WO2021158179A1 - SCHIZOPHYLLUM COMMUNE STRAIN PLNP13 AND β-GLUCAN OBTAINED FROM CO-CULTURING THE STRAIN WITH GANODERMA LUCIDUM - Google Patents

Abstract

The present invention relates to a process of mycelial co-cultivation of Schizophyllum commune strain PLNP13 and Ganoderma lucidum strain LZ, and to the β-glucan obtained therefrom. The invention provides a β-1,6-branched-β-1,3-glucan obtained from the co-culture of S. commune PLNP13 and G. lucidum LZ in a liquid medium. The β-glucan exhibits anti-oxidant, anti-inflammatory, and anti-diabetic properties. The invention further provides compositions comprising the β-glucan for cosmeceutical and therapeutic applications.

Description

SCHIZOPHYLLUM COMMUNE STRAIN PLNP13 AND b-GLUCAN OBTAINED FROM CO-CULTURING THE STRAIN WITH GANODERMA LUCIDUM

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application serial number 62/971 ,855 filed on February 7, 2020, U.S. provisional patent application serial number 62/971 ,853 filed on February 7, 2020 and U.S. provisional patent application serial number 62/971 ,849 filed on February 7, 2020. The contents of the aforementioned applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to the field of natural products, and more specially, to Ganoderma lucidum extract and methods of preparation.

BACKGROUND OF THE INVENTION

[0003] It has been well documented that excessive physical/chemical stimuli and stress from external environments, dehydration, malnutrition, and the like results in deterioration of the normal function of skin thus causing skin-aging and skin-damage. In order to keep the skin healthy and beautiful by preventing such adverse events, a great deal of efforts have been made to effectively suppress the skin damage by the use of beneficial substances obtained from various animals, plants and microorganisms. Throughout the world, extensive research has focused on applications of mushroom-derived physiologically active substances, b-glucans, for the prevention of skin aging. For example, it has been reported that extracts of mushrooms have a moisture retention ability, inhibition ability of melanin-formation and screening ability of ultraviolet and thereby can be incorporated in a cosmetic composition to impart skin-whitening effect or anti-oxidative action.

[0004] Mushroom b-glucan is a carbohydrate polymer derived from the cell wall of mushrooms b-glucan is a polysaccharide of glucopyranose molecules linked though b (1 3), b (1 4) 0G b (1 6) linkages. A large number of closely related b-glucans exhibit a similar branching pattern such as schizophyllan, sderoglucan, pendulan, cinerian, laminarin, lentinan and pleuran, all of which exhibit a linear main chain of b- D-(1-3)-glucopyranosyl units with a single b-D- glucopyranosyl unit (1-6) linked to a b-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3.

[0005] Among them, schizophyllan is naturally derived b-1 ,6-branched b-1 ,3-glucan produced by Schizophyllum commune ( Rau 1999; Rau 2002), and is commercially known for its cosmetic applications, such as soothing the skin and decreasing inflammation, irritation, and other UV-induced damages {Kumari et ai, 2008). A composition comprising schizophyllan with long-acting moisturizing and whitening functions has been reported in CN Patent Publication No. 108096096A. Currently there are a few suppliers of commercial schizophyllan for cosmetic applications. High-purity schizophyllan is available commercially from some chemical companies. However, due to its high cost, it is solely used for research of pharmaceutical applications.

[0006] Ganoderan, another mushroom derived b-glucan obtained from Ganoderma lucidum, has been shown to be effective in inducing immune response of the host organism for prevention and reduction of malignant tumors {Wang etal., 2014). It also has pharmacological activities related to cosmetics, such as antioxidant activity, inhibition of melanin, antibacterial activity, and regulation of inflammatory mediators {Li, LD., Mao, PW., Shao, KD. etal. Ganoderma proteins and their potential applications in cosmetics. Appl Microbiol Biotechnol 103, 9239-9250 (2019)).

[0007] However, because of the high production cost involved in cultivating, isolating and purifying these bioactive compounds, the commercial grade b-glucan form Ganoderma lucidum and Schizophyllum commune is very expensive. Conventional methods have not produced yields that are suitable for use in commercial skin-care products. Therefore, an objective of the present invention is to provide a high-potency b-glucan from mushroom sources such as Ganoderma lucidum and Schizophyllum commune, which can achieve the desired results at low concentrations, thereby reducing its dosage and its cost in cosmetic product formulation. Another objective is to enhance the therapeutical properties of the beta- glucan through the cocultivation of Ganoderma lucidum and Schizophyllum commune, thereby improving its effectiveness in medical applications.

[0008] As such, many attempts have been actively made to identify novel high- performance Schizophyllum commune that can produce b-glucan in economically significant way and also shows better characteristics, for use in the application of cosmetic as well as medicinal applications.

[0009] To this end, the inventors of the present application have developed a novel method through the cocultivation of Ganoderma lucidum and Schizophyllum commune to generate a novel beta-glucan. The present inventors have also identified and characterized the novel b-glucan, obtained by cocultivation of a Schizophyllum commune strain with Ganoderma lucidum, which has several therapeutic properties, including ameliorating skin damage, enhancing skin cell rejuvenation and exhibiting anti-diabetic property, in addition to anti-aging, and anti inflammatory effects.

SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention relates to a b-glucan obtained from Schizophyllum commune PLNP13 that is about 80% glucose and 10.5% mannose.

[0011] In some embodiments, the b-glucan is obtained by submerged cultivation of Schizophyllum commune PLNP13 in a liquid culture.

[0012] In one aspect, the present invention relates to a process for obtaining a b-1 , e-branched-b-1 , 3-glucan, having molecular weight of 0.5 to 2.0 MDa, and exhibiting enhanced anti-oxidant, anti-inflammatory and anti-diabetic properties. The process can include steps of:

(i) co-culturing in a liquid medium the Schizophyllum commune PLNP13 with Ganoderma lucidum LZ under conditions allowing said microorganism to produce said b-glucan; and (ii) recovering the polysaccharide from the medium. [0013] In some embodiments, the liquid medium comprises from 1 to 20% by weight of glucose, from 0.1 to 5% by weight of yeast extract, from 0.1 to 5% by weight of malt extract, from 0.1 to 1% by weight of (NH HRO^ or of (NH bO^ from 0.1 to 1% by weight of KH2PO4 and from 0.1 to 0.5% by weight of MgSC>47H2O.

The process can include a step of adding glucose to the liquid medium in an amount of 1 to 20% after 3 to 5 days of culture and continuing the culture for an additional 1 to 5 days.

[0014] In some embodiments, liquid medium includes 5% by weight of glucose, 1% by weight of yeast extract, 2% by weight of malt extract, 0.2% by weight of (NH )₂HR04 or of (NH4)2SC>4, 0.2% by weight of KH2PO4 and 0.1% by weight of KH2PO4. The process can include a step of adding 5% of glucose to the liquid medium after 3 to 5 days of culture and continuing the culture for 1 to 5 additional days.

[0015] In some embodiments, the co-culture-derived b-glucan is obtained by a process comprising step of:

1) inoculating seeds obtained by culture of the mycelium of Schizophyllum commune PLNP 13 and Ganoderma lucidum LZ, in an amount of 5 to 20% (v/v), and more preferably 10% (v/v), into a liquid medium;

2) co-culturing the mycelial cells in the liquid medium for 10 to 14 days;

3) recovering, separating and purifying thus-obtained b-glucan from the liquid medium.

[0016] In some embodiments, the co-culturing process is carried out at a temperature of 20 to 35° C, an agitation rate of 100 to 400 rpm, and an aeration rate of 0.5 to 2 vvm.

[0017] In a further aspect, the present invention relates to an improved b-1 , 6- branched-b-1 , 3-glucan, obtained from the co-culturing process as defined above, having a molecular weight of 0.5 to 2.0 MDa and exhibiting enhanced anti-oxidant, anti-inflammatory and anti-diabetic properties. [0018] In another aspect, the present invention relates to a composition comprising the b-glucan as defined above for cosmeceutical and therapeutic applications.

