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.

Beta-glucans are bioactive polysaccharides with immunomodulatory and hydrocolloid properties. This study explores the potential of beta-glucans extracted from mushroom waste to enhance oyster (Crassostrea gigas) immunity and quality, providing a sustainable alternative to conventional aquaculture interventions. Beta-glucan supplementation led to a 100 % survival rate in treated groups compared to 70% in the bacteria-only group (p = 0.024). Flow cytometry revealed a significant reduction in viable hemocytes in the beta-glucan group post-infection, suggesting modulation of inflammation. Protein content in gills, mantle, and digestive tissues increased by up to 2-fold in beta-glucan-fed oysters versus controls. Antioxidant activity, assessed by FRAP assay, was 25-40% higher in the mantle and digestive tract if beta-glucan groups. Bacteriostatic assays demonstrated a marked reduction in Vibrio sp. Counts in tissues, particularly in the digestive tract, with bacterial load similar to uninfected controls. These findings highlight the dual functionality of beta-glucans as both immunomodulators and hydrocolloids, with implications for improving aquaculture sustainability and food safety. Further research should explore feeding behaviour, gut transit time, and nutrient absorption to optimize beta-glucan application in commercial shellfish production.

Stillage is an acidic residue from ethanol production that has a high carbon load. Here, Ganoderma isolates were assessed for the treatment of rum stillage while producing biomass and associated metabolites. Isolates grew in 25% raw stillage, removing up to 73% of soluble organic carbon, 77% soluble nitrogen, and 74% phenolic compounds. Isolate G2 demonstrated faster removal of organic carbon and nitrogen. Biomass and metabolite production were benchmarked against a nutrient medium. In stillage, maximum values of the following were obtained: 8.2 g·L⁻¹ biomass; 52.8% crude protein; 22.1 mg·g⁻¹ extractable protein; antioxidants of 17.2 mg TE·g⁻¹ (2,2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS) and 16.6 µmol Fe²⁺·g⁻¹ (ferric reducing antioxidant power, FRAP); 2.9 mg GAE·g⁻¹ phenolic compounds (gallic acid equivalents); 1.2% lipids; and 11% β-glucans. In the nutrient medium, the following were obtained: 6.9 g·L⁻¹ biomass; 56.4% crude protein; 38.7 mg·g⁻¹ extractable protein; antioxidants of 24.9 mg TE·g⁻¹ (ABTS) and 25.9 µmol Fe²⁺·g⁻¹ (FRAP); 6.0 mg GAE·g⁻¹ phenolic compounds; 0.7% lipids; and 13% β-glucans. To our knowledge, this is the first report detailing the biomass metabolite content of Ganoderma mycelium using rum stillage. The production of edible biomass containing bioactive products demonstrates the potential of using Ganoderma strains to valorize this residue.

The gut microbiome has a significant role in general health and well-being. Novel types of prebiotics, such as fungal polysaccharides, show potential for the formulation of new synbiotic formulations. However, little is known about the underlying mechanisms of the prebiotic effects of such compounds. This study investigated the prebiotic properties of fungal glucan extracts from Pleurotus ostreatus, Lentinula edodes, and Saccharomyces cerevisiae, employing a novel high-throughput method based on optical density measurements. This approach enabled the simultaneous screening of the effects of multiple extracts on six different strains of probiotic bacteria. Experiments were conducted to evaluate the effect of the extracts on the growth dynamics (the duration of the lag phase and the growth rate) of probiotic strains of the genera Lactobacillus and Lacticaseibacillus and on pathogenic bacteria. Fungal polysaccharide supplementation, particularly with their β-glucans, significantly shortened the lag phase by an average of 7–8 h in all tested strains and increased the growth rate by 2-fold in four strains of lactic acid bacteria. Different magnitudes of effects were observed across the various strain-extract combinations. This study lays the groundwork for elucidating the mechanism by which fungal β-glucans stimulate growth in probiotic bacteria and for the rapid screening of optimal combinations for formulating innovative synbiotics.

