β-Glucan Assay Kit (Yeast and Mushroom)

A robust and reliable method has been developed for the measurement of β-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H₂SO₄ and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. β-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the β-glucan were compared, and the best option was the method using H₂SO₄. For most samples, similar β-glucan values were obtained with both the optimized HCl and H₂SO₄ procedures. However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H₂SO₄ procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H₂SO₄ procedure.

Clade A APSES family transcription factor Swi6 functions alongside Mbp1 to form the MBF (MluI cell cycle box-binding factor) complex in ascomycetes. In the agaricomycete Pleurotus ostreatus, Mbp1 plays a crucial role in regulating β-glucan and chitin synthesis; however, the role of Swi6 has not been explored in this fungus. In this study, its involvement in cell wall synthesis regulation was analysed using swi6 disruption strains in P. ostreatus. The Δswi6 strains exhibited reduced growth rates and shorter aerial hyphae formation in both agar and liquid media, suggesting an essential role of Swi6 in normal vegetative growth. Furthermore, swi6 disruption affected cell wall thickness distribution, the expression of specific chitin synthase genes, the relative percentage of chitin, and sensitivity to calcofluor white, suggesting that Swi6 is required for normal chitin synthesis regulation in P. ostreatus. In contrast, no significant differences were observed between the wild-type and Δswi6 strains in the relative percentage of α- and β-glucan and the expression of α- and β-glucan synthase genes, suggesting its unimportant role in α- and β-glucan synthesis regulation. In conclusion, Swi6 is necessary for normal mycelial growth and chitin synthesis regulation in P. ostreatus. To the best of our knowledge, this study is the first report on the functional differences and overlaps between Mbp1 and Swi6 in the regulation of cell wall synthesis in agaricomycetes.

Hydrophobins are small-secreted proteins with both hydrophobic and hydrophilic regions, enabling the mycelium to break through the air-medium interface by reducing the medium surface tension. Over 20 putative hydrophobin-encoding genes have been predicted in the agaricomycete Pleurotus ostreatus. Three hydrophobin-encoding genes, vmh2, vmh3, and hydph16, were predominantly expressed in the vegetative mycelium. Despite these common properties, we have previously demonstrated the distinct functions of Vmh2 and Vmh3 in environmental stress resistance. In this study, we focused on hydph16 and found that Δhydph16 strains had sparser aerial mycelium than control strains. The cell wall thickness of Δhydph16 strains reduced by 40 % compared to that of control strains, but no significant differences were found in the relative chitin and glucan percentages or relative putative cell wall synthesis-related gene expression levels. Furthermore, unlike vmh2 and vmh3, hydph16 deletion did not change the hydrophobicity of the aerial mycelium. This study is the first to report that the lack of hydrophobin can lead to a significant change in aerial hyphae cell wall formation without altering the major cell wall polysaccharide composition. Additionally, this study revealed multiple roles for Hydph16, distinct from those of other highly expressed hydrophobins, Vmh2 and Vmh3. These results suggested that agaricomycetes, including P. ostreatus, have evolved to possess multiple hydrophobins with different functions.

This study aimed to optimize the cultivation of Lignosus rhinocerus mycelium at a laboratory level and evaluate the antioxidant activity of β-glucan extracted from the mycelium. Using the Response Surface Methodology (RSM) and Central Composite Design (CCD), optimal conditions for mycelium growth were identified at a temperature of 31.5 °C, 15 % potato powder, and 3.5 % glucose concentration, yielding a maximum mycelial biomass of 44.7 g/L and β-glucan production of 15.08 g/L. The β-glucan was extracted using a household microwave, significantly improving the extraction efficiency. The extracted β-glucan demonstrated notable antioxidant activity, with DPPH radical scavenging activity at approximately 53.31 % and ABTS radical scavenging activity around 47.31 %, albeit slightly lower than the 71.94 % activity of ascorbic acid. Comparative analyses highlighted the consistency in antioxidant capacities across different morphological stages, with aqueous methanol extracts of mycelium (LR-MH, LR-MT) and culture broth (LR-BH, LR-BT) showing either higher or comparable antioxidant activities to sclerotium extracts (LR-SC). Field Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared (FTIR) spectroscopy confirmed the structural integrity of the extracted β-glucan. The findings support the feasibility of using mycelium for β-glucan extraction, offering a sustainable alternative to traditional methods.

