WO2025114975A1 - An improved method of preparing extracts of lentinula edodes and extracts prepared by the method - Google Patents

Abstract

An alcohol-free method of extracting β-glucans such as lentinan from mushrooms is described. The method excludes the drying and pulverising of the mushrooms prior to extraction. The simultaneous use of enzymatic digestion and ultrasonication provides extracts with high levels of desired properties including 1,1-diphenyl-2-picryl hydrazyl radial scavenging activity, superoxide radical scavenging activity, iron chelating capacity, and ferric-reducing antioxidant power.

Description

AN IMPROVED METHOD OF PREPARING EXTRACTS OF LENTINULA EDODES

AND EXTRACTS PREPARED BY THE METHOD

TECHNICAL FIELD

The invention relates to methods of preparing extracts for use in the formulation of nutraceuticals. The extracts comprise increased yields of beta-glucan with high specific bioactivities. In particular, the invention relates to an alcohol-free method of preparing such extracts from the fruiting bodies of species of fungi such as Lentinula edodes (shiitake mushroom) .

BACKGROUND ART

Beta-glucans (f>-glucans) are a family of p-D-glucose polysaccharides naturally occurring in the cell walls of bacteria, cereals (barley, oats, etc. ) , fungi and seaweed. The term 'lentinan' is often used to refer to the [J-glucan extracted from shiitake mushrooms .

Typically, [J-glucans form a linear backbone with [31 — 3 glycosidic bonds, but vary with respect to their degree of branching, confirmation, and molecular weight. The publication of Tada et al (2008) disclosed that the physiochemical properties, i.e. , gelation, solubility, viscosity, etc. , of f>- glucans differ according to their structure. Many methods of extracting f>- glucans are known.

The publication of Bian et al (2006) discloses investigations of an optimal technique for ultrasound-assisted extraction of polysaccharide from Lentinus edodes using a particle size of material of 1 to 2 mm.

The publication of Chen (2010) discloses a comparison of the extraction of lentinan using hot water, microwave-assisted extraction, and ultrasonic- assisted extraction. The latter was determined to be the best method.

The publication of Chikari et al (2020) discloses the use of subcritical water extraction and simultaneous multi-frequency ultrasound-assisted alcohol/aqueous salt two-phase extraction for the efficient and selective isolation of polysaccharide from Lentinus edodes.

The publication of Dai et al (2023) discloses the optimization of the extraction, emulsifying properties, and biological activities of polysaccharides from Lentinula edodes. Hot water extracts showed the best emulsifying properties.

The publication of Gao (2016) discloses a method of extracting lentinan including the preparation of a paste-like slurry by a high-pressure treatment. The method requires the addition of a total of 14 to 16 times by weight of deionised water. Following enzymatic digestion, ultrasonication and filtration ethanol is used to precipitate lentinan (a f>-glucan) and separate it from other components of the extract. The publication asserts that the purity of the lentinan polysaccharide is high, reaching 98.6% or more, with a DPPH inhibition rate of 28.6 to 34.2%.

The publication of Gun et al (2017) discloses a method of preparing a shiitake mushroom extract by ultrasound extraction of pulverized, dried shiitake mushrooms to which a solvent has been added and treating the resultant product with ultrasound at a temperature of 25 to 70°C for 1 to 6 hours .

The publication of Huang et al (2017) discloses a study on the two-phase ultrasonic-assisted hot solvent extraction of Lentinus edodes. An extraction rate of 7.24% is reported.

The publication of Ke (2015) discloses incubating a powdered mushroom with a cellulase for 30 minutes, followed by ultrasonication for 14 minutes and then ethanol precipitation to obtain the mushroom polysaccharides.

The publication of Lan et al (2023) discloses a method of extracting plant polysaccharides in general. The method requires the material to be soaked prior to extraction and the addition of ethanol for the purification of the plant polysaccharides.

The publication of Lee et al (2014) discloses a method for preparing p-glucan by ultrasonic extraction and heat extraction of finely ground mushroom. The method avoids the need for alkaline neutralization.

The publication of Li et al (2023) discloses the evaluation of autoclaving followed by ultrasonication for the efficient extraction of polysaccharides (PS) from Lentinula edodes mushrooms. A yield of 16.3% was reported. The publication concluded that fractional ethanol precipitation of the extract obtained was an efficient strategy for isolation of the major immunostimulatory PS with reduced solvent expenditure.

The publication of Liu et al (2013) discloses a leeching temperature of 80°C as being the best for the ultrasonic-assisted water immersion extraction of lentinan. Yields of 3.93% were obtained.

The publication of Lu (2010) discloses an optimized method for the ultrasonic-assisted extraction of lentinan. An extraction rate of 4.39% was obtained using a material to liquid ratio of 1:25, ultrasonic treatment for a period of time of 15 minutes and extracting at 100°C for a period of time of 2 hours .

The publication of Lu et al (2019) discloses the extraction of four different kinds of polysaccharides from Lentinus edodes using different extraction methods; microwave, ultrasonic, hot water, and enzyme. The monosaccharide components of the four kinds of polysaccharides were substantially the same, but their molar ratios were significantly different. Microwave extraction was identified as the best method.

The publication of Morales et al (2019) discloses ultrasound-assisted or subcritical water extraction as being more efficient than hot water extraction for obtaining p-glucan-enriched fractions from shiitake mushrooms. Pre-treatment of the raw material with supercritical carbon dioxide was also employed. The quality of the polysaccharides was influenced by the extraction method with the use of subcritical water extraction alone or in combination with ultrasound-assisted or supercritical carbon dioxide extraction providing polysaccharides with higher glucose levels.

The publication of Peng et al (2011) discloses the optimization of ultrasonic-assisted boiling water extraction of lentinan.

The publication of Qin et al (2018) discloses the optimization of the ultrasonic-assisted enzyme extraction of lentinan from Lentinus edodes. An optimized extraction rate of 8.97% was achieved for ultrasonic extraction alone. An optimized extraction rate of 12.46% was achieved with enzymic hydrolysis .

The publication of Shi et al (2014) discloses a trichloroacetic acid extraction method as being superior to hot water extraction, hot water extraction after ultrasonic treatment, and ultrasonic treatment after hot water extraction.

The publication of Sun and Xu (2015) discloses the extraction of polysaccharide from Lentinus edodes using a combination of ultrasound and high temperature water. The influence of the process parameters water to material ratio, ultrasonic temperature, time and power were studied. The ultrasonic time was found to have the most profound effect on extraction rate with a maximum extraction rate of 15.85% being reported.