[0019] In some embodiments, the composition is for external application having a formulation of an emollient lotion, a nourishing lotion, a nourishing cream, a massage cream, a pack or a gel, a body lotion, an ointment, a gel, a cream, a patch, a shampoo-type cleanser, a skin cleanser or a spray.

[0020] In some embodiments, the composition is formulated as an oral solid form such as a powder, capsule, tablet, caplet or sachets.

[0021] In some embodiments, the composition is formulated into a cosmetic product.

[0022] In some embodiments, the composition is formulated into a therapeutic product.

[0023] In some embodiments, the composition is for use in skin-care products.

[0024] In some embodiments, the composition is formulated for use in controlling of blood sugar levels and reduction of glycemic index diabetic patients.

[0025] In another aspect, the present invention relates to the use of the b-glucan as defined above for manufacture of skin care or therapeutic products.

[0026] In some embodiments, the processes of preparation and use of beta-glucan includes the following steps: (i) cultivating monoculture of Ganoderma lucidum and Schizophyllum commune strains on potato dextrose agar (PDA) plates.

(ii) ten fully grown agar disks (5 mm) of Ganoderma lucidum and Schizophyllum commune are respectively inoculated in the seed culture medium and cultivated for 7 days (iii) beta-glucan is prepared by inoculating seed culture of Ganoderma lucidum and Schizophyllum commune strains, 10% (v/v) each, into the coculture medium, and cultivation for 10 days (iv) Beta-glucan is harvested by ethanolic precipitation and centrifugation, and water washing. (v)The prepared beta-glucan can thereafter be used in at least one skin care products of hydrating, moisturizing, anti-aging, anti stress, anti-oxidant, anti-pollution, anti-inflammatory, Ultra Violet (UV) protection or skin-regenerations (vi) The prepared beta-glucan can also be used in the formulation of anti-diabetic health supplement products.

[0027] In another aspect, the present invention relates to a method for treating skin damage due to aging, stress, pollution, UV radiation, and the like, by applying the composition as defined above.

[0028] In another aspect, the present invention relates to a method of cultivation of a mushroom coculture comprising Ganoderma lucidum and Schizophyllum commune strains. The seed culture of Ganoderma lucidum and Schizophyllum commune is conducted in the fermentation medium containing 20-40 g/L glucose, 1- 10 g/L peptone, 1 -10 g/L yeast extract and were cultured at 28 °C for 5 to 10 days. The seed culture of Ganoderma lucidum and Schizophyllum commune can be inoculated to the fermentation medium at 1-20% (v/v) to produce a novel beta- glucan. The beta-glucan can contain 45-90% of glucose, 10-20% galactose and 10- 20% mannose. The beta-glucan presents greater therapeutic benefits including higher anti-inflammatory and high anti-oxidant property than beta-glucan obtained by the monoculture of Schizophyllum commune.

BRIEF DESCRIPTION OF THE FIGURES

[0029] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the Figures. However, the disclosure is not limited to specific methods and instrumentalities disclosed herein.

[0030] FIG. 1 is a graph 100 showing the comparative yield of mycelia and exo polysaccharides (EPS), from mono-culture of Ganoderma lucidum (LZ), mono- culture of Schizophyllum commune (PLNP13), and co-culture of Ganoderma lucidum (LZ) and Schizophyllum commune (PLPN13), in accordance with the disclosed embodiments.

[0031] FIG. 2 is a graph 200 showing viscosity of co-culture broth of Ganoderma lucidum (LZ) and Schizophyllum commune (PLNP13) submerged fermentation, in accordance with the disclosed embodiments.

[0032] FIG. 3 is a graph 300 showing mycelia production by co-culture of Ganoderma lucidum (LZ) and Schizophyllum commune (PLNP13), in accordance with the disclosed embodiments.

[0033] FIG. 4 is a graph 400 showing b-glucan yield by co-culture of Ganoderma lucidum (LZ) and Schizophyllum commune (PLNP13), in accordance with the disclosed embodiments.

[0034] FIG. 5 is a graph 500 showing antioxidant activity of mono-culture of Schizophyllum commune (PLNP13) and its co-culture with Ganoderma lucidum (LZ), in accordance with the disclosed embodiments.

[0035] FIG. 6A and FIG. 6B are graphs 600 and 650 showing anti-inflammatory activity of mono-culture of Schizophyllum commune (PLNP13) and its coculture with Ganoderma lucidum (LZ), (A), the graph 600 inhibition to cyclooxygenase 1 (COX-1); (B), the graph 650 inhibition to cyclooxygenase 2 (COX-2), in accordance with the disclosed embodiments.

[0036] FIG. 7 is a graph 700 showing the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), at different concentrations on cell viability of the human telomerase reverse transcriptase (hTERT)-immortalized foreskin fibroblast cell line, BJ-5ta, in accordance with the disclosed embodiments. The commercial Schizophyllum commune beta-glucan, schizophyllan, was used as the control. [0037] FIG. 8 is a graph 800 showing the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after Ultra Violet (UV) irradiation, in accordance with the disclosed embodiments. The commercial Schizophyllum commune beta- glucan, schizophyllan, was used as the control.

[0038] FIG. 9 is a graph 900 showing the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after cell starvation, in accordance with the disclosed embodiments. The commercial Schizophyllum commune beta-glucan, schizophyllan, was used as the control.

[0039] FIG. 10A and FIG. 10B are graphs 950 and 960 showing the effects of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), at different concentrations on glucose stimulated insulin secretion using human pancreatic cell line, RIN-m5F, in accordance with the disclosed embodiments. RIN- m5F cells were either cultured in basal (4 mM) or stimulated (20 mM) glucose concentrations in the presence of beta-glucan samples at varying concentration (mg/ml). (A): beta-glucan from mono-culture of Schizophyllum commune (PLNP13); (B): beta-glucan from coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ).

[0040] FIG. 11 shows the Fourier-Transform Infrared (FTIR) spectrum 970 of commercial schizophyllan and the crude EPS obtained by the coculture of Ganoderma lucidum (LZ) and Schizophyllum commune (PLNP13).

[0041] FIG. 12 shows the details of the FTIR spectrum 978 of the crude EPS obtained by the coculture of Ganoderma lucidum (LZ) and Schizophyllum commune (PLNP13).

[0042] FIG. 13 is a flowchart 980 showing the steps of preparing, isolating and using the beta-glucan, in accordance with the disclosed embodiments.

DEFINITIONS

[0043] The following words and terms used herein shall have the meaning indicated:

[0044] The term “aeration” refers to introduction of air into a liquid such as a culture. The unit 'vvm' is used for bioreactor culture. The first V stands for volume of air (e.g. liter) ; the second V stands for per unit of medium (e.g. liter); 'm' stands for per unit of time (e.g. minute). For example, 2 vvm (l/l/m) means in 1 minute time there is 2 liter of air passing through 1 liter of medium.

[0045] The term “co-culture” or “microorganism co-culture” refers to the cultivation of two or more microorganisms in the same confined environment.

[0046] The term “beta-glucan” or “b-glucan” refers to a soluble fiber found naturally in some mushrooms. A polysaccharide, beta-glucan may offer a number of health benefits, including lowering cholesterol, improving blood sugar management, and boosting the immune system.

[0047] As used herein, reference to “isolated” means that the strain is removed from the environment in which it exists in nature. Thus, the isolated strain can exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an agricultural carrier.

[0048] The term "obtained or derived from" as used herein is meant to be used inclusively. That is, it is intended to encompass any biologically active substance obtained from the Schizophyllum commune strain PLNP13.

[0049] Unless specified otherwise, the terms “consisting of”, "comprising", "comprise" and “contain”, and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements. [0050] Throughout this disclosure, certain embodiments can be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.