Background and aim: A critical causative factor of oxidative stress and inflammation leading to several skin complications is ultraviolet-B (UVB) irradiation. Lignosus rhinocerus (LR), or tiger milk mushroom, is native to Southeast Asia. Cold water extract of an LR cultivar, TM02® (xLr®) is a promising anti-oxidant and anti-inflammatory source. However, the effects of xLr® on UVB-induced photoaging have never been elucidated. Experimental procedure: This study investigated the protective effects of xLr® and its high, medium, and low molecular weight (HLR, MLR, and LLR, respectively) fractions against UVB irradiation using in vivo Caenorhabditis elegans (C. elegans) model. Results and conclusion: The investigation revealed a significant lifespan extension of xLr® and its fractions in UVB-irradiated C. elegans, which could be mediated by the regulation of genes associated with anti-oxidant (daf-16 and sod-3) and apoptosis (cep-1, hus-1, ced-13, and egl-1) pathways. xLr® significantly reduced the ROS production in C. elegans and increased the DAF-16 nuclear translocation compared to untreated worms. Additionally, the SOD-3 expression was increased in the xLr®-treated worms. Hence, it suggests that the different components in xLr® work synergistically to protect against UVB irradiation. Our findings may be beneficial for the application of xLr® as a treatment against UVB-induced cellular damage and photoaging.

Pleurotus ostreatus (oyster mushroom) holds excellent promise worldwide, bringing several opportunities and augmenting the tool sets used in the biotechnology field, the food industry, and medicine. Our study explores the antimicrobial and probiotic growth stimulation benefits of freeze-dried P. ostreatus powders (OMP-TF, oyster mushroom powder from the total fresh sample; OMP-CSR, oyster mushroom powder from the cooked solid residue; OMP-CL, oyster mushroom powder from the cooked liquid), focusing on their bioactive compounds and associated activities. Our research examined polysaccharide fractions—specifically total glucans and α- and β-glucans—alongside secondary metabolites, including polyphenols and flavonoids, from freeze-dried mushroom powders. Additionally, carbon nanodots (CNDs) were also characterized. The growth inhibition was tested against Escherichia coli and Staphylococcus epidermidis, while the capacity for stimulating probiotic growth was evaluated using Lactobacillus plantarum and Lactobacillus casei. Evidence indicates that OMP-CL and OMP-CSR exhibit significant antimicrobial properties against S. epidermidis Gram-positive bacteria. OMP-CL notably promoted the growth of L. casei. OMP-CL, containing the most significant number of CNDs, has shown to be a valuable source for gut microbiota modulation, with its antimicrobial and probiotic-stimulating efficacy. However, further in vitro and in vivo studies should be performed to explore CNDs and their behavior in different biological systems.

Coccidiosis, infection with protozoan parasites of genus Eimeria, is a major problem in poultry husbandry world-wide. The disease is currently managed by coccidiostats and live vaccines, but these approaches are not sustainable. Hence, it is important to identify new means to control the infection and/or ameliorate its detrimental effects on gut health. Laminarin, a β-glucan found in marine brown algae, has prebiotic and bioactive properties that could be beneficial in coccidiosis control. The present study aimed to examine the potential of laminarin as an immunostimulatory and microbiota-regulatory compound in broiler chickens infected with E. tenella. Chickens were continuously fed a diet supplemented with a laminarin-rich algal extract (AE) from first feed and subsequently infected with E. tenella at 19 days old. The outcome of infection including caecal microbiota and some immune parameters were monitored during the experiment. Results showed that AE supplementation affected some lymphocyte subpopulations, with increased numbers of TCRγ/δ+CD8-, B-cells and CD4-CD8αβ+ cells and lower numbers of CD4+CD8αα+ cells in blood and increased proportions of CD4-CD8αβ+ spleen cells compared to those in control chickens. The AE diet did not affect parasite excretion, lesion scores or E. tenella specific T-cell responses. However, reductions of E. tenella induced contraction of Bifidobacteriaceae and expansion of Clostridiaceae in caecal microbiota were observed for AE fed chickens compared to chickens fed the control diet. Thus, AE feed supplementation induced some immunostimulatory activity in chickens and affected some of the alterations in caecal microbiota evoked by E. tenella infection.

The increasing contamination of cereals by micromycetes and mycotoxins during malting still poses an unresolved food safety problem. This study characterises the potential of the novel, rapidly developing food production technology of Pulsed Electric Field (PEF) to reduce the viability of Fusarium fungi and the production of mycotoxins during malting. Barley, artificially inoculated with four Fusarium species, was treated by PEF with two different intensities and then malted using a standard Pilsner-type technology. Concentrations of fungi were quantified by RT-PCR, expression of fungal growth-related genes was assessed using mRNA sequencing, and mycotoxin levels were analysed by U-HPLC-HRMS/MS. Despite the different trends for micromycetes and mycotoxins after application of variously intense PEF conditions, significant reductions were generally observed. The greatest decrease was for F. sporotrichioides and F. poae, where up to six fold lower levels were achieved for malts produced from the PEF-treated barley when compared to the control. For F. culmorum and F. graminearum, up to a two-fold reduction in the PEF-generated malts was observed. These reductions mostly correlated with a decrease in relevant mycotoxins, specifically type A trichothecenes.

