AU2021338585A1

AU2021338585A1 - Isolating intracellular polysaccharides from fungi

Info

Publication number: AU2021338585A1
Application number: AU2021338585A
Authority: AU
Inventors: Kat ALLIKIAN; Yihuai GAO; Rashad Ahmed SYED
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-16
Filing date: 2021-12-16
Publication date: 2022-06-30
Also published as: AU2023201749A1; WO2022130299A1; AU2023201749B2

Abstract

The disclosure relates to a method for isolating intracellular polysaccharides from fungi comprising inoculating fungi into a fermentation medium, wherein the fermentation medium is subjected to agitation; extracting one or more polysaccharides from the fermentation media by filtration, wherein the filtration has a pore size of ony one or more of 1 to 5 kDa, 10 to 20 kDa, and/or 100 to 200 kDa, wherein the polysaccharides are extracted after about 3 or about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

Description

ISOLATING INTRACELLULAR POLYSACCHARIDES FROM FUNGI FIELD OF THE INVENTION

[0001] The present invention relates to a method for isolating intracellular polysaccharides from fungi.

BACKGROUND TO THE INVENTION

[0002] Historically, manufacturing of pharmaceutical products has had a considerable social and environmental footprint. It has relied on the use of volatile organic solvents, many of which are carcinogenic and toxic. Further, many synthetic processes require large quantities of energy, and generate unnecessary waste.

[0003] Extracting the pharmacologically active component from natural sources offers a greener alternative to traditional synthesis. If the active ingredient can be harvested from a species of fungi or bacteria, it allows for the use of more efficient and sustainable natural processes such as fermentation.

[0004] Fermentation is traditionally performed in aqueous environments, making the discharge environmentally benign. It also requires considerably less energy, as the biochemical conversions are performed by. As corporate social responsibility becomes more prominent, and the world's environmental position more precarious, pursuit of such processes may be of increasing commercial value.

[0005] It is an object of the present invention to isolating intracellular polysaccharides from fungi, to overcome any of the above-mentioned disadvantages, or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

[0006] In a first aspect there is described a method for isolating intracellular polysaccharides from fungi comprising • inoculating fungi into a fermentation medium, wherein the fermentation medium is subjected to agitation, • extracting one or more polysaccharides from the fermentation media by filtration, wherein the filtration has a pore size of a) about 1 to 5 kDa, b) about 10 to 20 kDa, c) about100to200 kDa, d) or any combination of (a) to (c), and wherein the polysaccharides are extracted after about 3 to about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

[0007] In a further aspect there is described a method for isolating intracellular polysaccharides from fungi comprising inoculating fungi into a fermentation medium, wherein the fermentation medium is subjected to agitation, wherein the agitation exerts a shear force of about 100 to about 1000 s1, isolating mycelium from the fermentation medium, extracting one or more polysaccharides from the mycelium by filtration, wherein the filtration has a pore size of a) about 1 to 5 kDa, b) about 10 to 20 kDa, c) about100to200 kDa, d) or any combination of (a) to (c), and wherein the polysaccharides are extracted after about 3 to about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

[0008] In a further aspect there is described a method for isolating intracellular polysaccharides from fungi comprising • inoculating fungi into a fermentation medium, wherein the fermentation medium (i) is periodically dosed with a sugar at a rate of about 15 to 30 g/L of medium, (ii) is subjected to agitation, or (iii) both (i) and (ii), • extracting one or more polysaccharides from the fermentation media by filtration, wherein the filtration has a pore size of b) about 1 to 5 kDa, c) about 10 to 20 kDa, d) about100to200 kDa, e) or any combination of (a) to (c), and wherein the polysaccharides are extracted after about 3 to about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

[0009] In a first aspect there is described a method for isolating intracellular polysaccharides from fungi comprising • inoculating fungi into a fermentation medium that comprises about 30 to about 50 g/L of a sugar, wherein the fermentation medium is subjected to agitation, • extracting one or more polysaccharides from the fermentation media by filtration, wherein the filtration has a pore size of b) about 1 to 5 kDa, c) about 10 to 20 kDa, d) about100to200 kDa, e) or any combination of (a) to (c), and wherein the polysaccharides are extracted after about 3 to about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

[0010] In a further aspect there is described an isolate from a fungal biomass, the isolate comprising one or more polysaccharides having a molecular mass of a) about 1 to 5 kDa, b) about 10 to 20 kDa, c) about100to200 kDa, d) or any combination of (a) to (c).

[0011] In one embodiment the fermentation medium initially comprises 30, 35, 40, , 50 g/L of a sugar, and suitable ranges may be selected from between any of these values. Preferably the sugar is glucose.

[0012] In one embodiment the dosing of the media with sugar achieves a ratio of carbon to nitrogen of about 1:3.

[0013] In one embodiment the dosing of the media with sugar achieves a concentration of sugar in the media of about 20 g/L.

[0014] In one embodiment the sugar is provided to the media every 24 hours.

[0015] In one embodiment the sugar is first provided to the media after 24 hours from inoculation.

[0016] In one embodiment the sugar is glucose.

[0017] In one embodiment the agitation is high shear mixing.

[0018] In one embodiment the shear force is about 100 to about 1000 s- at 200 C.

[0019] In one embodiment the fermentation is carried out at about 25 to about 350 C, and suitable ranges may be selected from between any of these values.

[0020] In one embodiment the fungi is a mushroom species.

[0021] In one embodiment the mushroom species may be selected from Inonotus obliquus, Trametes versicolor, (also known as Coriolus versicolor), Lentinula edodes, Hericium erinaceus, Cordyceps sinensis, Grifola frondosa, Schizophyllum commune, Flammulina velutipes, Pleurotus ostreatus, Agaricus bisporus, Auricularia auricula, Coprinus comatus, Phellinus linteus, or Laetiporus sulphureus.

[0022] In one embodiment the mushroom species is selected from Ganoderma lingzhi.

[0023] In one embodiment the mycelia comes from seed culture, and wherein the seed culture is first expanded.

[0024] In one embodiment the concentration of seed culture at the beginning of fermentation is about 2 to about 6% by volume, and suitable ranges may be selected from between any of these values.

[0025] In one embodiment the seed culture is a combination of pre-existing and expanded seed culture.

[0026] In one embodiment the inoculated media is incubated for up to 7 days.

[0027] In one embodiment the inoculated media is stirred.

[0028] In one embodiment the inoculated media is stirred at about 80 to 160 RPM, and suitable ranges may be selected from between any of these values.

[0029] In one embodiment the media comprises: a) about 30 to 50 g/L glucose, b) about 2 to 6 g/L soy peptone, c) about 1 to 3 g/L KH2PO4, d) about 0.5 to 2 g/L MgSO4.7H20, or e) any combination of (a) to (d).

[0030] In one embodiment the method comprises the addition of insoluble ingredient.

[0031] In one embodiment about 0.5 to about 4% w/v of insoluble ingredient, and suitable ranges may be selected from between any of these values.

[0032] In one embodiment the insoluble ingredient is lotus ground rice bran.

[0033] In one embodiment the vessel used for fermentation is a bioreactor.

[0034] In one embodiment the method controls any one or more parameters selected from pH, dissolved oxygen, aeration, and shear force.

[0035] In one embodiment the mycelia is harvested before fruiting body formation.

[0036] In one embodiment the pH of the media is about 3 to 5, and suitable ranges may be selected from between any of these values.

[0037] In one embodiment the level of oxygen in the fermentation mixture may be about 20 to about 30%, and suitable ranges may be selected from between any of these values.

[0038] In one embodiment the addition of an anti-foam agent to the media.

[0039] In one embodiment the biomass is dried prior to extraction.

[0040] In one embodiment the method extracts at least 5% polysaccharide per dry weight of the biomass.

[0041] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

[0042] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0043] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The invention will now be described by way of example only and with reference to the drawings in which:

[0045] Figure 1 is a flow diagram showing the processing steps as described.

[0046] Figure 2 is a flow diagram showing the downstream processing steps.

[0047] Figure 3 is a graph showing the dry cell weights on days 3, 6, 9, and 12 for Example 1 referred to in paragraph [0139].

[0048] Figure 4 is a graph showing dry cell weights (DCW) for previous flask study (medium G and J) and this bioreactor study (medium G for GL01-GLOO3 and medium J for GLO04-GLO06).

[0049] Figure 5 is a graph showing a comparison of total carbohydrate (C) estimates with the previous study (G and J medium results).

[0050] Figure 6 is a graph showing dry cell weights of GL07-GL015 over the fermentation period.

[0051] Figure 7 is a graph showing residual glucose (g/L) present in the supernatant of GL007-GL15 sampled during the fermentation period.

[0052] Figure 8 is a graph showing estimated PS per dry gram of biomass based on HPLC peak areas (of target PS) and actual total dry PS.

DETAILED DESCRIPTIONOFTHE INVENTION

[0053] The present invention relates to a submerged fermentation process for the growth of mushroom mycelia, and more particularly a process to provide for one or more extractable intracellular polysaccharides (IPS).

[0054] Submerged fermentation may refer to any fermentation process where the reactive components are immersed in a liquid phase.

[0055] One benefit of the disclosed method may be that the harvested mycelia exhibited greater IPS content compared to mycelia produced by existing alternative methods. Another benefit may be that the speed of mycelia growth is greater relative to the art. A further benefit may be that the IPS extracted from the mycelia has greater variance in size relative to the art.

1. Upstream process

[0056] The process (as shown in general terms in Figure 1) as described can broadly be divided into an upstream and downstream process (shown in Figure 2.) The upstream process describes the selection of seed through to the production batch. The downstream process describes collection of the mycelial biomass through to the production of polysaccharides.

[0057] The present disclosure relates to the fermentation of mushrooms.

[0058] The mushroom species may be selected from Inonotus obliquus, Trametes versicolor, (also known as Coriolus versicolor), Lentinula edodes, Hericium erinaceus, Cordyceps sinensis, Grifola frondosa, Schizophyllum commune, Flammulina velutipes, Pleurotus ostreatus, Agaricus bisporus, Auricularia auricula, Coprinus comatus, Phellinus linteus, and Laetiporus sulphureus.

[0059] The mushroom species may be selected from Ganoderma lingzhi.

[0060] Fermentation is a metabolic process used by many species of bacteria and fungi. Through the action of enzymes, such species are able to biochemically convert a given substance into a composite metabolite, thereby releasing energy for use by the species.

[0061] Industry has found utility in different aspects of fermentation. One aspect of fermentation is that it provides a cost effective and sustainable way to convert a given substance into a given product. This ability has been utilised for instance by the food and beverage industries to convert precursors into desired consumable products.

[0062] Alternatively fermentation may be used as a way to grow desirable bacteria and fungi. The healthcare and pharmaceutical industry has for instance found many species of bacteria and fungi to contain useful, pharmacologically active, components. Therefore, rather than synthetically producing the desired compound, fermentation provides a more effective pathway to obtain the same result. The given species may be deliberately grown via fermentation, harvested, and have their useful components extracted for pharmaceutical commodification.

[0063] Mycelium is a subset of fungi that grows via fermentation. Many mycelia species offer health benefits as they contain pharmacologically active IPS. This is robustly documented across scientific literature, and have been long utilised by many indigenous cultures.

[0064] It is therefore desirable to design a fermentation process that efficiently grows mycelia with high IPS (intracellular polysaccharide) concentrations. Such a process may allow the production of mycelia, and the subsequent extraction of socially useful IPS, on an industrial scale.

[0065] There are many variables that may be optimised in the design of the ideal conditions for mycelial fermentation. The present method focussed broadly on submerged fermentation and considers controlling factors such as fermentation media (including control of sugar concentration), duration of fermentation, stirring, dissolved oxygen concentration, pH levels, temperature and scale of fermentation.

[0066] Through the submerged fermentation process, mycelia are grown from a seed culture. Therefore, prior to fermentation, a sufficient quantity of the desired seed culture must first be obtained.

[0067] The seed may be cultured at the beginning of fermentation at about 3.0, 3.5, 4.0, 4.5 or 5.0% v/v, and suitable ranges may be selected from any of these values, for example the necessary quantity therefore depends on the scale of fermentation.

[0068] It will be appreciated that the seed culture used may consist of a combination of pre-existing and expanded seed culture. That is, there may initially be an insufficient quantity of seed culture. A sufficient quantity may therefore be generated by performing a seed train.