[0051] The invention illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including",

"containing", and so on. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

DETAILED DESCRIPTION OF THE INVENTION

[0052] In a following description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific example in which the invention can be practiced. It is to be understood that other embodiments can be utilized and structural changes can be made without departing from the scope of the present invention.

[0053] Schizophyllum commune, a basidiomycetous fungus, is a common invader of rotten wood. This fungus colonizes diverse trees and rotting woods worldwide. Schizophyllum commune is a known producer of b-1 ,6-branched-p-1 ,3-glucan polysaccharide (“b-glucan”) having a homogeneous composition which is extracellularly secreted by liquid culture b-glucan has myriad potential therapeutic uses. For example, it can be used as a cosmetically for the prevention of senescence. Because of its sweetness and mildness in characteristics, it has been used in health improvement of people with weak constitution and in the treatment of various gynecological diseases including leucorrhea.

Separation and identification of the novel Schizophyllum commune PLNP13:

Isolation of Schizophyllum commune PLPN13:

[0054] Fresh fruiting bodies of mushroom identified as strain PLNP13 were collected from rain forest in Pulau Ubin, Singapore. A small piece of the fruiting bodies was transplanted onto potato dextrose agar (PDA) plates and was cultured at a temperature of about 25 ° C. for seven days to obtain mycelia, which were propagated and used as strains. The strain was propagated and maintained on PDA plates every week for analysis and use.

Morphology and Culture Characteristics of Schizophyllum commune PLNP13:

[0055] The isolated strain PLNP13 is a strain showing the characteristics of typical basidiomycete as follows:

• When cultured in a potato dextrose agar medium, the total color of the flora was white, and after 7 days of incubation, the diameter of the circular flora was about 6 cm.

• When cultured for 7 days or more, the mycelial growth medium becomes hard, and white mycelia are formed in the old flora.

The flora with studied using a microscopic. The hyphae were long and entangled in thread form, and binucleus mycelium was formed as a general characteristic of basidiomycetes.

Physiological Characteristics of Schizophyllum commune PLNP13:

[0056] As shown under Example 1 and FIG 1., the mycelial growth of the Schizophyllum commune PLNP13 strain reached its highest on the 10th day of culture, and the production of b-glucan reached maximum of 3.20 g/L on day 10. As an optimal condition for the production of b-glucan, the incubation temperature is 25 °C and slightly acidic pH 5.5, when the culture for 10 days at this optimum condition was maintained until the end of the culture to about 5.0. Optimum production conditions of exo-polysaccharides were found when the culture was aerated 1.0 vvm (l/l/m), while stirring at 20 rpm at a temperature of about 25 °C.

Identification of the Schizophyllum commune PLNP13:

[0057] As a result of analysing the morphological, cultural and physiological characteristics of the new strain PLNP13 according to the present invention, it was confirmed that the fungus belongs to the genus Schizophyllum sp. and is a Schizophyllum commune strain. This was confirmed through 18S rDNA sequencing.

[0058] As described below, the Schizophyllum commune strain PLNP13 disclosed herein is particularly useful for producing b-glucan for cosmeceuticals and anti diabetic therapy. However, it will be appreciated that the said strain PLNP13 and b- glucan produced therefrom can also be used in various other industries such as food stuffs, medicines and animal feeds.

B-qlucan obtained from Schizophyllum commune PLNP13:

[0059] In a further embodiment, the present invention also provides for the b- glucan secreted and isolated from the novel strain Schizophyllum commune PLNP13.

[0060] In one aspect, the b-glucan obtained from Schizophyllum commune PLNP13 has a molecular weight of 1.2 MDa. In a dried state, the b-glucan is an odorless white powder and it is highly soluble in water producing a brown aqueous solution at high concentrations.

[0061] The b-glucan can be obtained from Schizophyllum commune PLNP13 by conventional techniques, for example, from dried fruiting bodies or from submerged culturing. The general steps for obtaining b-glucan from Schizophyllum commune PLNP13 include:

1) culturing in a suitable medium the Schizophyllum commune PLNP13 strain, under conditions allowing said microorganisms to produce b-glucan; and

2) recovering the b-glucan from the medium through ethanolic precipitation. These steps are described in greater detail below.

[0062] The fermentation medium in the culture flasks was centrifuged at 10,000 rpm and 4 °C for 20 min to separate the biomass from the liquid medium. The precipitated biomass was washed twice with distilled water and freeze-dried for three days to get the dry mass of the biomass. For the isolation of crude EPS, the equal volume of 95 % (v/v) ethanol was added to each volume of the supernatant, stirred vigorously and left at 4 °C overnight. The precipitated EPS was collected by centrifuging at 4 °C and 12,000 rpm for 20 min and filtered through a 70 mm filter paper. The EPS pellets were washed with absolute ethanol and centrifuged. The step of ethanol washing step was repeated twice. Thereafter, the EPS pellets were freeze-dried for three days to obtain constant weight of EPS.

[0063] The b-glucan obtained from Schizophyllum commune PLNP13 demonstrated a better profile than conventional (i.e. commercially available) schizophyllan. The was a surprising result. For example, it exhibited less toxicity and was more effective at ameliorating skin damages and enhancing cell regeneration. These characteristics are described in the Examples 4, 5, 6 and FIGS. 7, 8 and 9.

[0064] Example 4 and FIG. 7 shows that the b-glucan isolated from the PLNP13 exhibits better cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ- 5ta, as compared to the conventional schizophyllan purchased from a known commercial supplier (i.e. Contipro). Moreover, the commercial schizophyllan, which is a cosmetic grade b-glucan, presented cell toxicities. This was particularly apparent when its concentration is greater than 1 mg/ml.

[0065] Example 5 and FIG. 8 shows that the b-glucan isolated from the novel strain

PLNP13 also exhibits excellent cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after UV irradiation, as compared to the commercial schizophyllan. In this study, the commercial schizophyllan also presented apparent cell toxicities, particularly at concentrations greater than 2 mg/ml.

[0066] Further, Example 6, FIG. 9 shows that the b-glucan isolated from Schizophyllum commune strain PLNP13 exhibits better cell viability of the hTERT- immortalized foreskin fibroblast cell line, BJ-5ta, after cell starvation, as compared to the commercial Schizophyllum commune b-glucan, schizophyllan. In this study, the commercial schizophyllan also presented apparent cell toxicities particularly at concentrations greater than 2.5 mg/ml.

[0067] These results suggest that b-glucan obtained from the Schizophyllum commune (PLNP13) is less toxic, more skin cell friendly and is preferable over the commercial Schizophyllan in skin care applications.

Improved 3-glucan obtained by co-culturing Schizophyllum commune (PLNP13) and Ganoderma lucidum·.

According to another aspect of the invention, there is provided an improved b-glucan that is obtained by co-culturing Schizophyllum commune strain PLNP13 with Ganoderma lucidum (LZ). Specifically, embodiments include a process for obtaining improved b-glucan. The b-glucan is more effective cosmetically for skin care and anti-aging. It is more effective as an anti-oxidant, has better anti-inflammatory effects as well as unforeseen anti-diabetic effects. For at least these reasons, it is preferred over commercially available b-glucan. The inventors have achieved this objective by co-culturing Schizophyllum commune PLNP13 and Ganoderma lucidum (LZ) by submerged fermentation.

[0068] Many processes for the preparation of b-glucans are known in the art. Each includes steps of cultivation and fermentation of microorganisms that are capable of synthesizing such biopolymers. For example, patent applications EP 271 907 A2, EP 504673 A1 and DE 40 12238 A1 describe conventional processes for their preparation. The preparation is effected by batchwise fermentation of the Schizophyllum commune with stirring and aeration. The culture medium generally includes glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water. Application No. EP 271 907 A2 describes a method for isolating the polysaccharide, in which the culture suspension is first centrifuged and the polysaccharide is precipitated from the supernatant with isopropanol. A second method comprises a pressure filtration followed by an ultrafiltration of the solution obtained, without details of the method having been disclosed.