This study explores the effects of dietary supplementation with passion fruit peel pectin (Passiflora edulis) and red yeast cell walls (Sporidiobolus pararoseus) on growth performance, immunity, intestinal morphology, gene expression, and gut microbiota of Nile tilapia (Oreochromis niloticus). Nile tilapia with an initial body weight of approximately 15 ± 0.06 g were fed four isonitrogenous (29.09-29.94%), isolipidic (3.01-4.28%), and isoenergetic (4119-4214 Cal/g) diets containing 0 g kg⁻¹ pectin or red yeast cell walls (T1 - Control), 10 g kg⁻¹ pectin (T2), 10 g kg⁻¹ red yeast (T3), and a combination of 10 g kg⁻¹ pectin and 10 g kg⁻¹ red yeast (T4) for 8 weeks. Growth rates and immune responses were assessed at 4 and 8 weeks, while histology, relative immune and antioxidant gene expression, and gut microbiota analysis were conducted after 8 weeks of feeding. The results showed that the combined supplementation (T4) significantly enhanced growth performance metrics, including final weight, weight gain, specific growth rate, and feed conversion ratio, particularly by week 8, compared to T1, T2, and T3 (P < 0.05). Immunological assessments revealed increased lysozyme and peroxidase activities in both skin mucus and serum, with the T4 group showing the most pronounced improvements. Additionally, antioxidant and immune-related gene expression, including glutathione peroxidase (GPX), glutathione reductase (GSR), and interleukin-1 (IL1), were upregulated in the gut, while intestinal morphology exhibited improved villus height and width. Gut microbiota analysis indicated increased alpha and beta diversity, with a notable rise in beneficial phyla such as Actinobacteriota and Firmicutes in the supplemented groups. These findings suggest that the combined use of pectin and red yeast cell walls as prebiotics in aquaculture can enhance the health and growth of Nile tilapia, offering a promising alternative to traditional practices. Further research is needed to determine optimal dosages for maximizing these benefits.

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.

Lanostene-derived triterpenoids and β-glucans are important metabolites in Ganoderma mushrooms associated with benefits to human health. The medicinal value of the Australian Ganoderma species remains unclear, with no data on triterpenoid distribution or glucan content. In the present study, 22 Australian Ganoderma specimens were analyzed for triterpenoid and glucan contents. Thirty-two triterpenoids were identified in the fruiting bodies of 19 of the specimens. Distinct patterns in triterpenoid distribution between laccate and matte fruiting bodies were observed, leading to the classification of four groups of Ganoderma. Most of the glucans in the Ganoderma fruiting bodies were β-glucans (~99%), with a nominal α-glucan content (~1%). The β-glucan content ranged from 19.5 to 43.5% (w/w). A range of antioxidant activities was observed for methanol extracts using the ABTS (1.8 to 8.4 mg GAE.g⁻¹), DPPH (1.7 to 9.4 mg GAE/g⁻¹) and FRAP (24.7 to 111.6 mmol FeSO₄.g⁻¹) assays, with four specimens presenting relatively high radical scavenging and reducing activities. For the first time, we demonstrated that Australian Ganoderma mushrooms contain medicinal triterpenoids, including ganoderic acid A, and we established a link between its distribution and the fruiting body morphology. However, further research is required to isolate diploid clones and determine factors that impact triterpenoid and glucan synthesis in these strains.

Ganoderma lucidum extract (GE) exhibited excellent antioxidant activity with a diverse range of bioactive constituents for enhancing the antioxidant status in animals. However, the challenge is intestinal-targeted delivery. Microencapsulation is widely employed to protect pharmacological substances by fabricating microcapsules. This study investigated the preparation of multilayer microcapsules. Sodium alginate (SA) was used as the primary wall followed by the creation of polyelectrolyte multilayers using chitosan (CS)-SA to obtain GE/SA-CS. GE had high levels of ergosterol, flavonoids, and triterpenoids as well as >80% of antioxidant properties. In vitro simulated digestion indicated that the multilayers of SA-CS displayed a great controlled release ability under intestinal conditions (82.15 ± 3.99%). SA-CS not only protected the constituents of GE from acid, bile, trypsin, and heat treatment but also extended the storage shelf life of GE. Thus, the development of microencapsulation based on the antioxidant delivery system in multilayer SA-CS microcapsules could retain active compounds in GE.