The publication of Vezaro et al (2022) discloses ultrasound-assisted extraction of shiitake [8-glucans using water and eutectic solvent. Extraction conditions of time and temperature were varied to evaluate the effect on yield and antioxidant activity. The publication of Wang and Feng (2022) discloses an allegedly low-cost method of preparing lentinan when compared to water extraction and alcohol precipitation. The first steps of the method are to prepare crushed mushroom material and infiltrate with water before enzymatic hydrolysis (without ultrasonication) . Ultrafiltration is used to concentrate the lentinan containing liquid before it is spray dried as a formulation.

The publication of Wang and Lin (2010) discloses the ultrasonic wave-assisted extraction of polysaccharides from Lentinus edodes (and the use of this crude extract in the preparation of a beverage) .

The publication of Wang et al (2008) discloses an applied study on ultrasonic-assisted extraction and best techniques for the extraction of lentinan from Lentinus edodes.

The publication of Wang et al (2019) discloses the ultrasonic-assisted, hot- water extraction of polysaccharide from Lentinus edodes of a specific provenance. The effect of ultrasonic time, solid-liquid ratio, extraction time and temperature on yield and bioactivity was studied.

The publication of Wang et al (2020) discloses the ultrasound-assisted hot water extraction of mushrooms. The optimal ratios of material to water, soaking and ultrasonic times for the extraction were determined.

The publication of Wang et al (2021) discloses the extraction of polysaccharide from spent Lentinus edodes substrate in an effort to valorise this by-product. Polysaccharide was ultrasonically extracted from the byproduct and fractionated using gradient ethanol precipitation. Under optimal conditions the total polysaccharide content reached up to 37 mg/g.

The publication of Yang et al (2015) discloses the optimization of the ultrasound-assisted extraction of polysaccharides from Lentinus edodes. Extraction temperature was found to have the largest effect. The optimum extraction conditions were found to be an extraction temperature of 76.6°C, an extraction time of 88.8 minutes and a ratio of water to raw material of 58.1 mL/ g .

The publication of Yin et al (2018) discloses the use of a combination of enzymes, microwaves and ultrasound to extract polysaccharides from Lentinus edodes. The yields and properties of the extract are compared with the yield and properties of extracts obtained by other methods. The different extraction methods are reported to have no significant effects on the type of glycosidic bonds and monosaccharides present in the extracts. However, differences were observed by scanning electron-microscopy and when testing antioxidant activity. The publication of You et al (2013) discloses the use of ultrasound (62°C, 50 min, 640 W) , followed by hot water (120°C, 40 min) for the extraction of lentinan polysaccharide. The properties of the polysaccharides extracted following the first step and the second step are compared.

The publication of Zhang et al (2011) discloses the promoting function of ultrasound on the hot water extraction of lentinan.

The publication of Zhang et al (2016) discloses a method of extracting lentinan from Lentinus edodes using both amylase and ultrasound. The optimum extraction conditions included 1% of enzyme and ultrasound at a temperature of 60°C for a period of time of 25 minutes. Observed yields are reported to be double those obtained by traditional hot water extraction.

The publication of Zhang et al (2018) discloses a two-step ultrasonic- enhanced subcritical water extraction of polysaccharides from Lentinus edodes. The maximum extraction rate of polysaccharides was 17.34% using optimized conditions. Both the bioactivity and monosaccharide composition of the polysaccharide extracts was significantly influenced by the method of extraction employed.

The publication of Zhang et al (2020) discloses the extraction of lentinan from Lentinus edodes using an ultrasonic method after pre-treatment with 95% ethanol. The effects of hydrogen peroxide, pH and time on the solubility in water of modified lentinan were investigated.

The publication of Zhang et al (2021) discloses optimizing the extraction of polysaccharides from Lentinus edodes. The effects of extraction time, solidliquid ratio, ultrasonic time and ultrasonic power on the extraction are considered. An extraction rate (yield) of 15.86%, stated to be consistent with the "established" value of 15.97%, is achieved.

The publication of Zhao et al (2018) discloses the extraction of polysaccharides from the fruiting bodies of Lentinus edodes using ultrasound and hot water. The extraction conditions are optimized and a yield of 9.75% of Lentinus edodes polysaccharides obtained. The crude extract was purified to provide two fractions, both of which possessed potent anti-hepatitis B virus activity when assayed in vitro.

The publication of Zou et al (2013) discloses the ultrasonic-assisted hot water extraction of lentinan, the optimization of this extraction method, and the evaluation of the bioactivity of the extracts obtained by the method. The chemical structure of the lentinan did not change significantly compared with that prepared by traditional hot water extraction. The [J-glucans extracted from the fruiting bodies of fungi are known to have antioxidant, antitumor and immunomodulatory activities and extracts of shiitake mushrooms have been extensively studied. Furthermore, lentinan and other [J-glucans extracted from mushrooms are generally recognized as safe (GRAS) according to the classification of the United States Food and Drug Administration (USFDA) and can be administered both enterally and parenterally. Accordingly, the publication of Shamtsyan (2016) discloses that [J-glucans have great potential for development as nutraceuticals.

The publication of Chen et al (2021) discloses that extracts comprising f>- glucan have therapeutic activity in the high-fat diet-induced hyperlipidemia mice model by blocking the formation of radicals.

The publication of Jedinak et al (2011) discloses studies indicating that the high content of [J-glucans in mushroom extracts from different sources plays a powerful physiological activity, reducing the release of TNFa and IL-6 by LPS-challenged monocytes in vitro, and in vivo in mice, after administration of the extracts.

The publication of Maruyama et al (2021) discloses a randomized parallel controlled trial with hyperlipidemic subjects comparing the effect of two different Japanese diets. The diet including mushrooms resulted in a greater decrease of serum LDL-cholesterol , triglyceride, and insulin.

The publication of Okazaki et al (1995) discloses that the [J-glucans extracted from mushroom activate macrophages to enter the body and promote the secretion of various cytokines, thereby activating the immune function of T cells and B cells.

The publication of Shi et al (2022) discloses that p-glucan can effectively stimulate dendritic cells in animals to secrete pro-inflammatory and antiinflammatory cytokines .

The publication of Wang et al (2019) discloses that p-glucan from edible mushrooms suppresses colitis.

The publication of Yin et al (2021) discloses studies showing that [J-glucans suppress the proliferation and recurrence of cancer cells by activating the immune function of normal cells through a non-specific immune response without directly attacking cancer cells.

Methods of extracting [J-glucans in high yield with high specific bioactivities are desirable to realise their potential for use as food additives and development as nutraceuticals.