[0069] Although cultivation of individual fungus is known, methods that us co culture are generally not used. It can be problematic to put the two different fungi in a single medium because of issues related to the reproducibility and sustainability of the microorganisms in the co-culture. Specifically, competition among fungi is expected to lead to antagonistic reactions, where single hyphae show hyphal and tip bursting, granulation, swelling or vacuolization resulting from cell permeabilization (Cox KD, Scherm H. Interaction dynamics between saprobic lignicolous fungi and Armillaria in controlled environments: Exploring the potential for competitive exclusion of Armillaria on peach. Biol Control. 2006; 37:291-300). At colony level, the induced morphological changes can be categorized with deadlock, in which none of the fungi can overgrow the other, and replacement, in which one of the fungi can overgrow the other ( Boddy L. Interspecific combative interactions between wood- decaying basidiomycetes. FEMS Microbiol Ecol. 2000; 31 :185-194. pmid:10719199). Mycelial combat between Schizophyllum commune and Trichoderma viride has also been reported ( Ujor VC, Monti M, Peiris DG, Clements MO, Hedger JN. The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride. Fungal Biol. 2012; 116:332-341. pmid:22289778). Therefore, reproducibility of fungi with exponential mycelial growth and exo-polysaccharides (EPS) production is uncertain.

[0070] Despite these challenges, the present inventors were successful in co culturing Schizophyllum commune PLNP13 with Ganoderma lucidum LZ and obtained b-glucan with enhanced functionality. As described in Example 1 and FIG 1 , the co-culture of fungi, the co-culturing of Schizophyllum commune PLNP13 with Ganoderma lucidum LZ according to the present invention indeed resulted in • comparable growth of mycelium and production of exo-polysaccharides as compared to the mono-culture of Schizophyllum commune PLNP13 according to the present invention, and

• enhanced mycelium growth and exo-polysaccharide production as compared to mono-culture of Ganoderma lucidum.

[0071] Specifically, the improved b-glucan with enhanced functionality is produced by a process that includes steps of:

1. inoculating seeds obtained by culture of the mycelium of Schizophyllum commune PLNP 13 and Ganoderma lucidum LZ, in an amount of 5 to 20% (v/v), and more preferably 10% (v/v), into a liquid medium;

2. co-culturing the mycelial cells in the liquid medium for 10 to 14 days;

3. recovering, separating and purifying thus-obtained b-glucan from the liquid medium.

[0072] The liquid medium can be adjusted so that it includes from 1 to 20% by weight of glucose, from 0.1 to 5% by weight of yeast extract, from 0.1 to 5% by weight of malt extract, from 0.1 to 1% by weight of (NH HRO^ or of (NH bO^ from 0.1 to 1% by weight of KH2PO4 and from 0.1 to 0.5% by weight of MgSC>47H2O.

The medium can be cultured for 10-14 days.

[0073] In a preferred embodiment, the liquid medium includes 5% by weight of glucose, 1% by weight of yeast extract, 2% by weight of malt extract, 0.2% by weight of (NH )₂HP04 or of (NH4)2SC>4, 0.2% by weight of KH2PO4 and 0.1% by weight of KH2PO4. Preferably, the co-culturing process is carried out at a temperature of 20 to 35° C, an agitation rate of 100 to 400 rpm, and an aeration rate of 0.5 to 2 vvm.

[0074] Depending upon the origin and production methods, mushroom b-glucan exhibits significant differences in uniformity of a sugar composition, degree of branching, molecular weight and tertiary structure. These differences in physical characteristics can result in differences in physico-chemical properties and in vivo functions.

[0075] For example, even though b-glucan of Ganoderma lucidum is extracted largely from cell walls, b-glucan is also extracellularly secreted in a small amount and has a bond of mannose, galactose and the like, in addition to glucose.

[0076] Schizophyllum commune derived b-glucan is a homogeneous b-1 ,6- branched-b-1 ,3-glucan, and has a b-1 ,6-residue for every three b-1 ,3 main chains.

As a result, the Schizophyllum commune derived b-glucan has a branched, homogeneous and unique structure, exhibits extracellular secretion of stable neutral polysaccharide and is composed only of glucose. Whereas the b-glucans from Ganoderma lucidum have heterogeneous sugar compositions and structures.

[0077] Conventionally known uses of Schizophyllum commune derived b-glucan and Ganoderma lucidum derived b-glucan include proliferative action on skin cells and collagen fibers, skin-regenerating action such as healing of sunburn, and anti inflammatory action. Further, such b-glucan was largely used as a cosmetic material.

[0078] The present inventors have identified and elucidated the fact that the b- glucan prepared by the above-mentioned method has not only a conventionally- known anti-aging activity of skin, but also exhibits improved skin-protective functions, that is, being effective ameliorating skin damage, enhancing skin cell rejuvenation and exhibiting anti-diabetic properties.

[0079] Example 2 and FIG. 5 of the application shows that the b-glucan obtained from the co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ exhibits better anti-oxidant activity by achieving better ABTS radical scavenging activity as compared to the mono-culture of S. commune PLNP13.

[0080] Example 3, FIG. 6 of the application shows that the b-glucan obtained from the co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ exhibits greater anti-inflammatory activity as compared to the mono-culture of S. commune PLNP13.

[0081] In addition, the b-glucan obtained from the co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ according to the present invention also exhibits better cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, as compared to the mono-culture of mono-culture of Schizophyllum commune PLNP13, Ganoderma lucidum and commercial schizophyllan. The commercially obtained schizophyllan, which is a cosmetic grade b-glucan, presented apparent cell toxicities at concentration higher than 1 mg/ml, which is not seen in the b-glucan according to the present invention (see Example 4 and FIG. 7).

[0082] Example 5, FIG 8 shows excellent cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after UV irradiation, as compared to the mono culture of mono-culture of Schizophyllum commune PLNP13, Ganoderma lucidum and commercial schizophyllan. The commercial obtained schizophyllan presented apparent cell toxicities at concentration higher than 2 mg/ml, which is not seen in the b-glucan according to the present invention.

[0083] Example 6, FIG 9 shows better cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after cell starvation, as compared to the mono culture of Schizophyllum commune PLNP13, Ganoderma lucidum and commercial schizophyllan. The commercial obtained schizophyllan presented apparent cell toxicities at concentration higher than 2.5 mg/ml, which is not seen in the b-glucan according to the present invention.

[0084] Furthermore, the b-glucan obtained from the co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ according to the present invention is shown to stimulate insulin secretion in the presence of 20 mM glucose. Whereas, b- glucan obtained from the mono-culture of Schizophyllum commune (PLNP13) presented no stimulation of insulin secretion at all under the same conditions.

[0085] These results suggest that b-glucan obtained from the co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ can be potentially applied in anti-diabetic therapy, in addition to its skin friendly cosmetic applications.

[0086] A weight-average molecular weight of such b-glucan obtained from the co culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ in accordance with the present invention is 1.2 MDa, which is almost identical to those of the b-glucans obtained by the mono-culture of Schizophyllum commune PLNP13. Composition

[0087] In yet another aspect, the instant invention is directed to a composition comprising b-glucan obtained from co-culturing Schizophyllum commune PLNP13 and Ganoderma lucidum LZ in accordance with the present invention, for cosmeceutical and/or therapeutic application.

[0088] In one embodiment, the composition is formulated for external application having a formulation of an emollient lotion, a nourishing lotion, a nourishing cream, a massage cream, a pack or a gel, a body lotion, an ointment, a gel, a cream, a patch, a shampoo-type cleanser, a skin cleanser or a spray, and so on.

[0089] In another embodiment, the composition is for internal application formulated in an oral solid dosage form selected from a fine powder, capsules, tablets, caplets, sachets, and so on.

[0090] In another embodiment, the composition comprising the b-glucan of the invention is useful for controlling blood sugar levels and reducing glycemic index in Type II diabetes mellitus patients.