Brown macroalgae contains complex polysaccharides including laminarins that have shown prebiotic potential. The aim of this study was to investigate if feeding algal products from Saccharina latissima to either broiler breeders or directly to their chickens could affect growth performance, gut- and immune development in the chickens. A total of 45 hens of the parent line of Ross 308 were used to obtain fertilised eggs. The hens were fed one of three experimental diets, a control, the addition of 0.6% algal meal or addition of 0.08% algal extract. The progenies of those hens were followed in an experiment using a split-plot design where eggs from the three hen treatments were distributed into 24 modules. Half of the modules were assigned a control diet, and half of the modules were a diet supplemented with 725 ppm algal extract. A total of 255 chicks remained after hatching and individual marking, they were weighed at hatch, on days 3, 7, 14, and 37 and blood samples for determination of leukocyte counts and serum antibody levels were drawn on days 3, 7 and 12. Chickens were killed to assess organ development at days 7, 14 and 37, and histological examination of ileal tissue was performed on day 7. The results showed that chicks fed the algal extract diet had higher (P < 0.05) BW on days 3, 7 and 37, a higher proportion of serum immunoglobulin Y (IgY) and a lower proportion of maternal antibodies to infectious bronchitis virus on day 12 (P < 0.05). Chicks fed algal extract showed higher numbers (P < 0.05) of CD4+CD8- helper T-cells and total T-cell receptor (TCR)γ/δ+ T-cells, and among the TCRγ/δ+ T-cell subpopulations, the TCRγ/δ+CD8- T-cells were increased, and lower (P < 0.05) numbers of cluster of differentiation (CD)4+CD8αα+ and TCRγ/δ+CD8αβ+ T-cells in the circulation. Feeding algal extract to the breeders resulted in higher chick BW on day 7, and the villus height−to−crypt depth ratio was higher (P < 0.05) for chicks from hens fed algal extract than for chicks from hens fed algal meal. In conclusion, feeding algal extract from Saccharina latissima directly to the chicken improved growth performance throughout the growing period and altered the composition of T-cell populations in the circulation and may have enhanced the chicks’ IgY production. Maternal supplementation of algal extract to breeder hens had positive effects on the chickens’ early growth performance and gut architecture. However, no synergistic effects of both maternal feeding and direct supplementation to the chicken were found.

Cosmeceutical industry is continually searching for new substances or extracts from natural sources that are nontoxic and can delay the aging process. Lentinus squarrosulus is a wild edible mushroom valued by local communities in Asian countries as food as well as its medicinal benefits. However, the bioactive substances and cosmeceutical biological properties of L. squarrosulus mycelia grown in submerged culture have not been demonstrated. This study aims to investigate the feasibility of producing L. squarrosulus mycelial biomass and cosmeceutical bioactive compounds through large-scale submerged fermentation. L. squarrosulus mushroom mycelia grown in a 5-L airlift bioreactor submerged culture was investigated as a new source of cosmeceutical biological substances. An ethanolic extract derived from L. squarrosulus mycelia possesses potential antioxidant agents capable of inhibiting dermal enzymes such as collagenase, elastase, and tyrosinase for simultaneously treating skin aging and hyperpigmentation. The Molisch test, FTIR analysis, and b-glucan assays revealed that the main polysaccharide present in the L. squarrosulus mycelial extract was b-glucan (64.3 %), which acts as a major contributor to its cosmeceutical properties. The L. squarrosulus mycelial extract was non-toxic to human skin keratinocytes (HaCaT). Cosmeceutical formulations developed with the mycelial extract, especially in essence form, preserved the extract’s bioactivities, presented a pale-yellow color and a pH of 6.0, which is considered suitable for cosmeceutical design. This study provides new information regarding submerged growth of L. squarrosulus mycelia in an airlift bioreactor, which has potential as a novel source of useful bioactive b-glucans for cosmeceutical applications. High mycelial biomass was produced over a short time in this airlift bioreactor that concomitantly had anti-aging and skin whitening properties. The results suggest that the mycelial extracts of L. squarrosulus could be further developed to produce safe natural ingredients for inclusion in cosmeceutical products.