[0069] The seed medium may comprise a range of ingredients that support seed expansion. For example, the seed medium may comprise a sugar. The sugar may be present at about 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 g/L, and suitable ranges may be selected from any of these values. The sugar may be selected from glucose, sucrose, maltose, lactose or fructose, or a combination thereof. The seed medium may comprise a rich nutrient source to support growth. For example, the seed medium may contain soy peptone, which is high in carbohydrates. The soy peptone may be present at about 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8 or 5.0 g/L, and suitable ranges may be selected from any of these values. It may also be necessary to include a buffering agent. The buffering agent may be selected from monopotassium phosphate. The buffering agent may be present at about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 g/L, and suitable ranges may be selected from any of these values. The medium may contain a source of magnesium. The magnesium source may be selected from MgSO4.7H20. The magnesium source may be present at 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 g/L, and suitable ranges may be selected from any of these values.

[0070] Mycelial cultures may form pellets when grown in soluble liquid medium, which creates difficulties for transfer of the expanded seed culture. When this occurs an insoluble ingredient may be included. This may provide an insoluble matrix to help diffuse mycelial growth. The media may include about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0% w/v of an insoluble ingredient, and suitable ranges may be selected from between any of these values, (for example about 0.5 to about 5, about 0.5 to about 3, about 0.5 to about 2, about 1to about4, about 1 to about 3.5, about 1 to about 2.5, about 1 to about 2, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 4, about 2 to about 3, about 2.5 to about 4, about 2.5 to about 3.5% w/v). Without wishing to be bound by theory the insoluble ingredient may assist in keeping the mycelia dispersed. The insoluble ingredient may be selected from rice bran. Without wishing to be bound by theory, the rice bran may be added to provide an insoluble matrix.

[0071] A seed train is a technique that may be used to expand a given culture. At each stage of the seed train the culture propagates. Seed train stages may be performed consecutively until the quantity of culture is sufficient for the purposes of the intended fermentation.

[0072] For fermentation procedures performed on an industrial scale, seed expansion via seed train may be preferred. It is likely more cost effective to purchase and expand a small quantity of seeds to the necessary quantity, rather than regularly purchase the necessary quantity of seeds.

[0073] It may be appreciated that once sufficient seed culture has been prepared, initiation of the fermentation process consists of a media being inoculated by the given seed culture.

[0074] Fermentation media is necessary in order to support the growth of the mycelia. Primarily the media provides the mycelia with a source of nutrients that can be metabolically converted into energy. Additionally, the media may serve further purposes, for instance pH regulation.

[0075] The level of inoculation may be about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8% v/v, and suitable ranges may be selected from any of these values. (for example, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 4, about 2.5 to about 8, about 2.5 to about 6.5, about 2.5 to about 4, about 3 to about 8, about 3 to about 6, about 3 to about 4 or about 4 to about 8 % v/v). For example, a 1,000 L bioreactor may be inoculated with 40 L of secondary seed.

[0076] In one embodiment the vessel used for fermentation is a bioreactor. This provides the ability to control parameters such as pH, dissolved oxygen, aeration, and shear force.

[0077] The disclosed fermentation process is scalable to an industrial scale. Therefore, the size of the bioreactor may vary.

[0078] The fermentation media in the bioreactor may comprise an anti-foam agent. The anti-foam agent may be used to reduce any foam that may arise from mycelial fermentation. The bioreactor may be equipped with a foam detector. Upon detecting foam the detector may dispense anti-foam to counteract the production of foam. An example of a suitable antifoaming agent includes XIAMETER M T AFE-1520 Antifoam Emulsion that is a 20% active, food-grade, silicone emulsion. The antifoaming agent may be water-dilutable.

[0079] The inoculated media may then be fermented for 5, 6, 7, 8, 9, 10, 11 or 12 days. The inoculated media may be fermented for 3, 4, or 5 days. The inoculated media may periodically be stirred. The inoculated media may be stirred at about 80, 90, 100, 110, 120, 130, 140, 150 or 160 RPM, and suitable ranges may be selected from between any of these values, (for example, about 80 to about 160, about 80 to about 150, about to about110,about90 to about160, about90 to about150, about90 to about120, about 100 to about 160, about 100 to about150, about 100 to about130, about110 to about 160, about110 to about 140 or about120 to about160 RPM).

[0080] As per the initial stage, once the media is inoculated, the mixture may then be incubated. The preferred duration may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days. The preferred duration may be about 3, 4, or 5 days. The inoculation temperature may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 350 C, and suitable ranges may be selected from between any of these values. The inoculated media may be stirred at about 300, 320, 340, 360, 380, 400, 420, 440, 460, 380 or 500 RPM, and suitable ranges may be selected from between any of these values.

[0081] The media may be incubated at about 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 400 C, and suitable ranges may be selected from between any of these values, (for example, about 20 about 40, about 20 about 36, about 20 about 32, about 20 about 30, about 22 about40, about 22 about36, about22 about32, about24 about40, about24 about 38, about24 about 24, about 26 about40, about 26 about38 or about26 about 32°C).

[0082] For seed trains that are intended to grow cultures of the Ganoderma genus, the composition of the preferred media may comprise any one or more of: • 30 to 50 g/L glucose,

• 2 to 6 g/L soy peptone,

• 1 to 3 g/L KH2PO4, or

• 0.5 to 2 g/L MgSO4.7H20.

[0083] The composition of the media may comprise any one or more of about

S40 to 60 g/L glucose,

5 to 20 g/L peptone, or

1 to 10 g/L yeast extract.

[0084] The media used for Ganoderma cultures may contain a generic sugar, a generic nutrient source, a generic buffering agent, and a generic desiccant.

[0085] If the given culture is not a species of the Ganoderma genus, it will be appreciated that the relevant media may be modified to suit that culture.

[0086] The media may comprise 30, 35, 40, 45 or 50 g/L glucose (or sugar), and suitable ranges may be selected from between any of these values, (for example, about about50, about 30 about45, about 30 about40, about35 about 50,about35 about , about 35 about 40, about 40 about 50 or about 40 about 45 g/L glucose (or sugar)).

[0087] The media may comprise a source of nutrients with a high carbohydrate content, such as soy peptone. The media may comprise such a nutrient at about 2, 3, 4, or 6 g/L, and suitable ranges may be selected from between any of these values, (for example, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 3 to about 6, about 3 to about 5, about 3 to about 4 or about 4 to about 6 g/L).

[0088] The media may comprise a buffering agent, such as KH2PO4. The buffering agent maybe present at about 1.0, 1.5, 2.0, 2.5 or 3.0 g/L, and suitable ranges may be selected from between any of these values, (for example, about 1 to about 3, about 1 to about 2.5, about 1 to about 2, about 1.5 to about 3, about 1.5 to about 2.5 or about 2 to about 3 g/L).

[0089] The media may comprise a magnesium source such as MgSO4.7H20. The magnesium source may be present at 0.5, 1.0, 1.5 or 2 g/L, and suitable ranges may be selected from between any of these values.

[0090] The pH level of the bioreactor may be an important variable with regards to mycelial growth, as there may be given ranges of pH that are more or less conducive to growth.

[0091] In one embodiment the pH may be controlled to be about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0, and suitable ranges may be selected from between any of these values.

[0092] The initial stage of fermentation is typically aerobic. Therefore, to ensure that fermentation can proceed, it may be important to maintain a concentration of dissolved oxygen in the bioreactor mixture.

[0093] The level of controlled oxygen may be controlled in the fermentation media. The level of oxygen in the fermentation mixture may be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30%, and suitable ranges may be selected from between any of these values.

[0094] In an alternate embodiment the levels of dissolved oxygen may not be controlled.

[0095] When conducting biochemical reactions it is generally preferable for the temperature to be high, in order to maximise the rate of reaction. However, when the biochemical reaction proceeds via an enzymatic pathway, such as is the case with the present method, the stability of the enzyme must be considered. Temperature may have a pronounced effect on enzyme activity; higher temperatures may cause enzymes to degrade.

[0096] In one embodiment the temperature of the fermentation media may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 350 C, and suitable ranges may be selected from between any of these values.

[0097] The fermentation mixture may be dosed with sugar during fermentation. The sugar may be selected from any sugar.

[0098] In particular, the sugar used to dose the fermentation mixture may be selected from glucose, fructose, maltose, sucrose, lactose, galactose, xylose, or a combination thereof. In particular the sugar is selected from glucose.

[0099] Regarding the timing of when the sugar doses are made, it may be appreciated that the doses may be made between fixed intervals. For instance, it may be suitable to dose the fermentation mixture with sugar once a day. The fermentation mixture may be dosed with sugar at about 15, 20, 25 or 30 g/L and suitable ranges may be selected from between any of these values.

[0100] In an alternate embodiment the timing of each dose of sugar may not be fixed. It may be desirable to monitor the sugar concentration of the bioreactor, and dose the mixture with sugar only when the concentration is found to have fallen below a given threshold. Further, the concentration of the dosage may be variable based on how low the concentration in the bioreactor has fallen, and what the desired sugar concentration is.

[0101] If after any seed train stage there remains an insufficient quantity of seed culture, a further, successive stage may be performed. A fresh media, of the same composition as the media used in the preceding stage or stages, may be inoculated by the expanded seed from the preceding stage. The concentration of the expanded seed culture relative to the media may preferably be about 2, 3, 4, 5, 6, 7 or 8% by weight, and suitable ranges may be selected from between any of these values.

[0102] The mycelia may attach to surrounding surfaces as they grow. The media may be subjected to high shear mixing. If the speed of high shear mixing is too fast, it may damage or break the mycelia. If the speed is too slow, it may be unable to sufficiently disturb the mycelia and prevent yield loss due to clinging. The preferred speed of mixing is about 300, 350, 400, 450, 500, 550 or 600 RPM, and suitable ranges may be selected from between any of these values.

[0103] The shear may be provided by a mixer such as a Rushton turbine or impeller. A Rushton turbine is an impeller. The impeller may comprise a flat disk with vertically mounted blades that radially extend from the flat disk. The impeller may comprise 3, 4, 5, 6, 7, 8, 9, or 10 blades. The tip speed of the distal region of the impeller should not be so fast as to damage or break the mycelia, nor too slow such that it does not sufficiently disturb the mycelia and prevent yield loss due to clinging. The tip speed of the impeller may be about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2 or 3.3 m/s, and suitable ranges may be selected from between any of these values (for example, about 1.5 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 1.6 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 1.7 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 1.8 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 1.9 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 2.0 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 1.5 to about 2.2, about 2.1 to about 3.3, about 1.5 to about 3.1, about 1.5 to about 2.9, about 1.5 to about 2.8, about 1.5 to about 2.7 or about 1.5 to about 2.2 m/s). The tip speed may be about 1.8-2.75 m/s.

[0104] Prolonged fermentations risk a decrease in the yield of the mycelia upon harvest. If left to ferment for too long, there may be fruiting body formation (that is, there is a decrease in the quantity of mycelia because the mycelia is converted into a fruiting body). Further, the mycelia may cling to the sides of the bioreactor, decreasing the quantity of mycelia that can be removed and harvested from the bioreactor at the end of fermentation.

[0105] It may further be preferable to harvest the mycelia before the point where the yield begins to decrease due to fruiting and clinging. The rate of mycelial growth decreases over time. If there are a limited number of bioreactors available for fermentation, it may be a more efficient allocation of resources to harvest the mycelia once the rate of growth falls below a given threshold, thereby allowing the given bioreactor to be used for a new fermentation. This maximises the rate of fermentation across available bioreactors, and hence mycelia production across production facilities.

[0106] There is the countervailing concern that by not allowing fermentations to run their full length, more starting material (media and culture) per unit of mycelia produced is required. Therefore, different durations may be preferred depending on for instance the cost of starting material versus bioreactor availability and value of harvested mycelia.

[0107] and suitable ranges may be selected from between any of these values, (for example, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 3 to about 6, about 3 to about 5, about 3 to about 5, about 4 to about 6 or about 4 to about 5 days).In one embodiment the duration of fermentation is three days.

[0108] In an alternate embodiment the duration of fermentation may be 2, 3, 4, 5, or 6 days.

2. Downstream processing

[0109] Upon completion of fermentation the grown mycelia may be physically removed from the bioreactor for downstream processing, culminating in extraction of the commercially valuable IPS.