It is an object of the present invention to provide a method of extracting p- glucans in increased yield. It is an object of the present invention to provide a method of extracting [J-glucans that does not use solvents other than water. It is an object of the present invention to provide extracts comprising [J-glucans with high specific bioactivities suitable for use as food additives or in the formulation of nutraceuticals. These objects are each to be read in the alternative with the object at least to provide a useful choice in the selection of such methods and extracts.

SUMMARY OF INVENTION

In a first aspect a dried extract of the fruiting bodies of a species of fungi is provided where the extract comprises greater than 25% (w/w) f>- glucan. Preferably the extract comprises greater than 30% (w/w) p-glucan. More preferably the extract comprises between 30 and 35% (w/w) p-glucan.

The [J-glucan content is expressed as the percentage by weight of the dried extract determined using the p-glucan assay kit (yeast and mushroom) (Product code K-YBGL, Megazyme International Ireland Limited, Wicklow, Ireland) according to the directions provided with the kit and disclosed in the publication of McCleary and Draga (2016) .

Preferably the dried extract has a moisture content less than 25% (w/w) . More preferably the dried extract has a moisture content less than 20% (w/w) . Yet more preferably the dried extract has a moisture content less than 17.5 % (w/w) . Most preferably the dried extract has a moisture content between 15.0 and 17.5 % ( w/w) .

Preferably the dried extract is a freeze-dried extract. Preferably the extract is in the form of a powder. Most preferably the extract is a freeze- dried powder.

Preferably the species of fungi is an edible or medicinal species of fungi. Preferably the species of edible or medicinal fungi is selected from the group consisting of: Auricula polytricha, Coprinus comatus, Flammulina velutipes, Ganoderma sichuanense, Grifola frondosa, Inonotus obliquus, Lentinula edodes, Morchella esculenta, Pleurotus djamor, Pleurotus ostreatus, Pleurotus pulmonarius , Tremella fuciformis and Trametes versicolor . More preferably the species of edible or medicinal fungi is selected from the group consisting of: Ganoderma sichuanense , Grifola frondosa , Inonotus obliquus , and Lentinula edodes. Most preferably the species of edible or medicinal fungi is Lentinula edodes.

Preferably a solution of the extract in water at a concentration of 1 mg per mL has a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity of greater than 20%. More preferably the solution has a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity of greater than 30%. Most preferably the solution has a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity between 30 and 45%. The 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity of the solution is determined according to the method disclosed in the publication of Sakanaka and Tachibana (2006) using glutathione as a positive control.

Preferably a solution of the extract in water at a concentration of 1 mg per mL has a superoxide radical scavenging activity of greater than 40%. More preferably the solution has a superoxide radical scavenging activity of greater than 60%. Most preferably the solution has a superoxide radical scavenging activity between 60 and 70%. The superoxide radical scavenging activity of the solution is determined according to the method disclosed in the publication of Sakanaka and Tachibana (2006) using glutathione as a positive control.

Preferably a solution of the extract in water at a concentration of 1 mg per mL has an iron chelating capacity of greater than 50%. More preferably the solution has an iron chelating capacity of greater than 70%. Most preferably the solution has an iron chelating capacity between 70 and 80%. The iron chelating capacity of the solution is determined according to the method disclosed in the publication of Dinis et al (1994) using ED7A as a positive control .

Preferably a quantity of 1 mg of the extract has a f erric-reducing antioxidant power of greater than 1.5 ]aM Trolox equivalent. More preferably the quantity has a f erric-reducing antioxidant power of greater than 4.0 ]aM Trolox equivalent. Most preferably the quantity has a f erric-reducing antioxidant power between 4.0 and 4.5 ]aM Trolox equivalent. The ferric- reducing antioxidant power of the quantity is determined according to the method disclosed in the publication of Fogarasi et al (2015) using Trolox as a positive control.

In an embodiment of the first aspect a freeze-dried extract of the fruiting bodies of Lentinula edodes is provided where:

• the extract comprises between 30 and 35% (w/w) f>-glucan;

• a quantity of 1 mg of the extract has a f erric-reducing antioxidant power between 4.0 and 4.5 ]aM Trolox equivalent; and

• a solution of the extract in water at a concentration of 1 mg per mL has : a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity between 30 and 45%, a superoxide radical scavenging activity of between 60 and 70%, and an iron chelating capacity between 70 and 80% .

Preferably, the extract additionally has a total polyphenol content equivalent to 10 to 16% (w/w) gallic acid ( 10 to 16% (w/w) GAE ) . More preferably, the extract has a total phenol content equivalent to 12 to 14 % (w/w) gallic acid ( 12 to 14 % (w/w) GAE ) .

In a second aspect a nutraceutical comprising a quantity of the extract of the first aspect is provided . Preferably the quantity is between 1 and 50% (w/w) of the nutraceutical . More preferably the quantity is between 1 and 25% (w/w) of the nutraceutical . Yet more preferably the quantity is between 1 and 10% (w/w) of the nutraceutical . Most preferably the quantity is between 1 and 5% (w/w) of the nutraceutical .

In a first embodiment of the second aspect the nutraceutical is a water-based beverage consisting of a solution comprising between 1 and 5% (w/w) of the extract of the first aspect . In a second embodiment of the second aspect the nutraceutical is in the form of a tablet , e . g . , an efferves cent tablet , for use in preparing a water-based beverage . In a third embodiment of the second aspect the nutraceutical is a solid or semi-solid food comprising the extract of the first aspect . The solid food may comprise between 1 and 50% (w/w) of the extract of the first aspect . The semi-solid food may comprise between 1 and 25% (w/w) of the extract of the first aspect .

Preferably the nutraceutical is packaged with indications for use in suppres sing inflammation .

In a third aspect a method of preparing an extract of the fruiting bodies of a species of fungi where the extract comprises greater than 30% (w/w) f>- glucan is provided . The method comprises the blending with enzyme and water of undried fruiting bodies to provide an aqueous blend . The obj ective of the blending is to increase the surface area of the biomas s exposed to the enzyme . Other actions that achieve this obj ective include dicing, grating, shredding, slicing, etc .

The term "undried" will be understood in this context to mean that the fruiting bodies have not been purposefully dried, i . e . , not proces sed, and any partial desiccation is limited to that occurring naturally following harvest prior to the blending . Typically, the undried fruiting bodies will have a weight no les s than 90% (w/w) of the freshly harvested fruiting bodies .

Preferably the species of fungi is an edible or medicinal species of fungi .