Probiotic encapsulation enhances bacterial stability and viability, yet conventional materials like sodium alginate have limitations, particularly in acidic environments. This study explores the use of β-glucan-rich Saccharomyces cerevisiae cell walls, extracted via ultrasound-assisted enzymolysis, as an alternative microencapsulation matrix for probiotics. This study aims to improve encapsulation efficiency, enhance probiotic survival under stress conditions, and offer a sustainable solution for probiotic delivery. Among the tested strains, Levilactobacillus brevis C23 encapsulated in S. cerevisiae cell walls exhibited the highest encapsulation efficiency (90.42 ± 4.53 %) and cell viability (7.52 ± 0.45 log CFU/mL), significantly outperforming sodium alginate encapsulation (p < 0.05). Encapsulation in S. cerevisiae cell walls provided superior heat resistance (up to 65 °C) and enhanced survival in simulated gastrointestinal conditions, with viable counts of 2.266 ± 0.095 log CFU/mL in gastric juice (120 min) and 4.557 ± 0.086 log CFU/mL in intestinal juice (240 min). Fourier-transform infrared spectroscopy (FTIR) confirmed successful probiotic encapsulation. At the same time, storage studies showed that encapsulated cells maintained viability above 6 log CFU/mL for eight weeks at 4°C, though viability decreased at 25°C. These findings demonstrate the potential of S. cerevisiae cell walls as an effective and sustainable encapsulation matrix, offering enhanced probiotic protection for food and pharmaceutical applications.

The goal of this study was to establish an efficient protocol for producing bioactive-rich mushroom extracts for food ingredients development. For this, fresh mushrooms were dried, ground, and submitted to different extraction protocols-ethanolic (EE), fermentation-assisted (FAE), or enzyme-assisted extraction (EAE). In addition, ultraviolet B (UV-B) irradiation, coupled with the extraction protocols, was experimentally studied for potential improvement of extracts bioactivity and yields. The extraction yield (EY), bioactivity (total phenolic content TPC, phenolic acids, flavonoids, and antioxidant activity), α-, β-, and total glucan contents, and mineral content were evaluated. EAE yielded the highest EY (26.48%-31.30%) while EE resulted in the highest TPC, phenolic acids, flavonoids, and antioxidant activity (p < 0.05). The β-glucan content (0.13%-0.93%) was not affected by extraction type or UV-B irradiation (p > 0.05). All extracts were rich in essential minerals with low potential toxicity. Based on the high EY, phenolic load, antioxidant activity, glucans, and mineral contents, EAE proved to be an effective method to obtain bioactive-rich mushroom extracts. Overall, UV-B irradiation did not show significant results in improving bioactivity of mushroom extracts. Our results deliver a realistic roadmap to explore mushrooms as rich sources of phytochemicals for food and nutraceuticals applications.

Chaga (Inonotus obliquus) is an increasingly used natural product in botanical dietary supplements, valued for its bioactive compounds. However, inconsistent standardized analytical methods raise concerns over product authenticity, mislabeling, and quality control. This study employs a multi-analytical approach to differentiate wildcrafted chaga canker from North American chaga dietary supplements, particularly those containing mycelia fermented grain products. High-Performance Thin-Layer Chromatography (HPTLC), Liquid Chromatography with Evaporative Light Scattering Detection (LC-ELSD) or Photo/Diode Array Detection (LC-PDA/DAD), Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS), Nuclear Magnetic Resonance (NMR) spectroscopy, UV-Vis spectrophotometry, and iodine-starch assays were used to evaluate key markers, including triterpenoids, polysaccharides, and melanin. Whole chaga canker contained triterpenoids (inotodiol, trametenolic acid) and phenolics, like osmundacetone, while melanin absorbance at 500 nm differentiated it from fermented grain products. β-Glucan quantification and iodine-starch assays confirmed starch-rich composition in fermented grains and its absence in authentic chaga canker. NMR fingerprinting and LC-QToF-MS metabolomics demonstrated stark compositional deviations between wildcrafted chaga canker, I. obliquus mycelium, and fermented grain products. By integrating complementary techniques, we establish a framework that can reliably distinguish genuine chaga canker from misrepresented products, ensuring consumer safety and fostering trust in the functional mushroom, canker, and mycelium markets.

