallic acid equivalent/g and 18.50 mg quercetin equivalent/g, respectively). The ethyl acetate fraction (13.02%) showed the highest amount of total β-glucan, followed by the water (6.32%), chloroform (1.43%), and n-hexane fraction (0.85%). Therefore, it is suggested that the ethyl acetate fraction of Rhynchosia nulubilis cultivated with Ganoderma lucidum mycelium may be potential natural sources for nutritional and pharmaceutical applications.

Due to their immunomodulatory effect, 1,3-β-G from yeast are used as functional ingredients, but reliable methods for their detection in foods are lacking. We have adapted a method based on fluorescence detection with aniline blue to quantify the amount of five commercial yeast β-glucan preparations added to crisp or yeast-leavened bread. This assay detected yeast β-glucan preparations added to different breads with an average recovery of 90, 96, 99 and 105%, while one of the preparations was overestimated, with an average recovery of 157%. The presence of cereal 1,3-1,4-β-D-glucans did not interfere with assay performance. The addition of 1,3-β-G at 0.2 and 0.5 g/100 g is low compared to the recommended dose of 1,3-β-G per serving demonstrating assay sensitivity. However, more research is needed to fully understand 1,3-β-G conformation/structure on aniline blue interaction as well as the effect of baking on structure and dissolution properties of yeast β-glucans.