[0110] In one embodiment the bioreactors are equipped with a nozzle that may be connected to a hose. When a pressure is applied, the contents of the bioreactor may be harvested from the bioreactor into a further vessel for downstream processing.

[0111] The harvested contents of the bioreactor post-fermentation are a complex matrix. The desired biomass (the mycelia) must therefore be purified from the other components of the matrix. The first step in separation may be to concentrate the biomass from the liquid phase.

[0112] As a first step, the biomass may be sieved using a 50, 55, 60, 65, 70, 75, , 85, 90, 95 or 100 pm sieve, and suitable ranges may be selected from between any of these values (for example, about 50 to about 100, about 50 to about 90, about 50 to about80, about 50 to about70, about 55 to about100, about 55 to about85,about 55 to about70, about60 to about100, about 60 to about90, about60 to about80, about to about100,about65 to about90, about 65 to about80,about65 to about75, about70 to about100, about70 to about90, about70 to about80, about70 to about or about 80 to about 100 pm sieve).

[0113] The biomass may be concentrated by filtering the harvested contents of the bioreactor through a hollow fibre membrane (HFM). The pore size of the HFM may be about 450, 460, 470, 480, 490, 500, 510, 520, 530, 540 or 550 kDa, and suitable ranges may be selected from between any of these values.

[0114] In an alternate embodiment the biomass may be concentrated by rotating the harvested contents of the bioreactor in a centrifuge.

[0115] The process may include a further stage of purification where the liquid phase separated during the concentration step is used to wash the separated biomass. The washing may be performed by vacuum filtration, or any alternative technique used in the industry.

[0116] The IPS may be extracted from the isolated biomass.

[0117] For example, the biomass may first be dried, for example by heating.

[0118] The mycelia may first be broken or disrupted. This may assist the better release and separation of the IPS. The method of mycelia disruption may be high shear overhead mixing, for example, through the use of a Silverson high shear mixer.

[0119] The method of mycelia disruption may be high shear homogenisation. For example, using an emulsion homogeniser such as a microfluiser APVTM. For example, the biomass may be passed through at about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 bar, and suitable ranges may be selected from between any of these values. The biomass may be passed through the emulsion homogeniser multiple times. For example, the biomass may be passed through the emulsion homogeniser 2, 3, 4, or 5 times, and suitable ranges may be selected from between any of these values.

[0120] Water may be added to the biomass to assist disruption. That is, water may be added such that it accounts for 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40% by weight of the total amount of the biomass (i.e. including the water), and suitable ranges may be selected from between any of these values. Without wishing to be bound by theory, the addition of water may improve the fluid dynamics in order to assist with mycelial disruption.

[0121] The disrupted mycelia may then be heat treated at about 60, 62, 64, 66, 68, , 72, 74, 76, 78, or 80 °C, and suitable ranges may be selected from between any of these values. The media may then be cooled to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 °C prior to filtration, and suitable ranges may be selected from between any of these values.

[0122] In one embodiment the broken mycelia may be incubated in order to form a broth. The incubation may make it easier to extract the IPS. The fermentation temperature may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 °C, and suitable ranges may be selected from between any of these values.

[0123] The broken mycelia may be filtered to isolate the IPS using a HF membrane. The pore size of the membrane may be selected from

• about 10 kDa,

• about100 kDa, or

• about 500 kDa, or

• any combination thereof.

[0124] In an alternate embodiment any industry filtration technique may be used.

[0125] The permeate from filtration may be collected into a tank.

[0126] The process may collect IPS having a molecular weight of

• 100-200 kDa,

• 10-20 kDa, and

1-5 kDa.

[0127] The fractions may also comprise a-glucans and p-glucans.

EXAMPLES

[0128] A number of different examples are presented that evaluate the mycelia. Table 1 provides a summary of the upstream processing examined, and Table 2 summarises the downstream processing that has been examined.

Table 1. Upstream processing examined in Examples 1-5.

Step Media Conditions Expected outcomes/QC checks

Primary seed (4x 2 L 500-mL media: 40.0 Inoculate from DCW (g/L)= 18-20 flasks) g/L glycerol g/L. glucose, 4.0 g/L soy stock at 2% (v/v). Microscopy for growth peptone, 1.5 g/L and contamination KH2PO4, 1.0 g/L 30 °Cwith 120-140 check. MgSO4.7H2O and 2% RPM shaking over 7 (w/v) rice bran. pH days. not adjusted. Secondary seed 39-L media: 40.0 Inoculate 4% (v/v) DCW g/L of 14-18 (2x 20 L g/L glucose, 4.0 with primary seed. g/L. Microscopy for bioreactors; g/L soy peptone, growth and Sartorius Biostat 1.5 g/L KH 2 PO 4 , Agitation: 400-450 contamination DCU) and 1.0 g/L RPM (2.17 - 2.45 m/s check. MgSO 4 .7H 2 O. pH tip speed) not adjusted. Aeration: 1 Xiameter 1520 AFE (0.05 %(v/v in the VVM media; and as needed) to control DO and pH foaming not controlled

Upto 3 days

Production batch 960 L medium: 40.0 Inoculate 4% (v/v) DCW of 12-17 g/L. (1x 1,000 L g/L with bioreactor; glucose, 10 g/L soy secondary seed. Residual glucose of 0 Custom made; peptone, 5 g/L yeast 8 equipped with three extract, 1.5 g/L Agitation: 100-150 g/L. rushton impellers, KH 2 PO 4 , RPM (1.83 - 2.75 m/s ring and 0.5 g/L tip speed) Microscopy for growth sparger and baffles) MgSO4.7H20. pH not and contamination adjusted. Aeration: 0.2-1 VVM check.

Xiameter 1520 AFE DO and pH not Crude PS yield of (0.05 % (v/v) in the controlled. Target -11.5 media; and as >30% DO. kg needed) 3-5 days of Purified PS yield of tocontrolfoaming fermentation -1.15 kg

~ 6% (w/w)jp glucans in purified PS Tatua Soy peptone; Food grade glucose (interchem), DSM yeast extract and other chemicals from Sigma Aldrich

Table 2. Downstream processing steps examined in Examples 1-5.

Step Equipment Details Expected outcomes/ QC checks

Concentration 74 pm sieve and 500 Collect bulk solids Approx 250 kg of bulk kDa HFM (Koch TM using sieve, scrape solids and 157 kg of membranes) into a sterile 60-L concentrated biomass drum. Permeate (retentate). (from sieving) to be concentrated using HFM

Disruption Microfluidizer (APV TM ) Approximately dilute Visualise samples biomass 1:0.5 on after each pass weight basis with RO under microscope, water to improve and collect samples fluid dynamics. Pass for PS and beta through the glucan analysis. homogeniser three times at 600-900 Heat treatment Bioreactor or With low agitation temperature (if available), controlled sterile incubate for 60 min holding vessel at 70 °C.

Purification 500 kDa HFM Collect Sample for (Koch TM permeate microscopy and membranes) (product), and estimation of PS retentate to content and profiles waste using HPLC.

Freeze drying Any commercial Freeze dry upto 7 ~11.5 kg of crude and milling. freeze dryer. Robot days or until PS solids. Pack in coupe@ R45 or constant weight. vacuum sealed commercial milling Mill to bags. equipment. homogeneity.

1. Example 1: Initial flask growth and evaluation of Ganoderma lingzhi in submerged culture

[0129] This example examined the growth of Ganoderma lingzhi in ten different growth media.

1.1 Method

[0130] Shake flasks were harvested on days 3, 6, 9, and 12 and measured for pH and dry cell weight.

[0131] A subset of the dried mycelial samples was extracted in H20 at 70 °C disruption by sonication, and polysaccharides in the extracts were measured by size exclusion chromatography (SEC) using high-performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD).

[0132] The remaining mycelial samples were extracted in H20 at 70 °C with sonication, followed by SEC using HPLC and ELSD.

[0133] Ganoderma seed medium (GSM) was used to expand the seed culture of G. lingzhi, and included 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H20. Ground rice bran was included at a rate of 2% (w/v) to provide an insoluble matrix to support diffuse mycelial growth. A two-stage seed train was used to expand G. lingzhi. A 250 mL flask containing 50 mL GSM was inoculated with 1 mL of a glycerol stock vial of G. lingzhi. The flask was incubated at 30 °C with 120 RPM shaking for 6 days.

[0134] The primary seed culture was used to inoculate 500 mL GSM in a 2 L flask at a rate of 5% (v/v) (25 mL primary seed culture/500 mL GSM). The secondary seed culture was incubated at 30 °C with 120 RPM shaking for 4 days, at which point there was thick, diffuse mycelial growth.

[0135] Tested production media formulations are shown in Table 3.

Table 3. Formulations, in g/L, of experimental productionmedia

Medium A B C D E F G H I

glucose 35 25 40 38 50 45 25 20 50 sucrose 20 20 soy peptone 5 5 5 4 4.5 2 10 yeastextract 5 1 2 1 14 14 NH 4CI 4

KH 2 PO 4 1 1.5 0.75 0.5 1 1 1.5 1.5

K2 HP0 4 0.5

KH 2PO1H 20 1 2 0.75 0.45

MgSO 4 7H 20 0.5 1 1 0.5 0.26 0.26 1 0.5

corn oil 5

[0136] The production flasks were inoculated with the secondary seed culture at a rate of 4% (v/v, 2 mL seed culture/50 mL medium). The flasks were incubated at 30 °C, 120 RPM shaking for 12 days. One flask of each medium was harvested completely on days3,6,9,and 12.

[0137] Growth in many of the media was robust by day 3. In some media, there was little free liquid because biomass had grown to such high levels.

[0138] The pH of each harvested flask was measured. Generally, as aerobic microorganisms metabolize carbohydrates (e.g. glucose), organic acids are produced, which lowers the pH of the culture in uncontrolled systems. When available carbohydrate is exhausted, the culture pH tends to increase. A notable increase in pH was observed by day 12 in all media except B, E, and F.

[0139] To determine dry cell weight (DCW), the entire contents of each harvested flask were transferred to 50 mL centrifuge tubes and centrifuged at 4700 RPM (4816 g) for 20 min. The supernatant was removed, and the pellets were washed with up to 20 mL phosphate buffered saline to remove residual medium. The washed biomass was centrifuged again at 4700 RPM for 20 min, and the supernatant was removed. The tubes were placed, uncapped, in a 70° C drying oven for 3 days to dry the biomass to completion. Based on resulting dry pellet, DCW was calculated. The results are shown in Figure 3.

Extraction and polysaccharideanalysis

[0140] Extraction of dried mycelial biomass was conducted on all samples. The mycelial biomass was subjected only to aqueous extraction conditions. Reverse osmosis (RO) H20 was added to each sample at a ratio of 1:10 dried biomass:RO H20. All extractions were carried out in a shaking water bath at 70 °C with 200 RPM linear agitation. Samples were extracted twice with RO H20 for 2 h each. After the first extraction, the mixture was centrifuged at 4000 RPM (3488 g) for 5 min. The supernatant was removed, and an equal volume of RO H20 was returned to the biomass for the second extraction. The supernatant from the second extraction was added to the first extract, and the combined extract was filtered across a 0.2 pm membrane to ensure no viable G. lingzhi material remained.

[0141] Extracts were subjected to size exclusion chromatography (SEC) using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD) to assess the polysaccharide profiles of each sample.

1.2 Results

[0142] As shown in Table 4 three groups of peaks were observed in the mycelial extracts: early which are thought to be higher MW polysaccharides produced during fermentation; middle which are thought to be mainly associated with monomers and other media components; and late, which are unknown compounds. The areas of the peaks in each of these sections were calculated by integrating the area under the peaks.

Table 4: Early (A), middle (B), and late (C) peaks observed in the mycelial extracts. Table 2. Early, middle, and late peak areas of each extract.