The aim of this study was to develop a novel functional ingredient—goat’s skim milk enriched with Agrocybe aegerita (V. Brig.) Vizzini mushroom extract (ME/M)—using Central Composite Design (CCD). The optimized ME/M ingredient was evaluated for its physico-chemical, techno-functional, biological, and antimicrobial properties. Physico-chemical properties were analyzed using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy, Scanning Electron Microscopy (SEM), and Dynamic Light Scattering (DLS). The ingredient exhibited a polymodal particle size distribution and contained glucans, along with a newly formed polypeptide resulting from the selective cleavage of goat milk proteins. A 0.1% ME/M solution demonstrated good emulsifying and foaming properties. Additionally, ME/M showed strong antiproliferative effects on human cancer cell lines, particularly Caco-2 (colorectal) and MCF7 (breast) cancer cells. The ingredient also promoted HaCaT cell growth without cytotoxic effects, suggesting its safety and potential wound-healing properties. Furthermore, the addition of ME/M to HaCaT cells inoculated with Staphylococcus aureus resulted in reduced IL-6 levels compared to the control (without ME/M), indicating a dose-dependent anti-inflammatory effect. The optimized ME/M ingredient also exhibited antibacterial, antifungal, anticandidal, and antibiofilm activity in one-fourth of MIC. These findings suggest that the formulated ME/M ingredient has strong potential for use in the development of functional foods offering both desirable techno-functional properties and bioactive benefits.

Background: This study explores wound healing and the antimicrobial potential of a natural formulation containing a polysaccharide extract from Cerioporus squamosus, hyaluronic acid, and dexpanthenol. Methods: Wound healing effects were assessed using HaCaT keratinocytes, while antimicrobial activity was evaluated against human skin pathogens using a microdilution assay. In vitro cytotoxicity tests ensured formulation safety, whereas in vivo wound healing was further investigated using an animal model. Gene expression analysis was performed to assess the molecular mechanisms involved. Results: The unique glucan composition of C. squamosus (15.38% α-glucans and 7.91% β-glucans) deviated from typical mushroom polysaccharide profiles, warranting further exploration of its bioactivity. In vitro mushroom polysaccharides promoted 25.35% wound closure after 24 hours, while the three-component formulation achieved 35.81% closure. Antibacterial activity showed a minimum inhibitory concentration (MIC) of 0.44–1.75 mg/mL and minimum bactericidal concentration (MBCs) of 0.88–3.50 mg/mL, while antifungal activity ranged from 0.22 to 0.44 mg/mL (MICs) and 0.44 to 0.88 mg/mL (minimum fungicidal concentration—MFC). In vivo data showed that 60% of treated wounds fully closed by day 11, despite no statistically significant difference from the control. However, gene expression analysis highlighted VEGF and collagen upregulation, indicating an enhancement of wound healing on a molecular level. Conclusions: The novel three-component formulation demonstrated consistent wound healing and antimicrobial properties, supporting its potential as a safe and effective treatment for chronic and acute wounds.

Microalgae have gained significant attention as sustainable sources of high value compounds, such as bioactive polysaccharides that are usually rich in sulfated groups and exhibit antioxidant properties. Here, 14 new microalgae strains of the genera Tetraselmis, Dunaliella, and Nannochloropsis, isolated from Greek coastal lagoons were analyzed to quantify and characterize their polysaccharide content. Heterogeneity was observed regarding the content of their total sugars (5.5–40.9 g/100 g dry biomass). The strains with a total sugar content above 20% were analyzed concerning the content of total, α- and β-glucans. Tetraselmis verrucosa f. rubens PLA1-2 and T. suecica T3-1 were rich in β-glucans (11%, and 8.1%, respectively). The polysaccharides of the two Tetraselmis strains were isolated and they were mainly composed of glucose and galactose. The isolated polysaccharides were fractionated using ion-exchange-chromatography. The anionic fraction from T. verrucosa f. rubens PLA1-2 was rich in sulfated polysaccharides, had antioxidant capacity, and exhibited healing properties. The anionic polysaccharides from the two Tetraselmis strains did not negatively influence the viability of human cells, while exhibiting antiviral properties against the replication of Hepatitis C Virus (HCV), with median efficient concentrations (EC50) at a range of 210–258 μg/mL.

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.