[0091] These compositions may contain standard excipients and pharmaceutical acceptable carriers such as cellulose, methyl cellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and lubricating agents. The dosage of the composition will depend upon the usage and the desired results to be achieved.

[0092] The present invention also relates to method of treating skin degeneration due to aging, stress, pollution, UV radiation, and like, by applying the composition as defined above.

[0093] There is no particular limitation to formulations of the aforesaid composition. As examples of the formulations of the aforesaid composition, mention may be made of cosmetic compositions having formulations of emollient lotions (skin lotions), nourishing lotion (milk lotions), massage creams, packs or gels, and compositions for external application having formulations of body lotions, ointments, gels, creams, patches and sprays. Herein, in the external application composition of each formulation, other components with the exception of Schizophyllum commune derived b-glucan may be appropriately selected and mixed by those skilled in the art, depending upon formulations for external use and desired applications.

EXAMPLES

[0094] The present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1(a)

3-glucan production by co-culturinq Schizophyllum commune PLNP13 with Ganoderma lucidum LZ

[0095] The steps involved in the production of b-glucan by co-culturing Schizophyllum commune PLNP13 and Ganoderma lucidum LZ are described in more detail below:

[0096] In Step 1 , the mycelium of Schizophyllum commune PLNP 13 and Ganoderma lucidum are grown in the liquid culture medium, are aseptically homogenized and, are then inoculated in a concentration of 5 to 20% (v/v) and more preferably 10% (v/v) into a liquid culture medium.

[0097] In terms of the mycelial growth, it is preferred to use a liquid nutrient medium (pH 6.0) containing 1 to 20%, preferably 5% glucose, 0.1 to 5%, preferably 1% yeast extract, 0.1 to 5%, preferably 2% malt extract, 0.1 to 1%, preferably 0.2% ammonium sulfate ((NH4)2SC>4), 0.1 to 1%, preferably 0.2% potassium dihydrogen phosphate (KH2PO4), and 0.01 to 0.5%, preferably 0.1% magnesium sulfate (MgSC>4.7H20). [0098] In Step 2, the cells are cultured for 10 to 14 days. The cultivation of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ is carried out in a fermenter at a temperature of 20 to 35° C., preferably 28° C., an agitation rate of 100 to 400 rpm, preferably 300 rpm, and an aeration rate of 0.5 to 2 vvm, preferably 1.5 vvm, for 10 to 14 days, preferably 12 days.

[0099] In Step 3, the thus-obtained b-glucan from the culture solution is recovered, separated and purified.

[00100] The b-glucan solution is filtered through a filtration membrane. Thereafter, b- glucan is recovered by a conventional separation/purification process involving adding an ethyl alcohol to the filtrate two or three times to thereby precipitate, recover and dry the b-glucan.

Example 1(b)

3-glucan production by mono-culture of Schizophyllum commune PLNP13

[00101] The production of b-glucan from mono-culture of Schizophyllum commune PLNP13 is carried out in the same manner as in Example 1 (a).

Example 1(c) - Comparison of b-glucan from mono and co-culture b-glucan production by mono-culture of Ganoderma lucidum

[00102] The commercially available Ganoderma lucidum, purchased was used in this Example. The production of b-glucan from mono-culture of Ganoderma lucidum is carried out in the same manner as described above.

Concentration assay of b qlucan obtained from the above steps:

[00103] The content of beta-glucan was determined after ethanolic precipitation. The cultivation broth was harvested at periodic time. Equal volumes of 95 % (v/v) ethanol were added to each volume of the supernatant, stirred vigorously and left at 4 °C overnight. The precipitated exopolysaccharides (EPS) was collected by centrifuging at 4 °C and 12,000 rpm for 20 min and filtered through a 70 mm filter paper. The EPS pellets were washed with absolute ethanol and centrifuged. The step of ethanol washing step was repeated twice. Thereafter, the EPS pellets were freeze-dried for three days to obtain constant weight of exopolysaccharides (EPS). Each fermentation had triplicate flasks and the results were represented by mean ± SD (standard deviation). The thus-obtained results are shown in FIGS. 1, 2, 3 and 4.

[00104] FIG. 3 and 4 is a graph showing changes in time-dependent mycelial mass and exo-polysaccharide production (i.e. , productivity (yield) of b-glucan over time), upon co-culturing of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Example 1 (a). FIG. 1 is a comparative graph showing mycelial mass and polysaccharide production. The graph shows the productivity (yield) of b-glucan obtained from co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Example 1 (a), over the mono-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Comparative Example 1(c).

[00105] Referring to FIGS. 1, 3 and 4, it can be seen that 14-day-co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Example 1 (a) exhibits the highest productivity of mycelia and comparable production of b-glucan. The mono-culture of strain Schizophyllum commune PLNP13 as in Example 1 (b) also exhibits high production of mycelia and b-glucan as compared to the commercial Ganoderma lucidum as in Comparative Example 1 (c).

[00106] From the above, it can be seen that b glucan of Example 1(a) obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ exhibits a higher productivity of mycelia and comparable production of b-glucan, despite the concern of expected low productivity during co-culturing of different fungi.

[00107] FIG. 1 is an illustration of a graph 100 showing the yield of mycelia and exo polysaccharides, in accordance with the disclosed embodiments. The yield of mycelia and exo-polysaccharides in LZ is shown in bars 102, the yield of mycelia and exo-polysaccharides in PLNP13 is shown in bars 104 and the yield of mycelia and exo-polysaccharides in co-cultivation of PLNP13 and LZ mycelia is shown in bars 106. [00108] As shown in graph 100, co-cultivation of PLNP13 and LZ mycelia did not change the yield of mycelia; however, exopolysaccharides (EPS) were slightly decreased compared to the mono-culture of PLNP13. The yield for both EPS and mycelium for LZ was lower than that for PLNP13 mono-culture and its coculture with LZ. However, such decrease in the yield of EPS was insignificant.

[00109] FIG. 2 is a graph 200 of viscosity of coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) broth (cP) over time, FIG. 3 shows a graph 300 of Mycelia production of coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) over time, and FIG. 4 shows a graph 400 of beta-glucan yield over time, by coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). As shown in graphs 200, 300 and 400, beta-glucan reached maximum production of 3.20 g/L on tenth day, although viscosity and mycelia production of the broth continues increasing. This suggests that EPS can be harvested on day 10 of the coculture for application.

Example 2

Measurement of Antioxidant Effect of 3-glucan

[00110] The antioxidant activities of the b-glucans as obtained from the process as in Examples 1 (a) and 1 (b) were analyzed through ABTS+ assay following the conventional method (R. Re, N. Pellegrini, A Proteggente, A. Pannala, M. Yang, C. Rice-Evans Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radic. Biol. Med., 26 (9-10) (1999), pp. 1231-1237). The results are shown in FIG. 5.

[00111] Referring to FIG. 5, it can be seen that b-glucan obtained from co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Example 1 (a) exhibits higher anti-oxidant activity as compared to the b-glucan obtained from the mono-culture of novel Schizophyllum commune PLNP13 as in Example 1 (b).

[00112] This example shows that b glucan of Example 1(a) obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ possesses high antioxidant activity and free radical scavenging potentials. The data suggest that it can serve as a good source for safe natural antioxidants for skin care as well as pharmaceutical applications.

[00113] FIG. 5 is a graph 500 showing antioxidant activity of mono-culture of Schizophyllum commune (PLNP13) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), in accordance with the disclosed embodiments. A graph 502 shows the antioxidant activity of mono-culture of Schizophyllum commune (PLNP13) and the graph 504 shows the antioxidant activity of coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). Compared with mono-culture of Schizophyllum commune (PLNP13), the antioxidant activity of coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) is increased.

