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WO2025114975A1 - Procédé amélioré de préparation d'extraits d'edodes de lentinula et extraits préparés par le procédé - Google Patents

Procédé amélioré de préparation d'extraits d'edodes de lentinula et extraits préparés par le procédé Download PDF

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Publication number
WO2025114975A1
WO2025114975A1 PCT/IB2024/062068 IB2024062068W WO2025114975A1 WO 2025114975 A1 WO2025114975 A1 WO 2025114975A1 IB 2024062068 W IB2024062068 W IB 2024062068W WO 2025114975 A1 WO2025114975 A1 WO 2025114975A1
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extract
extraction
period
extracts
dried
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Hoon Sunwoo
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Vetanda Korea Ltd
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Vetanda Korea Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A23L31/10—Yeasts or derivatives thereof
    • A23L31/15—Extracts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/21—Removal of unwanted matter, e.g. deodorisation or detoxification by heating without chemical treatment, e.g. steam treatment, cooking
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • 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.
  • 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) .
  • Beta-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 .
  • [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.
  • 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 .
  • 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.
  • 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.
  • 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 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 .
  • Lu et al 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.
  • Vezaro et al 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 Yin et al (2016) 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.
  • 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.
  • Zhang et al 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.
  • 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.
  • 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.
  • a dried extract of the fruiting bodies of a species of fungi where the extract comprises greater than 25% (w/w) f>- glucan.
  • 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) .
  • 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) .
  • the dried extract is a freeze-dried extract.
  • the extract is in the form of a powder.
  • the extract is a freeze- dried powder.
  • the species of fungi is an edible or medicinal species of fungi.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the extract comprises between 30 and 35% (w/w) f>-glucan
  • 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% .
  • 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 ) .
  • a nutraceutical comprising a quantity of the extract of the first aspect.
  • 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 .
  • 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 .
  • the nutraceutical is in the form of a tablet , e . g . , an efferves cent tablet , for use in preparing a water-based beverage .
  • 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 .
  • the nutraceutical is packaged with indications for use in suppres sing inflammation .
  • 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 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 .
  • 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 .
  • the undried fruiting bodies will have a weight no les s than 90% (w/w) of the freshly harvested fruiting bodies .
  • the species of fungi is an edible or medicinal species of fungi .
  • 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 .
  • 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.
  • the subjecting the pH adjusted mixture to ultrasonication is in the absence of any prior incubation of the pH adjusted mixture.
  • the ultrasonication is at a frequency in the range 30 to 50 Hz.
  • 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 CELLUCLASTTM 1.5 L, PECTINEXTM XXL and VISOCZYMETM L (Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark) .
  • the proprietary blend supplied under the trade name VISOCZYMETM 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 .
  • 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.
  • 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.
  • 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.
  • convection drying conveyor, tray, tunnel
  • spray drying spray drying
  • flash drying fluidised bed drying
  • conduction or radiation drying drum, roller, vacuum
  • freeze drying 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.
  • a method of preparing an extract of the fruiting bodies of Lentinula edodes where the extract comprises greater than 30% (w/w) [J-glucan 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-
  • 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; “PECTINEXTM 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;
  • concentration or ratio specified is the initial concentration or ratio of the reagents.
  • pH or pH range specified is the initial pH or pH range.
  • 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.
  • Figure 1 A s chematic representation of the method of preparing extracts according to Example 1 .
  • 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 .
  • 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 .
  • 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 .
  • 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.
  • the extracts In addition to the high p-glucan content, the extracts also have a high total polyphenol content (GAE) .
  • GAE total polyphenol content
  • 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.
  • Example 1 fresh mushrooms
  • 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) .
  • 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 .