Day3 Day6 Day9 Day12 Medium Early Middle Late Early Middle Late Early Middle Late Early Middle Late A 26.4 23.7 17.5 6.6 4.9 4.8 8.5 1.3 7.3 8.7 1.7 7.1 B 14.4 25.0 13.3 11.7 10.8 10.2 14.7 11.9 12.3 17.6 10.1 14.6 C 8.5 6.8 6.6 6.0 11.5 5.9 7.6 0.8 7.4 8.3 1.5 7.9 D 11.2 13.8 6.7 6.3 10.4 5.0 5.0 5.7 3.1 7.4 0,5 6.6 E 12.2 17.4 7.9 6.4 9.8 5.3 10.9 12.8 10.3 6.4 7,3 6.0 11.1 15.1 7.5 10.6 9.2 8.6 5.9 4.7 4.7 6.3 5.8 4.8 G 11.4 10.9 5.6 7.3 8.0 4.1 6.6 2.5 4.1 7.1 2,4 4.5 H 12.0 27.4 6.7 7.1 10.0 5.4 12.4 12.1 9.2 9.4 12.2 6.7 1 9.8 13.8 8.2 10.5 11.6 10.0 12.3 0.6 11.9 10.0 0.3 10.2 J 12.5 14.2 6.2 8.2 22.3 4.6 7.7 10.8 7.0 6.3 4.0 4.5

[0143] The best growth was observed in media G and J, which both achieved final DCWs of ca. 26 g/L on day 12.

2. Example 2: Scale-up of Ganoderma lingzhi growth in submerged culture

[0144] This example looked at the growth of Ganoderma in 1 L bioreactors.

[0145] Two media and three process conditions were evaluated in 1 L bioreactors for growth and intracellular polysaccharide production by Ganoderma lingzhi. The process conditions include

Sa pH/dissolved oxygen (DO) shift,

• constant pH, and

• continuous fed-batch additions of glucose.

[0146] Two stage seed cultures were made in flasks, and a 3% v/v inoculation rate was used for the bioreactors.

[0147] Experiments in six bioreactors were run for 12 days, and were periodically sampled at 3, 6, 9 and 12 days.

[0148] Samples were analysed for dry cell weights (DCW) to assess growth, and both wet and dry extracts were used for SEC HPLC and total carbohydrate analysis as per methods developed in previous studies.

2.1 Methods

[0149] Ganoderma seed medium (GSM) was used to expand the seed culture of G. /ingzhi, and included, 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H2O. Ground rice bran was included at a rate of 2% (w/v) to provide an insoluble matrix to help diffuse mycelial growth. A two-stage seed train was used to expand G. Iingzhi. Primary (10) seed was prepared by inoculating 2 mL vial in 100 mL (2%, v/v) of GSMand incubated for 8 days at 30 °C and 120 rpm. Secondary (20) seed was prepared by inoculating 12 mL of 10 seed in 300 mL GSM (4 %, v/v) and incubating for 7 days at 30 °C and 140 rpm.

[0150] Two media recipes and three conditions in each were assessed for the growth and polysaccharide production in 1 L reactors over the period of 12 days (see Table 5). Production media (G and J) was prepared in bulk, and aliquoted into bioreactors. The reactors were then sterilised for 20 min at 121 °C and 15 psi. A 3% (v/v) inoculation was made. Samples were taken at four points (Days 3, 6, 9 and 12) for analysis. Temperature was maintained at 30 °C, and pH was controlled with manual additions of 1 M H 3 PO4 and 1 M NaOH. Xiameter 1520 AFE (0.05%) was added directly to the media for controlling foaming. Experimental conditions are shown below in Table 5.

[0151] Two 50 mL samples were pulled out at each point into tared 50 mL Falcon tubes. The weight of the culture was measured and then the samples were spun at 4,700 RPM for 20 min. The supernatant was discarded, and the pellet was washed twice with PBS (pH 7.4). One tube was stored at 4 °C to be used for wet extraction, and the other was dried at 60 °C for 3 days for dry extraction.

Table 5. Growth media and reactor conditionstrialled

Run ID (1 L Conditions bioreactors) pH controlled at 3 (Days 0-4); pH at 4.5 (until harvest) GL001 DO controlled at 25% (Days 0-6); DO at 10% (until harvest)

Constant pH at 4-4.5 throughout the run. DO not controlled. Agitation at 400 RPM. Aeration at1LPM. 61003 Repeated fed-batch addition of glucose (20 g/L). pH and DO not controlled. Agitation at 400 RPM. Aeration at1LPM. pH controlled at 3 (Days 0-4); pH at 4.5 (until harvest) DO controlled at 25% (Days 0-6); DO at 10% (until harvest) Constant pH at 4-4.5 throughout the run. DO not controlled. Agitation at 400 RPM. Aeration at 1 LPM. GRepeated fed-batch addition of glucose (20 g/L). pH and DO not controlled. Agitation at 400 RPM. Aeration at 1 LPM.

[0152] All fermentations ran for 12 days. The dry cell weights (DCW) are shown in Figure 4, also a comparison with media G and J from Example 1 is shown. As the run progressed there was formation of fruiting bodies in the bioreactor, particularly in bioreactors with Medium G (GL001 - GLO3), therefore the sampled volume is not representative of the total biomass, rather the submerged biomass. Formation of fruiting bodies could be a factor of pH, dissolved oxygen or tip speed. However, it was noted that the rice bran was not added to the media in reactors, which could have helped restrict the formation of fruiting bodies (as seen in flasks). Despite this, fewer fruiting bodies were seen in GL005, indicating that the medium and process parameters such as pH (4.5) and dissolved oxygen could also contribute to better submerged growth.

[0153] Glucose (20 g/L) was added on Day 3 for cultures GL003, as the pH dipped to ~ 3.4. Glucose bolus seems to have positive effect as seen in Day 6 DCW of the culture GL005.

Extraction and polysaccharideanalysis

[0154] Intracellular polysaccharides were extracted from dry cell pellets using the extraction method described in Example 1. A modified protocol was used for extraction from wet cell pellets. The total samples for extraction was 44 and the final filtered volumes of extracts ranged from 0.3 ml to 15 mL.

Dry cell extraction

[0155] Dry cell pellet weight was determined and reverse osmosis (RO) H20 added to each sample at a ratio of 1:10 dried biomass:RO H20. A minimum volume of 500 pL was used. The samples were incubated at 70 °C with 200 RPM linear agitation for 1 h. The samples were sonicated in a sonication water bath at 65 °C for 30 min, then returned to the shaking water bath for 1 h. The samples were centrifuged at 4,700 RPM for 5 min, and the supernatant transferred to a fresh tube. A second extraction was performed by adding the same volume of RO H20 and incubating for 2 h at 70 °C with 200 RPM linear agitation. The samples were centrifuged at 4,700 RPM for 5 min, and the supernatant combined with the first extraction. The extracts were filtered across a 0.2 pm membrane to remove viable G. lingzhi material.

Wet cell extraction

[0156] The same volume of RO H20 used for dry cell extraction was added to the wet cell pellets. The cells were disrupted by homogenization at 13,500 RPM for 1 min. The samples were incubated at 70 °C with 200 RPM linear agitation for 2 h. The samples were centrifuged at 4,700 RPM for 5 min, and the supernatant transferred to a fresh tube. A second extraction was performed by adding the same volume of RO H20 and incubating for 2 h at 70 °C with 200 RPM linear agitation. The samples were centrifuged at 4,700 RPM for 5 min, and the supernatant combine with the first extraction. The extracts were filtered across a 0.2 pm membrane to remove viable G. /ingzhi material.

SEC HPLC polysaccharideanalysis

[0157] Samples of filtered extracts were provided for each of 6 cultures (GL001 through to GL006) with individual samples at days 3, 6, 9 and 12. Only day 3 and 6 samples were collected for GL005.

[0158] Samples were either from "dry" extraction or "wet" extraction for a total of 44 samples. Samples were analysed directly as described previously for SEC HPLC chromatography.

2.2 Results

[0159] Early peaks indicate high molecular weight compounds, mid-peak may be monosaccharides or media components and late peak are unknown compounds. None of the cultures appear to be producing significant quantities of high MW carbohydrates which would appear as broad peaks around the early peak area in the chromatogram.

[0160] Figure 5 shows that both GL005 and GL006 had higher total carbohydrates when compared to the previous study of Example 1.

Total carbohydrate analysis.

[0161] The total carbohydrate analysis was performed using the phenol sulphuric acid colorimetric analysis method as described earlier. Samples were diluted 1 in 16 with distilled water. The results are shown in Table 6.

Table 6. Total carbohydratesas mg/ml glucose in WET and DRY extracts

Measurement Glucose (mg/mL) Sample Day Wet extract Dry extract GL001 3 2.7 1.6 6 1.9 9.9 9 4.7 15.4 12 13.5 7.4 GL002 3 12.7 24.4 6 1.3 1.8 9 1.2 2.1 12 3.9 1.5 GL003 3 11.2 11.3 6 3.8 6.1 9 3.1 6.2 12 1.8 1.2 GL004 3 10 13.1 6 9.3 18.5 9 2 13.4 12 3.5 9.6 GL005 3 12.1 15.8 6 23 16.5 GL006 3 11 12.3 6 19.9 18.1 9 14.2 18.2 12 9.4 16

[0162] G. /ingzhi showed good submerged growth in the bioreactors with Medium J. G. lingzhi grew well as a submerged culture at pH 4.5 when compared to the pH at 3.0.

[0163] The extracts were analysed by SEC HPLC to obtain fingerprint profiles of MW distribution as well as total carbohydrate analysis. The total carbohydrates as determined from total peak area in SEC HPLC and the colorimetric method appear to be in reasonable agreement. There is a higher level of total carbohydrates in the dry extracts, e.g. GLOO4 Wet extract -day 12 has 3.5 vs GL004 Dry extract- day 12 at 9.6 mg/mL. Otherwise the wet and dry extracts are somewhat similar in terms of changes through the fermentation period.

[0164] GL005 and GL006 appear to give an increase or relatively constant level of carbohydrates throughout the fermentation period and were higher than that seen in Medium J of Example 1.

3. Example 3: Bioreactor fermentations at 1 L scale

[0165] The example examined a set of nine fermentation runs over two batches (Stage 1 and 2) to study the impact of various process conditions on submerged growth and polysaccharides (IPS) formation. Stage 1 fermentations were run for 10 days, and Stage 2 for 6-days. The fermentation period was reduced in order to limit fruiting body formation.

[0166] In summary, the process conditions studied were aeration (0.5-1 litres/minute [LMP]), agitation (200-400 RPM), repeated glucose addition (to keep residual glucose levels 20-30 g/L, where practically possible), with and without pH control and DO control.

[0167] All fermentations were run successfully, and targets of higher submerged biomass and PS profiles were successfully achieved.

• GL008, GL12 and GL014 produced highest dry cell weights (DCW) of 18, 21 and 14 g/L, respectively. These values were higher than those reported in Example 2.

• All the fermentations showed submerged growth initially (except GLOO7), however due to the body formation, the submerged growth decreased over the fermentation period.

• The submerged biomass, supernatant and fruiting bodies were analysed for PS profile determination using SEC HPLC. $-glucan analysis was performed on the AG standard extract and the GLO14 biomass and extract (Day 3 and 6 sampled combined).

o All the fermentations (especially, GLO08, GLO11, GLO12 and GLO14) had similar profiles to that of AG standard (extract from AG fruiting bodies from China), however, the quantity of the PS varied amongst the fermentations.

o Supernatants and fruiting bodies had little to no target PS, confirming that the mycelial biomass retains most of the PS.

o Molecular weight determination using MALLS indicated that the submerged fermentations had similar molar mass of PS as seen in AG standard (5 KDa).

o Highest PS content was seen in GL14 followed by GLO11 and GL008.

o Biomass and extracts samples from GLO14 (Day 3 and 6) had 12.36% and 5.24% of P-glucans, when compared to 11.73% in the Standard extract.

Methods

[0168] "Ganoderma seed medium" (GSM) was used to expand the seed culture of G. lingzhi, and included, 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H20. Ground rice bran was included at a rate of 2% (w/v) to provide an insoluble matrix to help diffuse mycelial growth. A two-stage seed train was used to expand G. /ingzhi. Primary (10) seed was prepared by inoculating 2 mL vial in 100 mL (2%, v/v) of GSM and incubated for 7 days at 30 °C and 120 rpm. Secondary (20) seed was prepared by inoculating 12 mL of 10 seed in 300 mL GSM (4 %, v/v) and incubating for 7 days at °C and 140 rpm. Secondary seed had a DCW of 18.8 g/L.

Experimental setup in 1 L bioreactors

[0169] Medium J was prepared and sterilized as described in Example 1. Rice bran was not added to the reactors. A total of nine conditions were studied and the experimental conditions are presented in Table 7.