Example 3

Measurement of Anti-inflammatory Effect of b-glucan

[00114] The anti-inflammatory activities of the b-glucans as obtained from the process as in Examples 1 (a) and 1 (b) were analysed through COX-1 and COX-2 inhibitor screening assay. The inhibition of COX enzymes by crude EPS samples was determined in vitro using the Cyclooxygenase (COX) colorimetric inhibitor screening assay kit (Cayman Chemical, Ann Arbor, Ml, USA) following the manufacturer’s protocols. This kit contains two cyclooxygenase (COX) isoforms,

COX-1 from ovine and COX-2 from human. Briefly, 160 pL assay buffer and 10-pL hemin were added to the background wells, while 150 pl_ assay buffer, 10-pL hemin and 10-pL enzyme (either COX-1 or COX-2) were added to the 100% initial activity wells. Crude EPS samples (10 pL) in DMSO at varying concentrations (0.5-3.0 mg/mL) were added to the inhibitor wells and 10 pL DMSO was added to the 100% initial activity and background wells. The plate was carefully shaken for a few seconds and incubated for five min at 25 °C. The colorimetric substrate solution (20 pL) followed by arachidonic acid (20 pL) were added to each well. The plate was again shaken carefully for a few seconds and incubated for 5 min at 25 °C. The absorbance at 590 nm was read by the microplate reader. The inhibition of cyclooxygenase activity by EPS samples was calculated by the following formula,

Cyclooxygenase activity inhibition (%) = [(A100%-Asample)/(A100%-Ablank)] x100, where A100% was absorbance of the 100% initial activity wells without EPS samples, Ablank was the absorbance of the background wells without the enzyme and EPS samples. Asample was the absorbance of the sample wells with the enzyme. Triplicate independent reactions were conducted for each sample and average results were reported. The thus-obtained results are shown in FIG. 6A cyclooxygenase 1 (COX-1) and FIG. 6B cyclooxygenase 2 (COX-2).

[00115] Referring to FIGS. 6A and 6B, it can be seen b-glucan obtained from co culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as in Example 1(a) exhibits excellent anti-oxidant activity as compared to the b-glucan obtained from the mono-culture of Schizophyllum commune PLNP13 as in Example 1 (b).

[00116] This example shows that b glucan of Example 1(a) obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ possesses high anti inflammatory activity that could serve as the monotherapy or as adjuncts to standard therapeutic regimens for the management and prevention of skin damages due to stress and pollution.

[00117] Referring FIG. 6A a graph 600 the measurement of anti-inflammatory activity of mono-culture of PLNP13 and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) in inhibition to cyclooxygenase 1 (COX-1)

(A). The graph 602 refers the measurement of anti-inflammatory activity of mono culture of PLNP13. The graph 602 refers the measurement of anti-inflammatory activity of mono-culture of PLNP13

[00118] Referring FIG. 6B a graph 650 the measurement of anti-inflammatory activity of mono-culture of PLNP13 and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) in inhibition to cyclooxygenase 2 (COX-2)

(B). The inhibition to cyclooxygenase 2 (COX-2) (B) enzymes by beta-glucan obtained from PLNP13 and LZ coculture is significantly enhanced compared to that obtained from PLNP13 mono-culture.

Example 4

Cell viability assay on hTERT-immortalized foreskin fibroblast cell line, BJ-5ta

[00119] The influence of crude b-glucan from co-culture of Schizophyllum commune PLNP13 with Ganoderma lucidum as in Examples 1 (a) and mono-culture of Schizophyllum commune PLNP13 as in Example 1 (b), at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta (ATCC® CRL-4001TM), was tested using standard techniques in the art. The commercial Schizophyllum commune b-glucan, schizophyllan, was used as the control. The thus-obtained results are shown in FIG. 7.

[00120] Referring to FIG. 7, it can be seen b-glucan obtained from the co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) as in Example 1 (a) presented no toxicity to BJ-5ta cells. In addition, it displayed slightly higher cell proliferation activities than those obtained from the mono-culture of Schizophyllum commune (PLNP13) or Ganoderma lucidum (LZ). Similar effect can also be seen in mono-culture of S. commune PLNP13 as in Example 1 (b). Whereas the commercial Schizophyllum commune b glucan, schizophyllan, which is a cosmetic grade b- glucan, presented apparent cell toxicities, in particular when its concentration is higher than 1 mg/ml.

[00121] FIG. 7 is an illustration of a graph 700 the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), at different concentrations on cell viability of the hTERT- immortalized foreskin fibroblast cell line, BJ-5ta, in accordance with the disclosed embodiments.

[00122] The graph 702 shows the influence of crude beta-glucan from mono-culture of Ganoderma lucidum (LZ), graph 704 shows the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), and graph 706 shows the influence of crude beta-glucan from coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). The commercial Schizophyllum commune beta- glucan, schizophyllan, used as the control is shown in graph 708. Beta-glucan obtained from the coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) presented no toxicity to BJ-5ta cells. In addition, it displayed slightly higher cell proliferation activities that those obtained from the mono-culture of Schizophyllum commune (PLNP13) or Ganoderma lucidum (LZ). Whereas the commercial Schizophyllum commune beta glucan, schizophyllan, which is a cosmetic grade beta-glucan, presented apparent cell toxicities, in particular when its concentration is higher than 1 mg/ml.

[00123] This example shows that b glucan obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as well as mono-culture of Schizophyllum commune PLNP13 is more skin cell friendly and is more suitable in skin care applications than its commercial counterpart products.

Example 5

Cell viability assay on hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after

UV irradiation

[00124] The influence of crude b-glucan from co-culture of Schizophyllum commune PLNP13 with Ganoderma lucidum as in Examples 1 (a) and mono-culture of Schizophyllum commune PLNP13 as in Example 1 (b), at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta (ATCC® CRL-4001TM), after UV irradiation, is tested using standard technique. The commercial Schizophyllum commune b-glucan, schizophyllan, was used as the control. The thus-obtained results are shown in FIG. 8.

[00125] Referring to FIG. 8, it can be seen b-glucan obtained from the co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) as in Example 1 (a) showed no further damages to the cells after UV irradiation. Similar effect can also be seen in mono-culture of Schizophyllum commune PLNP13 as in Example 1 (b). Whereas the commercial Schizophyllum commune b glucan, schizophyllan, which is a cosmetic grade b-glucan, presented apparent cell toxicities, in particular when its concentration is higher than 2 mg/ml.

[00126] FIG. 8 is an illustration of a graph 800 the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and its coculture with Ganoderma lucidum (LZ) at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after UV irradiation, in accordance with the disclosed embodiments. The commercial Schizophyllum commune beta-glucan, schizophyllan, was used as the control. The graph 802 shows the influence of crude beta-glucan from mono-culture of Ganoderma lucidum (LZ), graph 804 shows the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13) and graph 806 shows the influence of crude beta-glucan from coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). The commercial Schizophyllum commune beta- glucan, schizophyllan, used as the control is shown in graph 808. Compared to the beta-glucan obtained from the mono-culture of Schizophyllum commune (PLNP13), beta-glucan obtained from the coculture of Schizophyllum commune (PLNP13) or Ganoderma lucidum (LZ) presented no further damages to the cells after UV irradiation. Whereas, the commercial Schizophyllum commune beta glucan, schizophyllan, which is a cosmetic grade beta-glucan, presented apparent cell toxicities, in particular when its concentration is higher than 2 mg/ml.

[00127] This example shows that b glucan obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as well as mono-culture of Schizophyllum commune PLNP13 is more skin cell friendly and is more suitable for ameliorating skin damages by stress, pollution and UV radiation, than its commercial counterpart products.

Example 6

Cell viability assay on hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after cell starvation

[00128] The influence of crude b-glucan from co-culture of Schizophyllum commune

PLNP13 and Ganoderma lucidum LZ as in Examples 1 (a) and mono-culture of

Schizophyllum commune PLNP13 as in Example 1 (b), at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta (ATCC® CRL-4001TM), after cell starvation, is tested using standard technique. The commercial Schizophyllum commune b-glucan, schizophyllan, was used as the control. The thus-obtained results are shown in FIG. 9.