  • the pH adjusted mixture When combined with ultrasonication the pH adjusted mixture was immediately, i.e. , within 10 minutes, transferred to an ultrasonic cleaner (POWERSONICTM 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.
  • 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.
  • the powdered biomass was mixed with a commercially available digestive enzyme (VISOCZYMETM L) at a ratio of l:10 4 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.
  • the pH adjusted mixture When combined with ultrasonication the pH adjusted mixture was transferred to an ultrasonic cleaner (POWERSONICTM 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.
  • 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.
  • 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) .
  • 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.
  • % 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.
  • 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 [ ( AA c /min-AA s /min) ] / (AA c /min) x 100
  • % 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10 4 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.
  • 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 (POWERSONICTM 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 (POWERSONICTM 420) at a frequency of 40 kHz for a period of time of 2 hours at a temperature of 50°C.
  • POWERSONICTM 420 ultrasonicated
  • 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 (POWERSONICTM 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.
  • a mixture of the immersed powder and VISCOZYMETM L was then agitated for a period of time of 30 minutes before ultrasonication (POWERSONICTM 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.
  • 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 (VISCOZYMETM L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONICTM 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 .
  • Example 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
  • 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 (VISCOZYMETM L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONICTM 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 .
  • 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 (VISCOZYMETM L & ultrasonication) or enzyme treatment (VISCOZYMETM L) alone showed significantly higher DPPH scavenging activity than the extracts obtained according to the Comparative examples 1 and 2 (dried, powdered biomass) .
  • the extracts obtained according to Example 1 using the simultaneous treatment showed significantly higher DPPH scavenging activity than the extracts obtained according to Example 1 using enzyme (VISCOZYMETM L) alone (p ⁇ 0.05) .
  • 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 (VISCOZYMETM L & ultrasonication) or enzyme treatment (VISCOZYMETM 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) .
  • 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) .
  • the iron chelation capacities of extracts of undried fruiting bodies prepared according to Example 1 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) .
  • the antioxidant iron chelation capacity of extracts prepared from undried fruiting bodies was significantly greater than that of extracts prepared from dried and powdered fruiting bodies (Comparative examples 1 and 2) (p ⁇ 0.05) .
  • the ferric reducing antioxidant power (FRAP) assay measures the ability of extracts of fruiting bodies to reduce Fe 3+ to Fe 2+ .
  • 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 (VISCOZYMETM 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
  • 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 (VISCOZYMETM L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONICTM 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p ⁇ 0.05.
  • POWERSONICTM 420 simultaneous ultrasonication
  • 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 (VISCOZYMETM L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONICTM 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 Terolox equivalent
  • prepared from dried and powdered mushrooms Comparative examples 1 and 2) (p ⁇ 0.05) .
  • Table 11 shows the [3-glucan and total polyphenol content determined for a quantity of 100 g of the extract.
  • 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 (POWERSONICTM 420) at a frequency of 40 kHz.
  • 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 (VISCOZYMETM L) , incubated with shaking for a period of time of 30 minutes followed by ultrasonication for a period of time of 2 hours (POWERSONICTM 420) at 50°C with a frequency of 40 kHz. Values in a column with different superscripts are significantly different at p ⁇ 0.05.
  • 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 (POWERSONICTM 420) at a frequency of 40 kHz.
  • Vezaro et al (2022) Ultrasound-assisted extraction of beta-glucans from Lentinula edodes using natural deep eutectic solvent and water Journal of Chemical Technology and Biotechnology 97 (12) , 3306-3316.

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Abstract

L'invention concerne un procédé sans alcool d'extraction de β-glucanes tels que le lentinane à partir de champignons. Le procédé exclut le séchage et la pulvérisation des champignons avant l'extraction. L'utilisation simultanée de la digestion enzymatique et de l'ultrasonication fournit des extraits ayant des niveaux élevés de propriétés souhaitées parmi lesquelles une activité de piégeage des radicaux de 1,1-diphényl-2-picryl hydrazyle, une activité de piégeage des radicaux de superoxyde, une capacité de chélation du fer et un pouvoir antioxydant de réduction ferrique.
PCT/IB2024/062068 2023-12-01 2024-12-02 Procédé amélioré de préparation d'extraits d'edodes de lentinula et extraits préparés par le procédé Pending WO2025114975A1 (fr)

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CN106117380A (zh) * 2016-06-22 2016-11-16 高枫 一种香菇多糖的提取方法
CN109160956A (zh) * 2018-11-16 2019-01-08 绩溪县徽菜宝生物科技有限公司 一种香菇多糖的提取方法
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