Preferably the species of edible or medicinal fungi is selected from the group consisting of : Auricula polytricha , Coprinus coma tus, Flammulina velutipes, Ganoderma sichuanense, Grifola frondosa, Inonotus obliquus, Lentinula edodes, Morchella esculenta, Pleurotus djamor, Pleurotus ostreatus, Pleurotus pulmonarius , Tremella fuciformis and Trametes versicolor . More preferably the species of edible or medicinal fungi is selected from the group consisting of: Ganoderma sichuanense , Grifola frondosa , Inonotus obliquus , and Lentinula edodes. Most preferably the species of edible or medicinal fungi is Lentinula edodes.

The method comprises subjecting a pH adjusted mixture of a first quantity of the aqueous blend and a second quantity of at least one digestive enzyme to ultrasonication at a temperature of 45 to 55°C for a period of time of one to two hours to provide an ultrasonicated digest. Preferably the subjecting the pH adjusted mixture to ultrasonication is in the absence of any prior incubation of the pH adjusted mixture. Preferably the ultrasonication is at a frequency in the range 30 to 50 Hz. Preferably the pH has been adjusted with acid to a value in the range 3.5 to 5.5. More preferably the pH has been adjusted to a value about 4.5. The digestive enzymes suitable for use in the method are cellulases, f-glucanases, hemicellulases, pectinases, proteases, and xylanases such as those supplied as proprietary blends under the trade names CELLUCLAST™ 1.5 L, PECTINEX™ XXL and VISOCZYME™ L (Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark) . The proprietary blend supplied under the trade name VISOCZYME™ L has been found to be particularly suitable for use in the method. The at least one digestive enzyme used in the method is preferably a blend of f-glucanases, pectinases, hemicellulases, and xylanases .

Preferably the method comprises subjecting the ultrasonicated digest to an enzyme deactivating treatment to provide a deactivated digest. The enzyme deactivating treatment is typically a heat treatment, e.g. , autoclaving for a short period of time of 5 to 30 minutes. Preferably the method comprises removing insoluble residues from the deactivated digest to provide a solution before drying the solution to provide the extract.

The solution may be dried by any method or combination of methods effective to remove water without denaturing the p-glucan. Such methods are well known in the field of bioprocessing and can be readily evaluated for use in preparing the dried extract and include convection drying (conveyor, tray, tunnel) , spray drying, flash drying, fluidised bed drying, conduction or radiation drying (drum, roller, vacuum) and freeze drying. The absence of solvents other than water excludes the risks associated with the use of organic solvents such as alcohol when preparing the dried extract.

It has been found that the enzymatic digestion of an aqueous blend of undried fruiting bodies provides for a higher concentration of p-glucan in the extract obtained than the enzymatic digestion of dried and powdered fruiting bodies. The concentration of p-glucan in the extract can be further increased by combining the enzymatic digestion with ultrasonication. The simultaneous enzymatic digestion and ultrasonication of an aqueous blend of undried fruiting bodies has been found to be optimal for the preparation of an extract comprising high concentrations of p-glucan with high levels of desirable properties ( 1 , l-diphenyl-2-picryl hydrazyl radical scavenging activity, superoxide radical scavenging activity, iron chelating capacity, f erric-reducing antioxidant power) .

In an embodiment of the third aspect a method of preparing an extract of the fruiting bodies of Lentinula edodes where the extract comprises greater than 30% (w/w) [J-glucan is provided. The method comprises: a) blending a quantity of undried fruiting bodies of Lentinula edodes with a volume of water to provide an aqueous blend; b) mixing a quantity of the aqueous blend with a quantity of digestive enzymes and adjusting the pH of the mixture to about 4.5 to provide a pH adjusted mixture; c) subjecting the pH adjusted mixture to ultrasonication at a frequency of about 40 Hz and a temperature of about 50°C for a period of time of about two hours to provide an ultrasonicated digest; d) heating the ultrasonicated digest to a temperature for a period of time sufficient to deactivate the digestive enzymes and provide a deactivated digest ; e) removing insoluble residues from the deactivated digest to provide a solution; and then f) freeze-drying the solution to provide the extract.

In the description and claims of this specification the following abbreviations, acronyms, phrases, terms and trade names have the meaning provided or refer to the substance described: "CAS RN" means Chemical Abstracts Service (CAS, Columbus, Ohio) Registry Number; "CELLUCLAST™ 1.5 L" refers to a proprietary blend of cellulase; "comprising" means "including", "containing" or "characterized by" and does not exclude any additional element, ingredient or step; "consisting essentially of" means excluding any element, ingredient or step that is a material limitation; "consisting of" means excluding any element, ingredient or step not specified except for impurities and other incidentals; "extract" means a preparation obtained by extraction without further fractionation (save for the removal of solids and solvents) ; "extraction" means the separation of components from a mixture, e.g. , a digest, by selective solubility, e.g. , by filtration or partition between solvents; "fractionation" means the dividing into components, e.g. , by chromatography; "letinan" means a p-glucan with [31—6 branching extracted from the fruiting bodies of Lentinus edodes; "mushroom" means the fruiting body of a species of fungus; "nutraceutical" means a food containing healthgiving additives; "PECTINEX™ XXL" refers to a proprietary blend of pectin lyase; "period of time" means a continuous, uninterrupted period of time; "specific activity" means activity per unit weight; "Trolox" means 6-hydroxy- 2 , 5 , 7 , 8-tetramethylchroman-2-carboxylic acid (CAS RN 53188-07-1) ; "ultrasonication" means sonication at a frequency in the range 15 kHz to 400 kHz; and "VISOCZYME™ L" refers to a proprietary blend of f-glucanases, pectinases, hemicellulases, and xylanases. A paronym of any of the defined terms has a corresponding meaning.

The terms "first", "second", "third", etc. used with reference to elements, features, integers, or other limitations, of the matter described in the Summary of Invention, or when used with reference to alternative aspects or embodiments are not intended to imply any order of preference. Elements, features, integers, or other limitations, of the elements described in the Summary of Invention are identified in order of preference by the introductory "preferably...", "more preferably...", "yet more preferably..." and so on. Preferred combinations of elements, features, integers, or other limitations, of the matter described in the Summary of Invention are similarly identified.

Where concentrations or ratios of reagents are specified the concentration or ratio specified is the initial concentration or ratio of the reagents. Similarly, where a pH or pH range is specified, the pH or pH range specified is the initial pH or pH range. Where values are expressed to one or more decimal places standard rounding applies. For example, 1.7 encompasses the range 1.650 recurring to 1.749 recurring.

Where a parameter is expressed as being "about" a specified range or value the term is used to indicate tolerance for some variation of the specified range or value (with the proviso that the parameter dependent effect is still achieved) . In the absence of any other proviso the term "about" should be understood to indicate a tolerance of no greater than 5% above or below the upper and lower limits, respectively, of the specified range or plus or minus 5% of the specified value.