Table 7. Experimental conditionsstudied (GL07-GLO15)

Run# Details Notes Stage 1 (10 days -fermentation) GL007 Constant pH (4-4.5); No DO control; 400 rpm; 1 Repetition of GL005 (SOW2a) LPM. GL008 Repeated glucose addition; No pH and DO control; Repetition of GL006 (SOW2a) 400 rpm; 1 LPM GL009 Repeated glucose addition; Constant pH (4-4.5); Same as GLO12 but with pH and DO Do control (>25% for 6 days; <10% until harvest) control GL010 Constant pH (4-4.5); DO control (>25% for 6 days; Same as GL007 but with DO control <10% until harvest) GL011 Constant pH (4-4.5); No DO control; 100-200 rpm; Same as GL007, but with lower 1 LPM. agitation GL012 Repeated glucose addition; No pH and DO control; Same as GLO8, but with lower 100-200 rpm; 1 LPM agitation Stage 2 (6 days -fermentation) GL013 Repeated glucose addition; No pH and DO control; Same as GLO8 but with lower 400 rpm; 0.5 LPM aeration rate GL014 Constant pH (4-4.5); No DO control; 100-200 rpm; Same as GL011 but with lower 0.5 LPM. aeration rate GL015 Constant pH (4-4.5); One-off glucose bolus (on Same as GL011 but with DO control Day 3), DO control (3 days - >25%; Until harvest (3 days - >25%; Until harvest <10%) <10%) Agitation and Aeration in cascade (50-400 rpm, 0.1-1 LPM)

[0170] A 5% (v/v) inoculation was added to the reactors from the secondary seed. Stage 1 fermentations were run for 10 days, whereas stage 2 fermentations for 6 days. Prolonged fermentations were avoided to limit fruiting body formation. The runs were periodically sampled for dry cell weight (DCW), residual glucose concentrations and polysaccharide (PS) analysis of both the biomass and supernatants. Fruiting bodies were collected at the end of the run and were analyzed for DCW and PS analysis. Sterile bolus of glucose was added to GL008, GL009, GL012 and GL013 to keep the glucose levels 20 g/L. In addition, one-off glucose bolus was added to GLO15, before changing the DO control to 510% on Day-3.

3.1 Results

[0171] All fermentations were run successfully. DCW for GL007-GL015 is presented in Figure 6.

[0172] By Day-3, the DCW of all fermentations was >4 g/L, except for GL009. By Day-6, the DCW of GL008, GL009, GLO11 and GL012 more than doubled. Amongst the stage 2 fermentations, GL013 produced 13 g/L DCW by Day 6, whereas the DCW decreased to 3.59 g/L from 9.68 g/L (Day 3) in GL013; and decreased to 4.21 g/L from 7.79 g/L (Day 3) in GLO15. Similar decrease in DCW was also evident in GL007, GL009, GLO10 and GLO11. By the end of fermentations (Day 10 for stage 1 and Day 6 for stage 2), GL008, GL012 and GL014 produced highest DCW of 18.1 g/L, 20.8 g/L and 13.1 g/L, respectively.

[0173] Residual glucose (g/L) in GL007-GL15 is shown in Figure 7. Except where glucose bolus was added and in GL014, the residual glucose was ~0 g/L by Day 6 indicating the complete exhaustion of the readily available C source.

[0174] All parameters were controlled as per the experimental plan. AFE Xiameter was added to control the foaming as required (foaming was more evident at the start of the runs). 1M NaOH and 1M H3PO4 was initially used to control pH in Stage 1 fermentations (GL007, GLOO9, GLO10 and GLO11). However, due to utilization of excessive amounts of both acid and base by GL007 and GLO10, the acid and base was upgraded to 5M NaOH and 5M H3PO4 on Day 2 for all the reactors (with pH control conditions). For stage 2 fermentations, 5M acid and base was used from the beginning.

[0175] Overall, the fruiting bodies were less evident in GL08, GLO11, GLO12 and GLO15. GLO15 produced the least amount of fruiting bodies.

Extraction, polysaccharide and 3-glucan analysis:

[0176] Intracellular polysaccharides were extracted from dry and wet cell pellets using the extraction method described in Example 2. For the HPLC profiles, samples are labelled in the following manner: 7W-day3 = Fermentation GLOO7, wet extract from day three).

SEC HPLC polysaccharideanalysis

[0177] Samples of filtered extracts were provided for each of 6 cultures (GLOO7 through to GL12) with individual samples at days 3, 6, and 10. Only day 3 and 6 samples were collected for GLOO13-15. Only dry extraction samples were analyzed directly as described previously for SEC HPLC chromatography.

[0178] Analysis was initially performed on all samples as described earlier using SEC HPLC. This method used the provided samples directly for analysis.

[0179] However, work on selected samples using molecular weight cut-off filters (MWCO) showed that a polysaccharide concentration step was necessary to enhance the polysaccharide concentration in each sample prior to analysis. The biomass and supernatant polysaccharides were concentrated using ethanol precipitation. Briefly the method used: 2 ml of sample was taken, and 8 ml of abs. ethanol was added, the sample shaken and centrifuged, the pellet was re-suspended in 0.5 ml water. After sonication and centrifugation, the resultant supernatant was used directly for analysis.

Results from SEC HPLC of polysaccharide concentrates of samples.

[0180] In general, all samples showed a broad polysaccharide peak as described above, some samples also showed significant large peaks at 10 minutes.

[0181] There was a lot of similarity between the biomass extracts and the standard material.

[0182] The estimated PS content was calculated based on the peak area, converting to pg polysaccharide using a standard curve for standard, then converted to mg/g biomass based on weight of biomass.

[0183] Using the above standard curve, the estimated PS data for all the fermentations is shown in Table 8 and Table 9. Table 8 is the estimated PS content per gram of dry biomass, whereas Table 9 is the estimated PS content per total dry biomass produced in a 1 L fermentation broth.

Table 8. Estimated PS content per dry gram of biomass. The values are based on the peak areas obtained from SEC HPLC

Sample Mg Polysaccharide

Day 3 Day 6 Day 10

GLOO7 90.3 76.6 0.0

GLOO8 63.0 40.0 35.2

GL009 76.8 40.1 0.0

GLO10 75.9 70.7 62.3

GLO1 89.5 71.8 53.5

GLO12 80.1 31.6 24.9

GLO13 51.7 69.9

GLO14 90.3 91.1

GLO15 70.3 109.0

Table 9. Estimated PS content per total dry biomass produced per litre of fermentation. The values are based on the peak areas obtained from SEC HPLC

Sample Mg Polysaccharide

Day 3 Day 6 Day 10

GLOO7 361.1 191.4 0.0

GLOO8 377.8 559.5 633.5

GLOO9 153.6 200.6 0.0

GLO10 531.0 494.9 218.2

GLO11 402.5 646.0 213.8

GLO12 376.7 474.4 523.6

GLO13 500.1 251.4

GLO14 828.6 1194.7

GLO15 548.1 459.7

[0184] The total PS content in a litre of fermentation indicates that GL14 produced up to 1,200 mg of PS, followed by GL11 and GL008.

Analysis of fruitingbodies and supernatant

[0185] Each of these samples from Day 10 was treated to produce a polysaccharide concentrate as described previously. Almost all of the supernatants and fruiting bodies have high levels of peaks ascribed to lower MW polysaccharides or non-polysaccharide material (Table 8 and Table 9). Therefore, it is suggested that the Figures for PS content from the peak areas are not comparable with the biomass extract numbers. The two exceptions are the fruiting bodies from 8 and 12 which show more typical polysaccharide type analytical profiles (Table 8). This demonstrates that the fruiting bodies and supernatants have no or very little polysaccharides.

Molecular weight determination using MALLS

[0186] The molecular weight was determined using the Multiangle laser light scattering (MALLS) detection system to confirm the similarity between the AG standard sample and the submerged fermentation samples. Only GL014 and GLO15 was run for confirmation. Results indicate that the molar mass distribution of GL014 Day 6 and GLO15 Day 6 samples were 4.7 KDa and 7.5 KDa respectively.

Determination of total 0-glucan content

[0187] p-glucan analysis was carried out on GL14 by pooling Day 3 and 6 samples.

[0188] The total s-glucan content was determined using the Megazyme assay kit based on the method by McCleary and Draga following the instruction given in the manual. This assay is based on the determination of the total glucan content and the a glucan content. The p-glucan content is then calculated by subtracting the a-glucan content from the total glucan content.

[0189] Briefly, for the determination of the total glucan content the samples (90 mg) were treated with 2 mL ice cold sulphuric acid (12 M) on an ice water bath for 2 h with occasional vigorous stirring on a vortex mixer. After addition of 4 mL water, the contents were vigorously mixed on a vortex mixer and another 6 mL water was added followed by mixing. The samples were heated at 100 °C for 2 h. After cooling down, the samples were transferred into 100-mL measuring flasks, 6 mL KOH (10 M) were added and the volume was made up to 100 mL with acetate buffer (200 mM). An aliquot of the solution was centrifuged at 1500 g for 10 minutes. The amount of glucose released from both a and P-glucans was determined by treating 100 pL sample solution with 100 pL enzyme mixture (containing exo-glucanase (20 U/mL) and P-glucosidase (4 U/mL)) at °C for 1 h. To the reaction mixture was added 3 mL of GOPOD reagent (mixture of glucose oxidase plus peroxidase and 4-aminoentipyrine in pH 7.4 buffer) and allowed to incubate for 20 min at 40 °C. 200 pL aliquots were transferred to a microtiter plate well and the absorption at 510 nm was determined against the reagent blank.

[0190] For the determination of the a-glucan content, the samples (100 mg) were treated with 2 mL KOH (2 M) for 20 minutes on ice followed by the addition of 8 mL of 1.2 M acetate buffer (pH 3.8). After addition of200 pL of enzyme (amyloglucosidase (1,630 U/mL) and invertase (500 U/mL)) the samples were incubated at 40 °C for 30 minutes. The volume was made up to 100 mL and an aliquot of the resulting solution was centrifuged 1500 g for 10 minutes. The amount of released glucose was determined as described above. The amount of p-glucan content was determined by subtracting the amount of a- glucan from the total glucan content. All samples were investigated in triplicates.

[0191] Both the extract obtained from the biomass and the biomass itself were tested for their p-glucan content as described under methods. As a control, a standard supplied by a-group was subjected to the analysis protocol. The results are summarised in Table 2

Table 10. Determination of the P-glucan content in the AG standard and GLO14 samples using the Megazyme assay kit

Sample % total glucan % a-glucan % P-glucan

Standard control extract 12.31 2.82 0.76 ±0.03 11.73 2.54

Extract from GL14 (day 3 and 6 5.53 0.26 0.35 ±0.12 5.24 0.35 combined)

Biomass from GL014 (day 3 and 6 20.3 1.07 8.47 ± 1.84 12.36 1.65 combined)

[0192] The total glucan content in the combined biomass generated during the fermentation process is 20.3% with an approximate ratio of s- to a-glucan 3:2. In comparison, the content of total glucan in the extract from this material is much lower (5.53%). However, the ratio between $- and a-glucan has increased with most of the glucan comprising p-glucan (approximately 95%) and is comparable to the ratio found for the standard control extract provided by the a-group.

[0193] In summary, all fermentations were run successfully. Compared to previous runs, fruiting bodies formation was limited. GL08, GLO12 and GLO14 showed highest submerged growth so far in the 1L bioreactors (18, 21 and 13 g/L, respectively). The fermentation period can be reduced to 6-days to avoid fruiting body formation. Agitation

(400 rpm), aeration (0.5-1 LPM), No pH and DO control and repeated glucose addition tends to support better submerged growth

4. Example 4: Scale-up in two 20 L bioreactors

[0194] The fermentation process was scaled up in 2 x 20 L bioreactors to validate the conditions developed in previous 1 L batch - GL027, and to develop large scale downstream process operations.

[0195] Seeds were expanded over two stages in flasks. Secondary seed reached a dry cell weight (DCW) of 17 g/L after four days of incubation at 30 °C and 140 RPM.

[0196] Two 20 L bioreactors (GL028 and GL029) were SIP (sterilised in place) and inoculated with 4% (v/v) of secondary seed. Anti-foam control was kept on auto. Dissolved oxygen (DO) and pH were not controlled. Concentrated glucose (500 g/L) was added at a rate of 20 g/L on Days 1, 2, 3 and 4 of fermentation.