[00129] FIG. 9 is an illustration of a graph 900 the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13), mono-culture of Ganoderma lucidum (LZ) and its coculture with Ganoderma lucidum (LZ) at different concentrations on cell viability of the hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, after cell starvation, in accordance with the disclosed embodiments. The commercial Schizophyllum commune beta-glucan, schizophyllan, was used as the control. The graph 902 shows the influence of crude beta-glucan from mono-culture of Ganoderma lucidum (LZ), graph 904 shows the influence of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13) and graph 906 shows the influence of crude beta-glucan from coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). The commercial Schizophyllum commune beta- glucan, schizophyllan, used as the control is shown in graph 908.

[00130] Referring to FIG. 9, it can be seen b-glucan obtained from the co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) as in Example 1 (a) was more beneficial to cell growth. Similar effect can also be seen in mono culture of Schizophyllum commune PLNP13 as in Example 1 (b). Whereas the commercial Schizophyllum commune b glucan, schizophyllan, which is a cosmetic grade b-glucan, presented apparent cell toxicities, in particular when its concentration is higher than 2 mg/ml.

[00131] This example shows that b glucan obtained by co-culture of Schizophyllum commune PLNP13 and Ganoderma lucidum LZ as well as mono-culture of Schizophyllum commune PLNP13 is more skin cell friendly and is more suitable in in skin cell rejuvenation applications than its commercial counterpart products.

Example 7

Effects of 3-glucan on glucose stimulated insulin secretion [00132] The influence of crude b-glucan from co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) as in Examples 1 (a) and mono-culture of Schizophyllum commune PLNP13 as in Example 1 (b), at different concentrations on glucose stimulated insulin secretion is tested using human pancreatic cell line, RIN- m5F, in accordance with the standard conditions. RIN-m5F cells were either cultured in basal (4 mM) or stimulated (20 mM) glucose concentrations in the presence of b- glucan samples at varying concentration (mg/ml). The thus-obtained results are shown in FIG. 10 (A): b-glucan from mono-culture of Schizophyllum commune (PLNP13); (B): b-glucan from co-culture of Schizophyllum commune (PLNP13) with Ganoderma lucidum (LZ).

[00133] Referring to FIG. 10, it can be seen b-glucan obtained from the co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) as in Example 1 (a) was able to stimulate insulin secretion in the presence of 20 mM glucose (B). Whereas, b-glucan obtained from the mono-culture of Schizophyllum commune (PLNP13) presented no stimulation of insulin secretion at all under the same conditions (A).

[00134] This example shows that b glucan obtained by co-culture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) can be applied in anti-diabetic therapy, in addition to its enhanced cosmetic effects.

[00135] FIG. 10A and FIG. 10B are illustration of graphs 950 and 960 showing the effects of crude beta-glucan from mono-culture of Schizophyllum commune (PLNP13) and coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) at different concentrations on glucose stimulated insulin secretion using human pancreatic cell line, RIN-m5F, in accordance with the disclosed embodiments. RIN-m5F cells were either cultured in basal (4 mM) or stimulated (20 mM) glucose concentrations in the presence of beta-glucan samples at varying concentration (mg/ml). (A): beta-glucan from mono-culture of Schizophyllum commune (PLNP13); (B): beta-glucan from coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ). [00136] Referring to FIG. 10A, the bars 952, 954, 956 and 958 represent the RIN- m5F cells that are either cultured in basal (4 mM) or stimulated (20 mM) glucose concentrations in the presence of beta-glucan samples at varying concentration (mg/ml), for mono-culture of Schizophyllum commune (PLNP13), for example, vehicle, PLNP132 (PLNP13 EPS at 2 mg/ml), PLNP134 (PLNP13 EPS at 4 mg/ml) and PLNP138 (PLNP13 EPS at 8 mg/ml), respectively.

[00137] Referring to FIG. 10B, the bars 962, 964, 966 and 968 represent the RIN- m5F cells that are either cultured in basal (4 mM) or stimulated (20 mM) glucose concentrations in the presence of beta-glucan samples at varying concentration (mg/ml), for coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ), for example, vehicle, PLNP132 (PLNP13 EPS at 2 mg/ml), PLNP134 (PLNP13 EPS at 4 mg/ml) and PLNP138 (PLNP13 EPS at 8 mg/ml), respectively.

[00138] It is apparent that the beta-glucan obtained from the coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) is able to stimulate insulin secretion in the presence of 20 mM glucose (B). Whereas, beta- glucan obtained from the mono-culture of Schizophyllum commune (PLNP13) presented no stimulation of insulin secretion at all under the same conditions (A). These results suggest that beta-glucan obtained from the coculture of Schizophyllum commune (PLNP13) and Ganoderma lucidum (LZ) can be potentially applied in anti diabetic health supplement formulation.

[00139] FIG.11 shows the Fourier-Transform Infrared (FTIR) spectrum 970 of commercial schizophyllan and the crude EPS obtained by the coculture of Ganoderma lucidum LZ and Schizophyllum commune PLNP13. Quite comparable spectrum was obtained, indicating their structures are very similar. The graph 972 represents Fourier-Transform Infrared (FTIR) spectrum of commercial schizophyllan and graph 974 represents Fourier-Transform Infrared (FTIR) spectrum of Schizophyllum commune (PLNP13) EPS.

[00140] FIG. 12 shows the details of the FTIR spectrum 978 of the crude EPS obtained by the coculture of Ganoderma lucidum LZ and Schizophyllum commune

PLNP13. The strong signals at 3285 cm^-1 corresponds to O-H stretching vibration of carbohydrates and the band at 2885 cnr¹ shows C-H stretching vibration. A strong absorption peak at 1636 cnr¹ could be related to the stretching vibrations of C=0 (carbonyl) bond of protein or polysaccharide-protein complex, which indicates that the protein molecules cannot be removed completely after deproteinization. The peak at 1457 cnr¹ was due to the CH2 in polysaccharides. The following five peaks in the region of 1420-1200 cm^-1 are close to each other and most of them are of low intensity. The signal at 1420 cnr¹ showed asymmetric and symmetric stretching vibrations of carboxylate, also related to protein structures. The peak around 1364 cm^-1 corresponds to the bending of C-H in CH3, peak 1316 cnr¹ was due to asymmetric CH2 bending of carbohydrates, the absorption peak at 1252 cnr¹ was derived from the O-H stretching vibration. The peak at 1200 cnr¹ was due to the C=0 stretching vibrations in pyranoid rings indicating the presence of polysaccharides as major component(s). The peak at 1151 cm-1 was due to the C- O-C asymmetric stretching of glycosidic linkage. The specific band at 1029 cm^-1 was due to the stretching vibration of C-0 of pyranose ring, suggesting that these polysaccharides possessed the pyranose ring skeleton. Furthermore, the characteristic absorptions in the range of 991 cnr¹, and at 887 cnr¹ arises from C-H variable angle vibration of beta-pyranosides, indicating the existence of carbon- glycoside bonds of beta-D-glucan.

[00141] Table 1 shows the monomer sugar composition of EPS samples obtained for the mono-culture and coculture of Ganoderma lucidum LZ and Schizophyllum commune PLNP13.

Table 1

[00142] Beta-glucan obtained by the coculture of Ganoderma lucidum LZ and Schizophyllum commune PLNP13 presents much less glucose content compared to EPS obtained by the monoculture of Schizophyllum commune PLNP13. It presents galactose, however, PLNP13 monoculture EPS does not. The content of mannose in the coculture EPS is also higher than that in the PLNP13 monoculture EPS.

Generally speaking, the EPS obtained from the coculture present a combination of monomer sugars from Ganoderma lucidum LZ and Schizophyllum commune PLNP13 monoculture EPS. The enhanced health benefits of the coculture EPS could be due to the contribution of LZ EPS.