The invention will now be described with reference to embodiments or examples and the figure of the accompanying drawings page. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 . A s chematic representation of the method of preparing extracts according to Example 1 .

DESCRIPTION

The physiological effect that extracts comprising p-glucan will have in humans will depend at least in part on the source of the extract and the method of its extraction . In conventional methods of extracting [J-glucans from mushrooms the biomas s is dried and milled to a powder before extraction . The powder is then heated in alcohol , typically ethanol , two or three times to dis solve fats and weaken the cell walls . The remaining residue is then heated in water to extract the [J-glucans . Efforts to optimise the extraction of [J-glucans are dis closed in the Background Art .

A method for extracting [J-glucans from mushrooms with minimal proces sing steps and production of harmful waste has now been developed . The method uses simultaneous enzymatic digestion and ultrasonication to improve the yield of [J-glucans . The method provides extracts with high levels of desirable properties such as 1 , l-diphenyl-2-picryl hydrazyl radial s cavenging activity, superoxide radical s cavenging activity, iron chelating capacity, ferric- reducing antioxidant power . The speci fic activities are higher than might be anticipated relative to the p-glucan content of the extracts .

An es sential feature of the method is the use of harvested fruiting bodies without the drying and milling of the biomas s to a powder as used in the conventional methods . Surprisingly, the yield of p-glucan obtained by combining the use of undried fruiting bodies with simultaneous enzymatic digestion and ultrasonication is two to three times that obtained when adopting an analogous method but including the drying and milling of the biomas s prior to extraction . Without wishing to be bound by theory it is believed that drying of the fruiting bodies , e . g . , by heating, prior to extraction may cause the [J-glucans to adhere strongly to other extra- or intra-cellular components , thereby decreasing their extractability . The method now provided obviates the need to dispose of alcoholic extracts and avoids the as sociated hazards . The need to accommodate larger volumes of biomas s in the extraction proces s is offset by the avoidance of these ris ks to workplace safety and environmental ris k .

Amongst other advantages the preparation of nutraceuticals of a predetermined [J-glucan content using reduced quantities of extract that retain high levels of desired properties is provided . The use of reduced quantities of extract is particularly advantageous in the preparation of nutraceuticals such as aqueous beverages or tablets used in their preparation where the solubility and compatibility of the extract with other ingredients of the formulation of the nutraceutical is an important consideration.

In addition to the high p-glucan content, the extracts also have a high total polyphenol content (GAE) . Recent publications have shown that Shiitake mushrooms contain polyphenols, flavonoids, phenolic acids (hydroxycinnamic acid, hydroxybenzoic acid, caffeic acid, chlorogenic acid, gallic acid) , tannins, chaicones, coumarins, ergothioneine and glutathione, which are also well-known for their antioxidant activity.

EXAMPLES

Example 1 (fresh mushrooms )

Referring to Figure 1 of the accompanying drawings pages a pH adjusted mixture of an aqueous blend of undried fruiting bodies and digestive enzymes is prepared in a first period of time of 5 to 15 minutes before ultrasonication for a second period of time of 1 to 2 hours. The first and second periods of time are contiguous, i.e. , the pH adjusted mixture is subjected to enzymatic digestion and ultrasonication simultaneously (not sequentially) .

Biomass

Shiitake mushrooms sourced from local markets were cut into pieces without any pretreatment (i.e. , undried) and blended in 5 volumes of distilled water for a period of time of 5 minutes to provide an aqueous blend of biomass. A quantity of 1 Kg wet weight of mushrooms was typically used in the preparation of a batch of extract according to the claimed methods .

Enzymatic digestion

Commercially available digestive enzymes were thoroughly mixed with the aqueous blend of biomass at a ratio of l:10⁴ based on the wet weight of the mushrooms and the final pH of the mixture adjusted to 3.5 to 5.5 with citric acid, acetic acid, hydrochloric acid, or guanic acid. Proprietary blends of digestive enzymes supplied under the trade names CELLUCLAST™ 1.5 L, PECTINEX™ XXL and VISOCZYME™ L were used alone or in combination.

U1 trasonica tion

When combined with ultrasonication the pH adjusted mixture was immediately, i.e. , within 10 minutes, transferred to an ultrasonic cleaner (POWERSONIC™ 420, Hwashin) operating at a frequency of 40 kHz for a period of time of up to 2 hours at a temperature of 50°C. The resulting ultrasonicated digest was then autoclaved (121°C) for a period of time of 5 to 30 minutes (or maintained at a temperature of 60°C for a period of time of 30 minutes) to deactivate the digestive enzymes. The deactivated ultrasonicated digest was then filtered and freeze-dried to provide a powdered extract.

Comparative example 1 (dried, mushrooms )

Biomass

Shiitake mushrooms sourced from the same location as used in Example 1 were chopped, dried by hot air convection (90°C) , and pulverized to obtain a quantity of dried and powdered biomass.

Enzymatic digestion

The powdered biomass was mixed with a commercially available digestive enzyme (VISOCZYME™ L) at a ratio of l:10⁴ based on the original wet weight of the mushrooms. The mixture was shaken for a period of time of 30 minutes and the final pH adjusted to 4.5 with citric acid.

U1 trasonica tion

When combined with ultrasonication the pH adjusted mixture was transferred to an ultrasonic cleaner (POWERSONIC™ 420, Hwashin) operating at a frequency of 40 kHz for a period of time of up to 2 hours at a temperature of 50°C. The resulting ultrasonicated digest was then autoclaved (121°C) for a period of time of 5 to 30 minutes (or maintained at a temperature of 60°C for a period of time of 30 minutes) to deactivate the digestive enzymes. The deactivated ultrasonicated digest was then filtered and freeze-dried to provide a powdered extract.

Comparative example 2 (dried mushrooms)

A conventional method of preparing an extract was adopted for the purposes of providing a comparison. A quantity of the dried and powdered biomass prepared according to Comparative example 1 was initially boiled in a volume of 80% (v/v) ethanol for a period of time of 3 hours. The residue obtained was then boiled with distilled water for a period of time of 3 hours to provide a f>- glucan rich water-soluble extract. The extract was then freeze-dried to provide a powder.

Evaluation

The yields and bioactivities of the extracts prepared according to the method described as Example 1 have been compared with those of the extracts prepared according to the methods described as Comparative example 1 and Comparative

Table 1 . Comparison of the methods of preparing the extracts. example 2.