4.1 Methods and results

[0197] Fermentations were carried out in 2 x 20-L parallel bioreactors for five days and biomass was concentrated using 500 kDa HFM pilot plant. Representative portions of concentrated biomass (~0.8 kg) were used to trial with different extraction experiments (GLLSE-01, GLLSE-02 and GLLSE-03) to assess the efficiency of extraction.

Fermentation process

[0198] Ganoderma seed medium (GSM) was used to expand the seed culture of G. /ingzhi, and included, 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H2O. Ground rice bran was included at a rate of 2% (w/v) to provide an insoluble matrix to help diffuse mycelial growth. A two-stage seed train was used to expand G. /ingzhi. Primary (10) seed was prepared by inoculating 2 mL vial in 100 mL (2%, v/v) of GSM and incubated for 7 days at 30 °C and 120 RPM. Secondary (20) seed was prepared by inoculating 16 mL of 10 seed in four 400 mL GSM (4 %, v/v) and incubating for 4 days at 30 °C and 140 RPM. The cultures from two secondary flasks each were combined and used for inoculating GL028 and GL029.

[0199] Fermentation process and media conditions were run in duplicates. Fermentation was carried out for five days with fed-batch glucose dosing (20 g/L) on Days 1, 2, 3 and 4. A concentrated glucose solution (500 g/L) was used for dosing.

[0200] The pH profile from this study is also similar to the previous 1 L batch (GL027), suggesting that the process did not behave differently when scaled-up.

Table 11. Fermentation Conditions studiedover Stage 3 and 4 fermentation batches

20 L batch# Details Seed conditions Similar to GL027 (SOW3 Stage 4). Similar to GL027. High aeration (400 RPM) and agitation (1 VVM), Two stage; Primary seed 60l28and029 modified batch medium (glucose-50 g/L, peptone- and secondary seeds (Duplicates) 10 g/L and yeast extract-5 g/L). Fed-batch glucose grown for 7 and 4 days, (20 g/L/day) respectively.

[0201] Duplicate 50 mL samples of submerged biomass were taken on Days 1, 2, 3, 4 and 5 and were centrifuged and dried in oven at 60 °C for 3-4 days to estimate dry cell weights (DCW) in g/L (Table 12).

Table 12. DCW of GL28 and GL29 during days 1-5, and the combined concentrate from GL28 and GL29. Day-5 DCW data point from previous study in 1 L bioreactor - GL027 (Example 3) is also included for comparison

Sample DCW g/L Day1 Day 2 Day3 Day4 Day 5 GL028 10.0±0.73 26.1±0.14 18.7±0.40 21.0±0.13 28.5±0.87 GL029 10.1±0.32 22.9±0.35 16.5±0.50 19.2±0.46 18.5±0.76

[0202] The DCW (g/L) for GL028 and GL029 are shown in Table 12. Error is given as standard deviation. For comparison, the Day 5 DCW of previous 1 L batch (GL027) was 15.0±0.6 and a concentrate as 29.5±0.34. After Day 1, the DCW of both GL028 and GL29 were similar and low at -10 g/L; an increase in biomass was seen from Day 2. High DCW of 26 g/L and 28 g/L were evident for GL028 on Days 2 and 5, respectively; whereas highest DCW (22 g/L) for GL29 was evident on Day 2 and then the DCW decreased by Day 5. Since the pH and DO profiles were similar for both batches, the difference in DCW may be attributed to the insoluble mycelia. In addition, more attached mycelia were found in GL029 compared to GL028, which further supports justification of the deviation. These mycelia were referred to as fruiting bodies in the previous reports, however they are best to be described as insoluble mycelia attached to the surfaces. Regardless, high DCW were evident in 20 L batches (average of 26 g/L) compared to the previous 1 L batch (15 g/L), suggesting that the process scaled-up successfully in terms of submerged biomass yield.

[0203] Due to technical issues, the residual glucose was not calculated during the fermentation, however Day 1-5 samples for both GL028 and GL029 were analyzed using HPLC at later stage.

Table 13. Residual glucose concentrationsas measured by HPLC during the five-day fermentation period of GL028 and GL029. * indicates, fed-batch addition of glucose (20 g/L) was after sampling.

Sample Residual glucose g/L Day 1 Day 2 Day3 Day4 Day 5 GL028 25.3 35.8 38.1 46.8 52.9 GL029 33 36.5 42.7 60.4 55.5

[0204] The glucose concentrations were between 25-60 g/L during the fermentation. In contrast, the residual glucose for the previous 1 L batch (GL027) was consistently 0 g/L during days 2-5.

Downstream process

[0205] About 38 kg was collected and combined from both 20 L batches (GL028 and GL029) and concentrated using pilot scale 500 kDa HFM plant. About 32.75 L of permeate (equivalent to supernatant) was collected and only 2.25 kg of retentate (concentrated biomass) was collected. An additional 3 L of the collected permeate was used to wash the retentate line of the HFM which resulted in a total collected concentrated biomass of 5.25 kg. A portion of the estimated biomass (~3 kg) could not be collected, due to the difficulties with processing low volumes using a pilot HFM plant. This challenge can be overcome by simply washing the membrane filter with phosphate buffer saline (PBS). The concentration step can be re- validated in the next stage of scale-up.

[0206] Representative samples of the concentrate were taken to estimate DCW and polysaccharide (PS) analysis using previously established analytical techniques. The rest of the concentrated biomass was aliquoted into ~0.8 kg batches and stored at -20 °C for extraction experiments.

[0207] Three extraction experiments were carried out (Batches: GLLSE-01, -02 and -03). In contrast to the previous analytical methods, drying of biomass post concentration was not carried out for these three extraction experiments. To break the mycelia, SilversonTM high shear overhead mixer was used for 30 min for two experiments (GLLSE-01 and GLLSE-02), and a high shear homogeniser (APV TM) was used for the third experiment (GLLSE-03), where three passes at 600 bar pressure was used to disrupt the mycelia. Both experiments using the Silverson high shear mixer, (GLLSE-01 and GLLSE 02) were similar except that the biomass was washed before extraction for GLLSE-02 batch. Each 0.8 kg biomass batch was thawed and extracted either using Silverson mixer or homogeniser and incubated at 70 °C for 1 hr to allow polysaccharides to dissolve in the broth. The extracts were then filtered using 500 kDa and 100 kDa HFM. Further separation up to 10 kDa was carried out for GLLSE-03 to compare the PS profile. Samples from each fraction were analysed for PS (profile and yields), beta glucans, triterpenes and MALLS (to estimate the size of the PS).

Polysaccharide and 0-glucan analysis

[0208] Previous work on selected samples using molecular weight cut-off filters (MWCO) showed that a polysaccharide concentration step was necessary to enhance the polysaccharide concentration in each sample prior to analysis. However, this may not be required during the manufacturing process. The methods used for PS analysis were as described in the previous reports. Briefly, the method used: 2 mL of sample was taken, and 8 mL of absolute ethanol was added, the sample shaken and centrifuged, the pellet was re-suspended in 0.5 mL water. After sonication and centrifugation, the resultant supernatant was used directly for analysis.

[0209] A standard curve was prepared using a sample of the standard polysaccharide at 10 mg/mL. The GL028 and GL029 samples show similar profiles, a large peak at ca. 10 min is more obvious in the Day 1 samples than Day 5. The profiles of all extracts are somewhat different from the standard in showing a broad peak ("hump") centered around 8 min, this may be due to higher levels of slightly larger polysaccharide than the Standard extract. There was no obvious difference between the membrane samples from GLLSE- 01 to -03 experiments, except that the 500 kDa and 100 kDa fractions from GLLSE-03 had spikes of peak ca. 7 min. Also, overall peak areas were larger than other samples indicting that more of the PS was extracted using the homogenizer.

[0210] Figure 8 shows PS yields of both estimated based on HPLC peak areas (6-11 min) and total dry PS solids per gram of dry biomass. It is important to note that for some samples (GLLSE-02) the actual yield of "solids PS" after freeze drying is only around half the value estimated from the standard curve using the AG standard. The AG standard does not all dissolve in water leading to over estimation of PS, and the PS response will vary according to molecular weight.

[0211] Estimated PS for analytical samples of both GL028 and GL029 varied during the five-day fermentation period. Day 3 tends to accumulate higher estimated PS per dry g biomass. Interestingly, the 100 kDa fractions of GLLSE-03 is more than two-fold when compared to the GLLSE-01, indicating that mycelial disruption using the homogenizer is results in a better subsequent extraction. Both estimated and solids PS for 10 kDa fractions are about half of 100 kDa fractions, indicating that large MW PS are lost during separation.

Molar mass analysis

[0212] Molar mass analysis was performed to understand the differences between the fractions of the PS extracts from GLLSE01-03 experiments and to confirm the presence of target PS. Ethanol precipitated samples were used for GLLSE-02 100 kDa, GLLSE-03 500 kDa, 100 kDa and 10 kDa fractions, whereas crude PS (without ethanol extract) was used for GLLSE01 100 KDa fraction. All samples were prepared at 10 mg/mL.

[0213] Results indicated that all 100 kDa and 500 kDa fractions were somewhat similar. The GLLSE-03100 kDa and 500 kDa are almost identical. The GLLSE-03 10 kDa sample had less of the higher MW early eluting peak. The GLLSE-03 100 kDa sample also had lower levels of high MW peaks. These chromatograms show that all samples are significantly different from the AG standard extract. The most obvious feature is that the GLLSE-01 100 kDa sample is dominated by a low MW peak, possibly a media component. Since crude PS of GLLSE-01 100 kDa (without ethanol precipitation) was used for analysis, the resolution of the profile is not clear. This reiterates that an ethanol precipitation step is necessary before carrying out analysis using HPLC/Refractive index systems. The standard shares with most samples a peak at 29 min but has a second larger peak at 30 min. The standard shows only a slow increase in RI after 22 min rather than the distinct peaks of the GLLSE-02 and GLLSE-03 samples. The GLLSE-02 and GLLSE-03 samples were quite similar while the GLLSE-01 100 kDa sample again is different with weaker UV absorption. Using pullulan standards the fractions are predicted to have PS of MW 100-200 kDa (at 19-21 min), 10-20 kDa (at 25-26 min) and 1-5 kDa (at 29-30 min)

Glucans analysis

[0214] Methods previous described were used to measure glucans [7] with an exception that crude PS (without ethanol precipitation) were used in this study. Results indicate that about 7-10% p-glucans were present in all the fractions. A large % ofc glucans were also present and this could be due to elimination of ethanol precipitation step, as this removes majority of low MW compounds (include media components) present in the PS extract liquid samples. Therefore, the % of 0-glucans could be much higher if the ethanol precipitation step is used. However, it is important to note that ethanol precipitation step may be necessary if media components and other low MW compounds are not desired in the product.

Table 14. Glucans analysis (avgsd) on samples. The % is relative to the crude PS solids except for GL027 (1-L batch)* where ethanol precipitateddry PS solids were used

Alpha glucan % (w/w) Beta-glucan % (w/w) GLLSE-01100KDa 30.9 10.1 GLLSE-02 100 KDa 30.5±2.1 9.5±2.2 GLLSE-03 500 KDa 31.1±3 10±3 GLLSE-03 100 KDa 29.2±0.5 6.7±3.8 GLLSE-03 10 KDa 30.9±1.5 8±2.3 GL027 (previous 1-L batch) * 1.58±0.07 11.32±1.13

Triterpenes analysis

[0215] A qualitative analysis has been performed to compare the triterpene content of the standard extract (SE) with the samples from the recent fermentation work. The triterpenes are known to have UV absorption at 254 nm and can be seen in the crude extract as peaks between 8 and 12 min. These peaks are also seen in an ethyl acetate partition of the water soluble SE. LCMS shows this set of peaks have masses in the range expected for triterpenes. No such peaks are seen in the fermentation samples. Even when the crude supernatant is partitioned with a small amount of ethyl acetate no triterpenes are seen. It may be concluded the triterpenes are not present in the submerged fermentation samples. On the contrary, the crude terpene fraction represents approx. 12% of the total extract weight of the SE.