[00143] FIG. 13 is a flowchart 980 showing the steps of preparation and use of beta- glucan, in accordance with the disclosed embodiments. As at step 982, monoculture of Ganoderma lucidum LZ and Schizophyllum commune PLNP13 strains are cultivated on potato dextrose agar (PDA) plates. Then, as at step 984, ten fully grown agar disks (5 mm) of Ganoderma lucidum LZ and Schizophyllum commune

PLNP13 are respectively inoculated in the seed culture medium and cultivated for 7 days. At step 986, beta-glucan is prepared by inoculating seed culture of Ganoderma lucidum LZ and Schizophyllum commune PLNP13 strains, 10% (v/v) each, into the coculture medium, and cultivation for 10 days. At step 988, beta-glucan is harvested by ethanolic precipitation and centrifugation, and water washing. The prepared beta- glucan can be used in at least one skin care products of hydrating, moisturizing, anti aging, anti-stress, anti-oxidant, anti-pollution, anti-inflammatory, Ultra Violet (UV) protection or skin-regenerations at step 990. As at step 992, the prepared beta- glucan can also be used in the formulation of anti-diabetic health supplement products.

[00144] In another aspect, the present invention relates to a method of cultivation of a mushroom coculture comprising Ganoderma lucidum and Schizophyllum commune strains. The seed culture of Ganoderma lucidum and Schizophyllum commune is conducted in the fermentation medium containing 20-40 g/L glucose, 1 - 10 g/L peptone, 1 -10 g/L yeast extract and were cultured at 28 °C for 5 to 10 days. The seed culture of Ganoderma lucidum and Schizophyllum commune is inoculated to the fermentation medium at 1 -20% (v/v) to produces a novel beta-glucan. The beta-glucan contains 45-90% of glucose, 10-20% galactose and 10-20% mannose. The beta-glucan comprises higher anti-inflammatory and high anti-oxidant property than beta-glucan obtained by the monoculture of Schizophyllum commune.

[00145] Thus, the present invention is related to a novel beta-glucan produced through the co-cultivation of Schizophyllum commune PLNP13 and Ganoderma lucidum (LZ). The advantage of the cocultivation of PLNP13 with LZ is that novel beta-glucan was produced and its anti-oxidant and anti-inflammatory activities were enhanced. Beta-glucan obtained from coculture could have better skin benefits.

[00146] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

What is claimed is:

1. A b-glucan obtained from Schizophyllum commune PLNP13 comprising: a) about 80% glucose; and b) about 17% galactose.

2. The b-glucan of claim 1 , further comprising: c) about 15% mannose; d) about 5% fucose; e) about 4% xylose; and f) about 6% rhamnose.

3. The b-glucan of claim 1 or 2, wherein the molecular weight is about 1.2 MDa,

4. A method of preparing a b-glucan comprising steps of: a) co-culturing in a liquid medium of Schizophyllum commune PLNP13 with Ganoderma lucidum under conditions allowing growth and production b-glucan; and b) recovering the b-glucan from the liquid medium.

5. The method of claim 4, wherein the liquid medium comprises: i) 1 to 20% w/w of glucose; ii) 0.1 to 5% w/w yeast extract; iii) 0.1 to 5% w/w malt extract; iv) 0.1 to 1% w/w (NH₄)₂HP04 or 0.1 to 1% w/w of (NH₄)2S0₄; v) 0.1 to 1% w/w KH2PO4; and vi) 0.1 to 0.5% w/w MgSC 7H2O.

6. The method of claim 5, further comprising steps of: c) adding 1 - 20% w/w glucose to the liquid medium after three to five days of culture; and d) continuing the culture for an additional 1 to 5 days.

7. The method of claim 4, wherein the liquid medium comprises:

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SUBSTITUTE SHEET RULE 26 i) about 5% w/w of glucose; ii) about 1% w/w yeast extract; iii) about 2% w/w malt extract; iv) about 0.2% w/w (NH HRO^ or about 0.2% w/w of (NH bO^ v) 0.1 to 1% w/w KH2PO4; and vi) 0.1 to 0.5% w/w MgSC 7H2O.

8. The method of claim 7, further comprising steps of: c) adding about 5% w/w glucose to the liquid medium after 3 to 5 days of culture; and d) continuing the culture for an additional 1 to 5 days.

9. A method of preparing a b-glucan by co-culturing a liquid medium of Schizophyllum commune PLNP13 with Ganoderma lucidum, the method comprising steps of: a) inoculating seeds obtained by culture of mycelium of Schizophyllum commune PLNP 13 and Ganoderma lucidum, in an amount of 5 to 20% (v/v), and more preferably 10% (v/v), into a liquid medium; b) co-culturing the mycelial in the liquid medium for 10 to 14 days; and c) recovering, separating and purifying b-glucan from the liquid medium.

10. A method of preparing a b-glucan by co-culturing a liquid medium of Schizophyllum commune PLNP13 with Ganoderma lucidum, the method comprising steps of: a) growing a monoculture of Ganoderma lucidum and Schizophyllum commune strains on potato dextrose agar (PDA) plates; b) inoculating agar disks with Ganoderma lucidum and Schizophyllum commune respectively in seed culture medium and cultivating for about seven days; c) preparing b-glucan by inoculating 10% (v/v) of seed culture of Ganoderma lucidum and Schizophyllum commune strain into a coculture medium, and co- culturing for about then days; and d) harvesting b-glucan by ethanolic precipitation, centrifugation, and water washing.

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SUBSTITUTE SHEET RULE 26

11. The method of either of claims 9 or 10, wherein the step of co-culturing is carried out at a temperature of 20 to 35° C, an agitation rate of 100 to 400 rpm, and an aeration rate of 0.5 to 2 vvm.

12. A b-glucan, obtained from the method of claims 9 or 10, wherein the b-glucan has a molecular weight of 1.2 MDa and exhibits one or more of anti-oxidant, anti inflammatory and anti-diabetic properties.

13. The b-glucan of claim 12, further comprising one or more of a humectant, an emulsifier, an emollient, or a combination thereof.

14. The b-glucan of claim 13, wherein the b-glucan is formulated as an emollient lotion, a nourishing lotion, a nourishing cream, a massage cream, a pack or a gel, a body lotion, an ointment, a gel, a cream, a patch, a shampoo-type cleanser, a skin cleanser or a spray.

15. The b-glucan of claim 12, wherein the b-glucan is formulated for internal application formulated as an oral solid dosage, a fine powder, a capsule, a tablet, a caplet or a sachet.

16. The use of the b-glucan according to any one of claims 1 or 3 for manufacture of skin-care and/or therapeutic products.

17. A method of treating skin damage due to aging, stress, pollution or UV radiation comprising a step of applying the composition of claim 14 to a skin surface.

18. A method of cultivating a mushroom co-culture comprising Ganoderma lucidum and Schizophyllum commune strains.

19. The method of claim 18, wherein a seed culture of Ganoderma lucidum and Schizophyllum commune is fermented in medium comprised of 20 - 40 g/L glucose, 1 - 10 g/L peptone and 1 - 10 g/L yeast extract.

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SUBSTITUTE SHEET RULE 26

20. The method of claim 18, comprising a step of culturing seed culture of Ganoderma lucidum and Schizophyllum commune at 28 °C for five to ten days.

21. The method of claim 20, wherein the seed culture is inoculated with fermentation medium at 1 - 20% (v/v).

22. The method of claim 18, further comprising a step of purifying b-glucan.

23. The method of claim 22, wherein the b-glucan comprises 45 - 90% of glucose.

24. The method of claim 22, wherein the b-glucan comprises 10 - 20% galactose.

25. The method of claim 22, wherein the b-glucan comprises 10 - 20% mannose.

26. A b-glucan comprised of less than 60% glucose.

27. The b-glucan of claim 26, further comprised of about 17% galactose.

28. The b-glucan of claim 26, further comprised of greater than 10% galactose.

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SUBSTITUTE SHEET RULE 26