Beta-glucan yield

Water-soluble extracts obtained according to Example 1 and Comparative examples 1 and 2 were separated from the biomass residue by filtration and freeze-dried. The weights of the powdered extracts obtained were determined gravimetrically . The [3-glucan content of the powder obtained was determined using a [3-glucan assay kit (yeast and mushroom) (Product code K-YBGL, Megazyme International Ireland Limited, Wicklow, Ireland) according to the directions provided with the kit and disclosed in the publication of McCleary and Draga (2016) .

Bioactivities

• Antioxidant activity

1 , 1-Diphenyl- 2 -picryl hydrazyl (DPPH) radical scavenging

The DPPH radical scavenging activity of extracts at a concentration of 1 mg/mL was analysed in triplicate according to the method disclosed in the publication of Sakanaka and Tachibana (2006) . Glutathione (GSH) was used as the positive control.

Radical scavenging activity of the extracts was calculated according to the following equation:

% DPPH free radical scavenging = 1-(A_S/A_C) x 100 where A_s and A_c are the absorbance of the sample and control, respectively.

Superoxide radical scavenging

A volume of 80 pL of extract (1 mg/mL) was mixed with 80 pL of 50 mM TrisHCl buffer (pH 8.3) in a 96-well microplate, followed by the addition of 40 pL of 1.5 mM pyrogallol in 10 mM HC1. The rate of superoxide-induced polymerization of pyrogallol (AA_s/min) was measured as the increase in absorbance at 320 nm for a period of time of 5 minutes at a temperature of 23°C according to the method disclosed in the publication of Sakanaka and Tachibana (2006) . GSH was used as a positive control and Tris-HCl buffer was used in control experiments (AA_c/min) .

Superoxide scavenging activity of 3 independent replicates was calculated using the following equation:

% superoxide scavenging activity = [ ( AA_c/min-AA_s/min) ] / (AA_c/min) x 100

Iron chelating

The chelation of ferrous (Fe²⁺) ions by extracts (1 mg/mL) was estimated in triplicate according to the method described in the publication of Dinis et al (1994) . EDTA, a strong metal chelator, was used as a positive control.

Ferrous ion chelating capacity was calculated using the following equation:

% iron chelating capacity = (B_c-B_s) /B_c x 100 where, B_s and B_c represent the absorbance of the sample and control (excluding extract) , respectively.

Ferric-reducing antioxidant power

The antioxidant power of extracts (1 mg/mL) was estimated according to the ferric-reducing antioxidant power (FRAP) assay procedure disclosed in the publication of Fogarasi et al (2015) . Trolox (1.0 to 200 pM) was used as the positive control. The results of triplicate assays were corrected for dilution and expressed as pM of Trolox equivalents per mg sample.

Total phenolic content

The total content of polyphenols was determined for quantities of extract by the Folin-Ciocalteu method as described, for example, in the publication of Perez et al (2023) . Gallic acid ( 3 , 4 , 5-trihydroxybenzoic acid) (Sigma Aldrich, South Korea) was used as a positive control and results were expressed as mg gallic acid equivalent (GAE) /100 g of the extract.

The Folin-Ciocalteu method was used with slight modification. Reagents and sample solutions were prepared as follows: The F-C reagent was diluted to 1:10 with distilled water just before use. Sodium carbonate (7.5% w/v) was also prepared in distilled water. Stock solutions of samples (1000 pg dried extract/mL) and gallic acid (500 pg/mL) were prepared in methanol (95% (v/v) ) . Dilutions of samples (200 and 400 pg dried extract/mL) and gallic acid (50 and 100 pg/mL) were prepared from these stock solutions.

Following the general procedure, a volume of 0.5 mL of each of the dilutions or blank (methanol, 95% (v/v) ) was transferred to a screw-capped tube and mixed with a volume of 2 mL of the F-C reagent using a vortex mixer for a few seconds. After a period of time of at least 3 minutes, but no greater than 8 minutes, a volume of 4 mL of NaaCOs was added and mixed well. The sample was then transferred to a quartz cuvette and scanned in the wavelength range of 400 to 900 nm from 5 to 60 min at 5 min intervals. The obtained spectra and absorbance data were used to determine the X_max and the optimum reaction time. The total phenolic content in each sample was calculated using the formula:

C=cV/m where, C is the total phenolic content (mg GAE/g dried extract) , c is the concentration (mg/mL) of gallic acid obtained from the calibration curve, V is the volume (mL) of sample, and m is the quantity (g) of extract.

• Cytotoxicity and nitrite production

Cell culture

The RAW264.7 murine macrophage cell line was used in all in vitro studies. Cells were cultured in DMEM containing 10% FBS and 1% penicillin- streptomycin-fungizone and incubated at 37 °C under 5% CO2. All cell-based studies were conducted in at least 5 independent trials. Cell passages 7 to 15 were used in all experiments.

Cytotoxic! ty

The cytotoxicity of extracts was measured using the ( 3- ( 4 , 5-dimethylthiazol- 2-yl ) -2 , 5-diphenyltetrazolium bromide) (MTT) assay described in the publication of Mosmann (1983) . RAW264.7 murine cells ( 10⁴ cells/mL) were seeded into a 96-well culture plate and incubated for a period of time of 24 hours. The cells were then treated with extracts at 1 mg/mL for a period of time of 20 hours. To observe cell viability, 20 pL of a 500 pg/mL MTT solution was added to each well in the dark and the plate was incubated for another period of time of 4 hours. The purple formazan precipitate that developed was dissolved in 100 pL of dimethyl sulfoxide, and the absorbance was recorded at 560 nm. Cytotoxicity for each sample was expressed as a percentage of absorbance of tested samples compared to the negative control (cells grown in medium only) .

Nitrite production

The effect of extracts on the production of nitrite (as an index of nitric oxide [NO] ) was evaluated in cells of the RAW264.7 murine cell line using the Griess assay described in the publication of Kim et al (1999) . RAW264.7 murine cells (10⁵ cells/mL) were seeded into a 48-well culture plate and incubated for a period of time of 24 hours under 5% CO2 at a temperature of 37°C. Cells were then treated with a volume of 300 pL of medium only (negative control) , 1 pg/mL bacterial LPS (positive control) , or 10 pg/mL of mushroom p-glucan extracts along with 1 pg/mL LPS. Following incubation for a period of time of 24 hours, volumes of 100 pL of supernatant and 100 pL of Griess reagent were mixed in the 96-well plate and left for a period of time of 10 minutes at room temperature. Absorbance was determined at 540 nm. After generating the standard curve using the stable conversion product of NO, nitrite, the concentration of nitrite was calculated and compared to the negative and positive controls.

Statistical analysis

All experiments were performed using one pooled sample of extracts in at least three independent trials. The results were reported as means ± SD. Results were subjected to analysis of variance using SAS software (SAS Institute, Cary, NC) . Statistical significance of differences (P < 0.05) was evaluated by the least significant difference procedure.