Conclusions

[0216] The fermentation process was successfully scaled-up to 20 L bioreactors. Biomass yields were higher than those reported in the previous example. Mycelial disruption using the homogeniser (APV T M unit resulted in efficient extraction of the PS product. SEC-HPLC analysis indicated that the PS profiles of all the samples were slightly different from the standard extract, especially regarding a hump at ca. 8 min and absence of second peak at ca. 10 min. Regardless, high PS yields were produced in this study. Molar mass analysis confirmed the presence of low (1-5 kDa), medium (10-20 kDa) and high MW (100-200 kDa) PS in the fractions, and using a suitable membrane (500 kDa, 100 kDa and 10 kDa) desired MW PS can be extracted. Triterpenes were not found in the biomass extracts, supernatants or dry PS solids.

5. Example 5: Scale-up in two 200 L bioreactors

[0217] The purpose of this example is to examine a scaled up process. The fermentation process was scaled up in 2 x 200 L bioreactors, run in parallel, and the efficacy of previously developed downstream process operations were investigated.

5.1 Methods

[0218] Fermentations were carried out in 2 x 200 L parallel bioreactors. GL030 was run for 3 days and GLO31 for 5 days. The biomass was then concentrated using hollow fiber membranes (HFM) and mycelial disruption was carried out using high shear homogenizer (M110P Microfluidizer@). Disrupted mycelial solution was then heated at °C for 1 hr and filtered using a 500 kDa HFM. The crude PS was then freeze dried, milled and stored in sealed bags with N2 headspace at -20 0 C.

Fermentation

[0219] Ganoderma seed medium (GSM), was used to expand the seed culture of G. Iingzhi, and included, 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H20. Ground rice bran (Lotus) was included at a rate of 2% (w/v) to provide an insoluble matrix to help diffuse mycelial growth in flasks. A three-stage seed train was used to expand G. Iingzhi. 4% (v/v) inoculum was used throughout except for primary seed where 2% (v/v) inoculum was used. Primary (10) seed was prepared by incubating the 250 ml flask for 7 days at 30 °C and 140 RPM. Secondary (20) seed was prepared in four 1 L (400 mL working volume) flasks by incubating for 7 days at 30 °C and 140 RPM. Tertiary seed (30) was prepared in a 20-L bioreactor using the same seed media except rice bran wasn't added. The culture was agitated at 400 RPM and aerated at 20 LPM for hrs (~2.5 days).

[0220] Production was carried out in 200 L bioreactors in duplicate as per Table 15. To allow addition of condensate during SIP and addition of glucose bolus during the runs, the final weights of both batches were lower than 200 L. Post-inoculation, the weights of GLO30 and GL31 were 190 kg and 171 kg, respectively.

Table 15. Fermentation conditions

200 L # Conditions Fermentation period GLO30 Agitation (200 RPM) and aeration (200 3 days LPM), modified batch medium (glucose-50 g/L, peptone-10 g/L and yeast extract-5 g/L). Bolus of glucose (20 g/L/day) when residual glucose drops <20 g/L GLO31 Duplicate of GLO30 5 days

[0221] By the end of fermentation, the pH of tertiary seed dropped to ~3.5, similarly the final pH of GL030 and GL31 were 3.67 and 4.59, respectively. The pH of GL31 dropped to 3.73 on day-3 and then increased to 4.59 by the end ofthe run (day ). This potentially indicates dimorphic growth of G. Iingzhi, where the fungus switches carbon sources. The fermentation profiles of both GL030 and GL031 were almost similar, except GLO30 had slightly longer lag phase.

[0222] Duplicate 50 mL samples of submerged biomass were taken on days 1, 2, 3, 4 and 5 and were centrifuged and oven-dried at 60 °C for 5-7 days to estimate dry cell weights (DCW) in g/L. DCW (g/L) of seeds were as follows, 1° seed = 18.53 g/L, 20 seed = 17.88 and 30 seed = 17.81 g/L.

[0223] The DCW (g/L) for GL030 and GL031 are shown in Table 16. The DCW for GL30 and GL31 were similar for day 2 and day 3, but in GL31 the mycelial biomass gradually increased to 23 g/L by the end of fermentation (day 5). Given, GLO31 (5 day fermentation) yielded higher biomass when compared to GL030 (3 day fermentation), a day fermentation period is recommended. In general, for a typical 5 day fermentation period, the dry biomass yields from this study are comparable to previous scaled-down studies. The average DCW from 20 L study was 23 g/L, whereas 1 L study yielded 15 g/L of dry biomass.

Table 16. DCW (g/L) of GLO30 and GL31 over the productionperiod

DCW g/L Day GLO30 GLO31 1 4.9±0.15 10.1±0.15 2 15.9±0.24 17.7±0.09 3 15.7±0.12 15.3±0.14 4 18.5±0.10 5 22.4±0.19

[0224] Error shown in Table 16 is standard error. Mycelial mat formation was evident in all the bioreactors (Figure 6), however much less foaming was noticed compared to previous runs [6][7][8]. This indicates that antifoam 204 may be more suitable for limiting foaming. Except for initial antifoam addition in batch media at 0.05 % (v/v), no antifoam addition was made during the run. In addition, antifoam 204 doesn't foul the HFM filters during concentration, whereas previously used silicon-based Antifoam (Xiameter 1520) tended to foul the membrane filters. Antifoam 204 is therefore recommended for future runs.

[0225] Data on residual glucose present in the supernatant samples of GLO30 and GL31 is shown in Table 17. Glucose bolus additions were not made during the run. By the end of fermentation period, GL030 had 29.2 g/L of residual glucose and GLO31 had

13 g/L. Similar profiles were seen in the previous 20 L study [8], where glucose was not exhausted during the fermentation period.

Table 17. Residual glucose concentrationsas measured using glucose measurement kit during the fermentation period in GLO30 and GLO31 samples.

Day DCW g/L GLO30 GLO31 1 45.6 40.5 2 32.4 36.8 3 29.2 34.8 4 26.2 5 13.0

Downstream process

[0226] GLO30 was harvested on day-3 and GL31 on day 5. The biomass was concentrated using a single, clean 500 kDa hollow fiber membrane (HFM). To collect the residual biomass (retentate), the lines and membrane filter was rinsed with sterile phosphate buffer solution. The biomass was then disrupted using a homogenizer (Microfluidizer@ M110p) at 2000 bar with a single pass. Disrupted mycelia were observed under the microscope for confirmation of the extent of disruption. Disrupted mycelia were then heated at 70 °C for 1 hr to allow PS to dissolve in the aqueous phase, which was followed by filtration using a clean 500 kDa HFM. Permeate (crude PS - liquid) was collected and PBS was used to wash the retentate and the hose lines. The 500 kDa fraction of liquid containing crude PS was then freeze dried (FD) in open trays using a pilot scale freeze dryer over four batches. Due to presence of residual glucose, the solids were very hygroscopic. FD crude PS solids were then milled and stored in sealed bags with N2 headspace at -20 °C. GLO30 yielded ~1.8 kg of crude PS, whereas GL031 yielded ~2.9 kg of crude PS (Table 18).

Table 18. Residual glucose concentrationsas measured using glucose measurement kit during the fermentation period in GLO30 and GLO31 samples.

GLO30 GLO31 Total wet biomass (kg) 190 170 Concentrated wet biomass (kg) 60.47 63.76 Permeate (PS 500 kDa) wet (500 kDa) 71.57 74.62 (kg) Freeze dried crude PS solids (kg) 1.84 2.99 Estimated FD crude PS solids (kg) per 9.7 17.5 1000 L batch

[0227] It was noted during the biomass concentration of GLO30, about 8-fold concentration was achieved, however this led to accumulation of biomass in the retentate side of the membrane filter.

[0228] Recirculation with PBS had little effect on removing the residual biomass. On the other hand, GLO31 biomass was concentrated only 3-4-fold in order to limit loss of biomass from the retentate side of the membrane filter. Regardless, it was not possible to collect all biomass from GLO31 either. Therefore, to limit losses during concentration, it is suggested to use alternate methods of concentration such as batch/continuous centrifugation.

[0229] Representative samples (50 mL) of the biomass were taken on days 1-5, at harvest and post concentrate and were processed using previously described analytical methods to estimate target polysaccharide (PS) using HPLC method.

[0230] Microscopy images of samples taken during the fermentation period suggested no evidence of contamination. The concentrated samples look stressed when compared to the samples at harvest indicating that the shear of membrane tubules might be partially causing disruption of the mycelia resulting in loss of PS.

Polysaccharide analysis using SEC-HPLC

[0231] Previous work on selected samples using molecular weight cut-off filters (MWCO) showed that a polysaccharide concentration step was necessary to enhance the polysaccharide concentration in each sample prior to analysis. However, this may not be required during the manufacturing process. The methods used for PS analysis were as described in the previous reports. Briefly, to 2 mL of sample, 8 mL of absolute ethanol was added, the sample was shaken and centrifuged, the pellet was resuspended in 0.5 mL water. After sonication and centrifugation, the resultant supernatant was used directly for analysis.

[0232] Defatted extract of AG Standard extract was used to prepare the standard curve. Some of AG standard was dissolved in 5 mL water and 3 mL ethyl acetate was added and shaken. After centrifuging, lower aqueous layer was used as standard. Previous reports showed that about 12% of the weight of the AG standard is lipid like, and the concentration was adjusted accordingly.

[0233] The chromatograms of GLO30 and GL31 samples on different days show a similar profile but different levels. The PS levels in post-concentration are significantly lower than that in samples at harvest indicating that some of PS might have been lost during the concentration step.

[0234] Samples processed at pilot scale were also analyzed using SEC-HPLC for comparison. The chromatograms profiles clearly show that the crude PS fractions has the target PS along with several low-medium molecular weight compounds.

[0235] The ethanol precipitated PS isolate is much cleaner as the ethanol wash removes other low molecular weight compounds present in the crude PS.

[0236] The PS yields (mg per g of dry biomass) on days 3 and 5 of GL030 and GL31 were found to be in the range of 120-150 mg/g and are comparable to previous reports. PS yield dropped by ~50% in both GL030 and GL031 concentrate samples indicating loss of PS during concentration of biomass using HFM.

[0237] Using the SEC-HPLC method, it is estimated that a 1,000 L batch over 3 and day fermentation period will produce 0.94 kg and 1.7 kg of "target PS", respectively.

Glucans analysis

[0238] Both GL030 and GL031 had approximately 26-27 % (w/w) of total glucans, but no or very low amounts of p-glucans (0-1% w/w). The crude PS was then purified using ethanol precipitation and was used for total glucans analysis using the same kit. Results indicated that GL030 had 1.78% (w/w) $-glucans and 7.18% (w/w) of P-glucans, whereas GL031 had 1.38% (w/w) $ -glucans and 6% (w/w) $-glucans as shown below in Table 19.

Table 19. Glucans analysison crude and (ethanol) purified PS samples of GLO30, GLO31 in comparison with the previous study. * estimated based on -glucanspresent in crude PS.

GLO30 GLO31 GL029 (previous study) a-glucans(%) in crude PS 27 25 31 P-glucans(%) in crude PS <1% <1% 10% a-glucans (%)in purified PS 1.8 1.4 p-glucans (%)in purified PS 7.2% 6% Totalcrude PS g/1,000 L batch 9700 17500 3600 Total purified PS g/1,000 L batch 942 1729 Estimated total -glucans g/1,000 L 68 104 360* batch

[0239] When compared to the previous study, the yields of 0-glucans were lower in GLO30 and GL31 batches and it is anticipated due to loss of biomass during the concentration step and (2) inefficient extraction of p-glucans during mycelial disruption. Both GLO30 and GL31 mycelium had gone through one pass of disruption (at 2000 bar) when compared to previous batch (GL029; GLLSE03) which went through three passes (at 600 bar).

Conclusions

[0240] The fermentation process was successfully scaled-up to 200 L bioreactors. GL030 yielded 15 g/L of dry biomass over 3 days of fermentation, whereas GL030 yielded 23 g/L of dry biomass over 5 days of fermentation. Concentration of biomass using HFM may not be a suitable option due to the difficulties of collecting biomass and potential loss of PS during filtration as strongly evidenced by PS yields.