Beta-glucan (β-glucan), a naturally occurring complex polysaccharide, has drawn attention for its diverse health benefits, including immune system modulation. β-glucan was extracted from two fungi, Monascus purpureus (Mp) and Monascus kaoliang (Mk), cultured in Saccharina japonica via submerged fermentation. The yield, solubility, total sugar, reducing sugar, protein content, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric analysis (TGA), Scanning Electron Microscopy (SEM), in vitro free radical scavenging activity, and cytotoxicity were analyzed. A significant yield of β-glucans, with the contents of 51.30 ± 1.54% in Mp and 44.24 ± 1.18% in Mk was observed on a dry weight basis. Water solubility slightly varied, measuring 36.25 ± 1.14% in Mp and 31.25 ± 0.94% in Mk. Total sugar and reducing sugar content in Mp and Mk derived β-glucans were 114.75 ± 2.54 mg/g and 100.25 ± 1.86 mg/g, 7.38 ± 0.78 mg/g, and 8.39 ± 0.46 mg/g, respectively. FTIR spectra resembled the standard, and TGA confirmed heat stability. XRD patterns indicated that the extracted β-glucans, including the standard one, showed the most prominent diffraction peaks in the lower 2θ range, suggesting similar crystalline phases; however, they differed in crystallinity and degree of amorphous content. SEM images displayed characteristic rough and fibrous shapes and surfaces for extracted β-glucans but it was uniform and of a regular shape in the standard sample. The isolated β-glucans exhibited in vitro free radical scavenging and no cytotoxicity was observed in the MTS assay. Therefore, utilizing S. japonica as a substrate in the fermentation process by Monascus spp. presents a unique opportunity in the production and utilization of β-glucans.

The chitosan production process from fishery waste is already established in industrial scale, whereby fungal chitosan is produced in lower amounts. Since fungal chitosan could be isolated from under-valorized vegan streams while exhibiting slightly different characteristics, it has also potential for other applications. Within this publication, we focus on the chitosan production from Aspergillus niger. This study provides a detailed determination of the biomass composition, adapting and comparing different analytical tools, with special focus on the chitin and chitosan content. The major content of the dried biomass is composed of glucans (48.6 ± 1.4%), followed by proteins with an amount of 22.2 ± 0.7%. Chitin and chitosan provide 16.0 ± 0.8% of the biomass. Within our chitosan production studies, we compared the effect of different process strategies including steps as deproteinization (DP), acid extraction (AE), deacetylation (DA), as well as purification. Initially, we obtained poor values (lower than 73.6%) for the chitosan purity. A direct DA step followed by purification resulted in a chitosan purity of up to 89.6%, a recovery of 30.5% and a yield with regard to the biomass of 5.5%. The DA degree of the resulting chitosan is similar to chitosan derived from fishery waste, whereas the molecular weight is lower. The results achieved so far are consistent with the literature, extending beyond, the data emphasized that a chitosan production from residual fungal biomass after fermentation is suitable by direct DA and purification. However, further adaption is necessary so that other matrix compounds could be also obtained.

It is estimated that more than one-third of people aged over 16 reported living with inflammation-related chronic illness. Fucoidan, sulfated polysaccharide, has been proven to modulate immune response and inhibit inflammation in both in vitro and in vivo research. In the present study the effect of commercially available fucoidan from Fucus vesiculosus (with purity 97%) on immune response was investigated. Two cell lines, human fibroblasts (HDFa) and mouse macrophages (J774A.1) were used in the study. Cells were activated with pro-inflammatory stimulus (LPS/IFN-γ or PMA) and treated with fucoidan. Results of NO inhibition assay revealed that fucoidan has anti-inflammatory activity and significantly reduces the level of NO produced by the cells. A significant decrease in secretion of IL-6 from both cell lines was observed. Moreover, fucoidan inhibits IL-12 production by mouse macrophages. The obtained results indicate that fucoidan not only acts as anti-inflammatory compound, but also has immunomodulatory activity. Enhanced secretion of TNF-α and IL-8 from macrophages and fibroblasts, respectively, was also noted. The antioxidant activity of fucoidan has also been studied. Fucoidan demonstrates dose-dependent scavenging properties by DPPH assay. Fucoidan's simultaneous ability to reduce inflammation, activate macrophages, and remove free radicals may be used as a tool to fight chronic inflammation disorders. These all contribute to the effective immunoprevention of cancer or other diseases related to the impaired function of the immune cells.