Results

Yields

The results of investigating the optimal pH to extract p-glucan from undried fruiting bodies according to Example 1 are presented in Table 2. No significant difference was observed within the range pH 3.5 and 5.5 recommended by the supplier (Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark) of the proprietary blends of enzymes used in the preparation of the extracts. A p-glucan content of 34.68% was obtained at pH 4.5 using VISOCZYME™ L.

It was investigated whether the extraction of p-glucan could be influenced by the use of different acids. As shown in Table 3, although there was no significant difference, hydrochloric acid and citric acid showed high values of [8-glucan extraction.

Table 2. Effect of pH on extraction of p-glucan by enzymatic and ultrasonic treatment (Example 1) . All values are expressed as mean ± SD of triplicate determinations. Fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to between 3.5 and 5.5 using hydrochloric acid. The pH adjusted blend was ultrasonicated (POWERSONIC™ 420) at a frequency of 40 kHz for a period of time of 2 hours at a temperature of 50°C. Values with the same superscript are not significantly different at p < 0.05.

Table 3. Effect of acid on extraction of (3-glucan by enzymatic and ultrasonic treatment (Example 1) . All values are expressed as mean ± SD of triplicate determinations. Fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using acetic acid, citric acid, hydrochloric or guanic acid. The pH adjusted blend was ultrasonicated (POWERSONIC™ 420) at a frequency of 40 kHz for a period of time of 2 hours at a temperature of 50°C.

Table 4. Optimal incubation time of enzymatic and ultrasonic treatment to extract [3- glucan (Example 1) . All values are expressed as mean ± SD of triplicate determinations. Fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was ultrasonicated (POWERSONIC™ 420) at a frequency of 40 kHz for a period of time of 30, 60 or 120 minutes at a temperature of 50°C. Values with different superscripts are significantly different at p < 0.05.

Table 5. Comparison of yields of [3-glucan extracted from undried (fresh) (Example 1) and dried and powdered (Comparative examples 1 and 2) fruiting bodies. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Dried and powdered fruiting bodies were immersed in acidic water (pH of 4.5) for a period of time of 10 minutes. A mixture of the immersed powder and VISCOZYME™ L was then agitated for a period of time of 30 minutes before ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz for a period of time of 2 hours. Values in a column with different superscripts are significantly different at p < 0.05.

The effect of incubation time on the extraction of [3-glucan using simultaneous enzymatic and ultrasonic treatment is presented in Table 4. The extraction of [3-glucan was significantly increased at 1 and 2 hours compared to 30 minutes (p < 0.05) . The results indicate the longer the exposure to

Table 6. Effect of blends of enzymes to extract p-glucan. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Values with the same superscripts are not significantly different at p < 0.05.

Table 7. Antioxidant DPPH free radical scavenging activity of extracts of undried (Example 1) and dried (Comparative examples 1 and 2) fruiting bodies. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at a temperature of 50°C with a frequency of 40 kHz. Values with different superscripts are significantly different at p < 0.05. simultaneous enzymatic and ultrasonic treatment the higher the yield of [3- glucan .

The data presented in Table 5 shows that the simultaneous enzymatic and ultrasonic treatment used in Example 1 significantly (p < 0.05) increased the yield of [3-glucan over that obtained according to the methods used in Comparative examples 1 and 2. In addition, the simultaneous enzymatic and ultrasonic treatment showed a significant (p < 0.05) increase relative to

Table 8. Antioxidant superoxide radical scavenging activity of extracts of dried and undried fruiting bodies. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p < 0.05. enzymatic treatment alone. Surprisingly, the yield of [3-glucan from the enzymatic and ultrasonic treatment of dried and powdered fruiting bodies ( cf . the sequential treatment disclosed in the publication of Ke (2015) ) was significantly (p < 0.05) lower than that obtained for undried (fresh) fruiting bodies using enzymatic treatment alone. The yield of [3-glucan from undried (fresh) fruiting bodies was significantly (p < 0.05) higher than that obtained for dried and powdered fruiting bodies when using either enzymatic treatment alone or a sequential enzymatic and ultrasonic treatment. The use of blends of enzymes to extract [3-glucan showed similar results regardless of whether one, two or three enzymes were used. As shown in Table 6 high yields of [3-glucan are obtainable when VISCOZYME™ L is mixed with other enzymes such as CELLUCLAST™ 1.5 L or PECTINEX™ XXL or when VISCOZYME™ L is used alone.

Bioactivities

The radical scavenging activities of extracts were evaluated using DPPH and superoxide radical scavenging assays. DPPH is a relatively stable free radical, widely used for in vitro evaluation of antioxidant activity. Table 7 shows that the extracts obtained according to Example 1 using either the simultaneous treatment (VISCOZYME™ L & ultrasonication) or enzyme treatment (VISCOZYME™ L) alone showed significantly higher DPPH scavenging activity than the extracts obtained according to the Comparative examples 1 and 2 (dried, powdered biomass) . Furthermore, the extracts obtained according to Example 1 using the simultaneous treatment (VISCOZYME™ L & ultrasonication) showed significantly higher DPPH scavenging activity than the extracts obtained according to Example 1 using enzyme (VISCOZYME™ L) alone (p < 0.05) . Regardless of the method of extraction, the antioxidant DPPH free radical scavenging activity of extracts from undried mushrooms was significantly greater than that of extracts of dried and powdered mushrooms (p < 0.05) .

Table 8 shows that the extracts obtained according to Example 1 using either the simultaneous treatment (VISCOZYME™ L & ultrasonication) or enzyme treatment (VISCOZYME™ L) alone showed significantly higher superoxide radical scavenging activity than the extracts obtained according to Comparative examples 1 and 2 (dried, powdered biomass) . The combination of enzymatic and ultrasonic treatment provided a f>-glucan extract with a significantly higher superoxide scavenging activity than that provided by the use of the enzyme treatment alone (p < 0.05) . Regardless of the extraction method, the antioxidant superoxide radical scavenging activity of extracts of undried fruiting bodies was significantly greater than that of dried and powdered mushrooms (p < 0.05) .

Metal chelation

Using the positive control of EDTA, the iron chelation capacities of extracts of undried fruiting bodies prepared according to Example 1 (VISCOZYME™ L) with (73.5 %) and without (55.4%) ultrasonication were significantly higher than those of extracts of dried fruiting bodies prepared according to Comparative example 1 (30.6%) or Comparative example 2 (21.3%) at 1.0 mg/mL (Table 9) . The iron chelation capacity of extracts prepared according to Example 1 was comparable to the positive control EDTA (75%) at 10 pg/mL. The chelation capacity of extracts prepared according to Example 1 was also significantly higher than that of the extract prepared according to Comparative example 1 with ultrasonication (p < 0.05) . Regardless of the method of preparation, the antioxidant iron chelation capacity of extracts prepared from undried fruiting bodies (Example 1) was significantly greater than that of extracts prepared from dried and powdered fruiting bodies (Comparative examples 1 and 2) (p < 0.05) .

Reducing capacity

The ferric reducing antioxidant power (FRAP) assay measures the ability of extracts of fruiting bodies to reduce Fe³⁺ to Fe²⁺. Table 10 shows a wide range of FRAP values from 0.56 to 4.2 ]aM TE/mg sample. Extracts prepared according to the ethanol-hot water method (Comparative example 2) showed a very low FRAP value at 0.56 ± 0.08 ]aM TE/mg. A significant difference in the FRAP values for extracts prepared according to Example 1 (VISCOZYME™ L & ultrasonication) was observed (p < 0.05) . Once again, regardless of the extraction method, the FRAP value for extracts prepared from undried fruiting bodies (Example 1) was significantly greater than that observed for extracts

Table 9. Antioxidant iron chelation capacity of extracts of fruiting bodies. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz.

Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p < 0.05.

Table 10. Fer ric reducing antioxidant power (FRAP) value of mushroom (3-glucan extract. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p < 0.05. TE (Trolox equivalent) . prepared from dried and powdered mushrooms (Comparative examples 1 and 2) (p < 0.05) .

Total polyphenolics

Dried extracts were prepared according to the method described in Example 1.

Table 11 shows the [3-glucan and total polyphenol content determined for a quantity of 100 g of the extract.

Table 11. Bet a-glucan and total polyphenol content of a quantity (100 g) of mushroom [3-glucan extract prepared according to the method described in Example 1. All values are expressed as mean ± SD of triplicate determinations.

Table 12. Viability of macrophage cells (RAW264.7) following exposure to mushroom [3- glucan extract. All values are expressed as mean ± SD of triplicate determinations. Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p < 0.05.

In vitro cytotoxicity

The viability of macrophage cells (RAW264.7) was maintained following incubation in the presence of each of the extracts of fruiting bodies (Table

12) .

In vitro anti-inflammatory activity

The LPS-stimulated nitic oxide (NO) production was suppressed following incubation in the presence of each of the extracts of fruiting bodies (Table

13) . The use of [3-glucan containing extracts as anti-inflammatory agents is suggested .

Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specified elements, features, integers, or other limitations, of the matter described in the Summary of Invention, such equivalents are incorporated as if specifically referred to

Table 13. Nitrite content of macrophage cell (RAW264.7) supernatants following exposure to mushroom (3-glucan extract. All values are expressed as mean ± SD of triplicate determinations . Undried fruiting bodies and enzymes were mixed in a blender for a period of time of 10 minutes and the final pH adjusted to 4.5 using citric acid. The pH adjusted blend was then incubated for a period of time of 120 minutes at a temperature of 50°C with or without simultaneous ultrasonication (POWERSONIC™ 420) at a frequency of 40 kHz. Alternatively, dried and powdered fruiting bodies were either extracted with ethanol and water or immersed in acidic water (pH 4.5) for a period of time of 10 minutes before adding a quantity of digestive enzymes (VISCOZYME™ L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONIC™ 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p < 0.05. in this specification. Variations and modifications to the embodiments or examples that include elements, features, integers, or other limitations, disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description included in this specification may be provided in the alternative or in combination in these different embodiments of the invention.

INDUSTRIAL APPLICABILITY

Methods of preparing extracts of mushrooms comprising high concentrations of [3-glucan and high levels of desired properties ( 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity, superoxide radical scavenging activity, iron chelating capacity, f erric-reducing antioxidant power) of use in the preparation of nutraceuticals are provided.

INCORPORATION BY REFERENCE

For the purposes of 37 C.F.R. 1.57 of the United States Code of Federal Regulations the disclosures of the following publications (as more specifically identified under the heading "Referenced Publications") are incorporated by reference: Dinis et al (1994) , Fogarasi et al (2015) , McCleary and Draga (2016) and Sakanaka and Tachibana (2006) . REFERENCED PUBLICATIONS

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Claims

1) An alcohol-free method of preparing a dried extract of Lentinula edodes comprising between 30 and 35% (w/w) p-glucan, the method comprising:

(a) blending a quantity of undried fruiting bodies of Lentinula edodes with a volume of water to provide an aqueous blend;

(b) mixing a quantity of the aqueous blend with a quantity of digestive enzymes and adjusting the pH of the mixture to a value in the range 3.5 to 5.5 to provide a pH adjusted mixture;

(c) subjecting the pH adjusted mixture to ultrasonication at a temperature of 45 to 50°C for a period of time of about one to two hours to provide an ultrasonicated digest;

(d) heating the ultrasonicated digest to a temperature for a period of time sufficient to deactivate the digestive enzymes and provide a deactivated digest;

(e) removing insoluble residues from the deactivated digest to provide a solution; and then

(f) drying the solution to provide the dried extract, where a quantity of 1 mg of the dried extract has a f erric-reducing antioxidant power between 4.0 and 4.5 ]aM Trolox equivalent and a solution of the dried extract in water at a concentration of 1 mg per mL has a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity between 30 and 45%, a superoxide radical scavenging activity of between 60 and 70%, and an iron chelating capacity between 70 and 80%.

2) The method of claim 1 where the adjusting the pH of the mixture is to a value about 4.5.

3) The method of claim 1 or 2 where the ultrasonication is at a frequency of about 40 kHz.

4) The method of any one of claims 1 to 3 where the temperature is about 50°C and the period of time is about two hours.

5) The method of any one of claims 1 to 4 where the drying is freeze-drying.

6) A dried extract of the undried fruiting bodies of Lentinula edodes comprising between 30 and 35% (w/w) f>-glucan where a quantity of 1 mg of the extract has a f erric-reducing antioxidant power between 4.0 and 4.5 ]jM Trolox equivalent. 7) The extract of claim 6 where a solution of the extract in water at a concentration of 1 mg per mL has:

• a 1 , l-diphenyl-2-picryl hydrazyl radial scavenging activity between 30 and 45%,

• a superoxide radical scavenging activity of between 60 and 70%, and

• an iron chelating capacity between 70 and 80%.

8) The extract of claim 6 or 7 where dried extract is a freeze-dried powder.

9) A nutraceutical comprising the dried extract of any one of claims 6 to 8.

10) The nutraceutical of claim 9 where the nutraceutical is a beverage.