[0241] The SEC-HPLC profiles of all samples were similar but differed in the levels of PS. The crude PS includes PS that falls within the target molecular weight range in addition to other low-medium molecular weight compounds. GLO30 yielded 1.8 kg dry crude PS, whereas GL31 produced 2.9 kg dry crude PS. It is estimated that a 1,000 L batch over 5-day fermentation can produce 17.5 kg crude dry PS and 1.7 kg of target PS (post ethanol wash). Both GL030 and GL031 had <1% $-glucans in crude PS samples but on the contrary had 6-7% of $-glucans in (ethanol) purified PS samples indicating that ethanol purified PS samples must be used for estimation of $-glucans.

6. Example 6: Scale-up in 1,000 L bioreactor

[0242] The purpose of this example is to examine a scaled up process. The fermentation process was scaled up in 1 x 1,000 L bioreactor with downstream processing operations to process the material to extract polysaccharides.

[0243] Since small molecules are of interest, it was decided to target crude polysaccharide of 500 kDa without any ethanol washing step. In a previous study the fermentation process was scaled up and validated in 2 x 200 L bioreactors run in parallel that differed in the harvest point. Fermentation until day 3 resulted in 15 g/L DCW, whereas 5 days fermentation resulted in 23 g/L dry biomass. Delayed growth was identified, potentially due to impact of antifoam 204. Concentration of mycelial biomass using tangential flow filtration (TFF), a 500 kDa hollow fibre membrane (HFM) was not efficient and lots of biomass could not be collected. About 6% (w/w) $-glucans were present and it was anticipated that the number of passes though microfluidizer could potentially increase the overall PS and $-glucans yield.

[0244] In this study, the fermentation process was scaled up to 1,000 L and demonstrated the downstream process at pilot scale. Due to high wet solids content (20%/, w/w), continuous centrifuge was deemed not feasible. Instead, an integrated approach was used by collecting bulk of mycelium using 74 pm sieve and HFM. About a third of the biomass was retained for future trials. The remaining portion was processed to extract PS.

6.1 Methods

[0245] Primary seed was cultured in 2 L flasks over 7 days at 30 °C and 140 RPM. Secondary seed was cultured in 2 x 20 L bioreactors over approximately 3 days.

[0246] Primary seed reached a dry cell weight (DCW) of 20.2±1 g/L and secondary seed achieved a DCW of 14.2±6.4 g/L. Primary seed has a higher DCW due to addition of 2% (w/v) rice bran to keep the mycelia dispersed. 4% (v/v) inoculation was used throughout.

[0247] The 1,000 L bioreactor was sterilised in place (SIP) and inoculated with 40 L of secondary seed. Foaming was autocontrolled using Xiameter 1520 AFE, and pH and DO were not controlled. Fed-batch addition of glucose was not made. Fermentation was carried out for five days with periodical sampling.

[0248] Upon harvest, biomass was sieved using a 74 pm sieve to collect bulk of the mycelium, and the rest of the material was passed through a 500 kDa hollow fibre membrane (HFM) to collect remaining of the biomass.

[0249] Mycelial slurry was disrupted using a microfluidizer over three passes at 600 900 bar, and then heat treated at 70 °C, and passed through a 500 kDa HFM to collect the polysaccharide (PS), which was then freeze dried, milled and vacuum packed.

[0250] Samples were analysed for DCW, polysaccharide (PS) content and profile and 1-glucans.

[0251] Fermentation was carried out in 1 x 1,000 L bioreactor over five days as per conditions in Table 20. The downstream process is briefed in Figure 1.

Table 20. Fermentation conditions

1,000 L # Details Seed conditions GL032 Similar to GLO31 (SOW6). Similar to GLO30/31. Two Agitation (upto 150 RPM) and aeration stage; Primary seed in flasks, (up to 800 SLPM), modified batch medium and secondary seed in 2x 20 L (glucose - 40 g/L). pH and DO were not bioreactors (400 RPM, 20 SLPM, controlled. Auto foam control using pH and DO not controlled, seed Xiameter 1520 AFE. media without rice bran.

Fermentation process

[0252] Ganoderma seed medium (GSM) was used to expand the seed culture of G. /ingzhi, and included 40 g/L glucose, 4 g/L soy peptone, 1.5 g/L KH2PO4, and 1 g/L MgSO4.7H2O. Ground rice bran was included at a rate of 2% (w/v) to provide an insoluble matrix to help diffuse mycelial growth. A two-stage seed train was used to expand G. /ingzhi. Primary (10) seed was prepared by inoculating 4x 500-mL GSM (2-L flasks) with 2% (v/v) glycerol stock, and incubated for 7 days at 30 °C and 140 RPM. Secondary (20) seed was prepared in 2x 20-L bioreactors by sterilising in place (SIP) using the GSM but without rice bran. The secondary seed was grown for ~3 days at °C, 400 RPM, 20 SLPM, no pH and DO control.

[0253] Fermentation process and media conditions were as per Table 20. Fed-batch glucose dosing was not made. Fermentation profiles of secondary seeds and production batch (GL032) are shown in Figures 2 and 3.

[0254] Duplicate 50-mL samples of submerged biomass were taken on days 1, 2, 3, 4 and 5 and were centrifuged and dried in an oven at 60 °C for 4-5 days to estimate dry cell weights (DCW) in g/L.

[0255] Highest DCW was seen on day 2 (17 g/L), and then it gradually decreased to 12.6 g/L by the end of fermentation period. The DCW of primary seed was higher due to the presence of 2% (w/v) rice bran in the medium. Mycelial mat formation was evident during the course of fermentation.

[0256] Using the CDR Beer lab kit, residual glucose was measured in supernatant samples (Figure 5). By the end of the fermentation period, about 8 g/L of glucose was present.

[0257] Microscopic analysis of samples over the fermentation period was performed. Contamination was not evident and typical mycelial growth was observed.

Downstream process

[0258] Downstream processing was started with 942 kg of mycelial biomass in the bioreactor (Table 21). Using a 74 pm sieve, bulk of solids (mycelia) were collected and the rest was passed through a 500 kDa HFM to collect and concentrate biomass. About 2/3rds of the concentrated biomass (250 kg) was used for disruption by adding 104 kg of water with a combined mass of 354 kg (16.8 g/L DCW) to improve the fluid dynamics in order to assist with mycelial disruption. The biomass was passed through a Microfluidizer (APV T M ) at 600-900 bar three times. Sampleswere collected at the end of each pass to confirm disruption under microscopy and later analysed for 0-glucans. The disrupted mycelia were then heat treated at 70 °C and then cooled to 30 °C before passing it through a 500 kDa HFM. The permeate was collected into a sterile tank, representative samples were taken for analysis and stored in 60-L sterile drums and frozen. About 2 drums were retained for future trials, and the rest four drums were defrosted and freeze dried over four batches in four weeks. The FD bulk material was then milled to homogeneity in Robot Coupe@ (R45) and packed in about 500-600 g vacuum sealed bags to avoid moisture absorption of the product over storage.

Table 21. Downstream processing yields. *RO water (104 kg) was added to the biomass (250 kg) to improve fluiddynamics

Downstream processing Processed (kg)

Biomass at harvest 942

Concentrated biomass (sieved + HFM) 409(250+157)

Concentrated biomass used fordisruption* 250

Total PS extracted 210

Crude PS solids (freeze dried) 4.6

Estimated crude PS from a 1000 Lfermentation 11.5

Estimated purified PS from a 1000 Lfermentation 1.15

Polysaccharide andf-glucans analysis

[0259] Previously described methods were used for extraction and analysis of PS using HPLC, as described previously. Absolute ethanol was added to each sample to precipitate polysaccharides (80% ethanol, v/v). Samples were centrifuged and the PS pellet re-dissolved in water for analysis. Samples were re-dissolved in 2 mL water and the concentrate samples (O-Q) re-dissolved in 10 mL water. Analysis was performed by HPLC using a size exclusion gel column with ELS detection. The amount of PS in each sample was calculated from a standard curve prepared from the Alpha Group standard PS sample. The standard was prepared by precipitation of the PS with 80% (v/v) ethanol.

[0260] Estimated PS mg/g of biomass is shown in Figure 8. PS production was evident from day 1 and peaked on day 3 and was lowest on day 5. A GL32 concentrate contained 82.7±7.2 (SD) mg/g PS.

Table 22. Estimated PS per dry gram of biomass based on HPLC peak areas (of target PS) and actual total dry PS.

Sample Estimated PS mg/g Primary seed 56.0±2.8 Secondary seed 157.5±17.7 GL032 Day 1 158.0±15.6 Day 2 180.0±35.4 Day 3 208.0±39.6 Day 4 181.5±2.1 Day 5 94.5±3.5

Glucans analysis

[0261] Methods previously described were used to measure glucans. Results indicated that about 6% (w/w) P-glucans were present in the purified PS samples, whereas masking effect was seen in crude PS. The yields of P-glucan were similar to those of Example 4 but lower when compared to those of Example 3 (~11%, w/w).

Table 23. Glucans analysis (avg±sd) on samples

Sample Total glucan (/o, a-glucan (%, w/w) p-glucan ( %, w/w) w/w) G1032 purified PS 6.75 0.30 0.64 0.02 6.11 0.72

G1032 crude PS 16.89 0.55 18.42 0.19 n.d.

G1032 1 st pass 5.85 0.68 0.81 0.08 5.05 0.62 of disruption

G1032 2 nd pass 5.75 0.82 0.76 0.09 4.99 0.67 of disruption

G1032 3 rd pass 6.05 0.05 0.65 0.07 5.40 0.39 of disruption

6.2 Results

[0262] The fermentation process was successfully scaled up in 1,000 L bioreactor. High DCW was evident on day 2, where the DCW decreased to 12 g/L by day 5. High PS mg/g biomass was evident on day 3 and it halved by day 5. The PS profile was almost similar to the AG standard. Microscopy indicated no contamination and typical mycelial morphology was observed as seen in previous studies. Three passes of mycelial disruption is recommended at 600-900 bar.

[0263] Even though the batched media have lower concentrations of glucose than the last study (50 vs 40 g/L), the residual glucose by the end of day 5 was at about 8 g/L, suggesting that more studies at 20 L or 1 L scale could allow the study of the impact of lower concentrations of glucose in batched media on biomass and PS yield. Another option is to perform ultrafiltration of the PS sample to remove glucose, however this step may remove other small molecules present in the crude PS. Detailed analysis of components that are essential in the target PS product is recommended to be performed by the Client before commencing manufacturing.

[0264] Results indicated that:

• The DCW of submerged biomass varied during the five day fermentation period. Highest DCW was seen on day 2 (17±0.1 g/L) and the submerged biomass decreased over the next few days to 12.6±1.5 g/L by day 5.

• Batched glucose was consumed linearly over the course of fermentation period, and by the end of day 5, about 8 g/L of residual glucose was present. • About 2.4 fold concentration was achieved using sieving and HFM, and about 240 kg was used to disrupt, extract, purify and freeze dry to produce 7 kg of crude PS, an estimated 11.5 kg crude PS from a 1,000 Lbioreactor.

• HPLC analysis of pure PS profiles indicated similarity with standards, and were inconsistent with previous runs.

• High PS content was evident from day 2 to 4 (180-200 mg/g), however it halved by day 5 (94 mg/g).

• Analytical purification of crude PS with ethanol indicated that a pure PS is approximately 10 % (w/w) of crude PS. This is estimated to be 1.15 kg of pure PS from a 1000-L fermentation.

• About 6.1% (w/w) of 1-glucans, and 0.14% (w/w) of a-glucans were present in the pure PS.

Claims

WE CLAIM:

1. A method for isolating intracellular polysaccharides from fungi comprising • inoculating fungi into a fermentation medium, wherein the fermentation medium is subjected to agitation, • extracting one or more polysaccharides from the mycelium by filtration, wherein the filtration has a pore size of a) about 1 to 5 kDa, b) about 10 to 20 kDa, c) about100to200 kDa, d) or any combination of (a) to (c), and wherein the polysaccharides are extracted after about 3 to about 12 days of fermentation, to produce greater than 2% polysaccharide by weight of mycelium biomass.

2. A method of claim 1 wherein the agitation exerts a shear force of about 100 to about 1000 s-.

3. A method of claim 1 or 2 comprising isolating mycelium from the fermentation medium.

4. A method of any one of claims 1 to 3 wherein the dosing of the media with sugar achieves a ratio of carbon to nitrogen of about 1:3.

5. A method of any one of claims 1 to 4 wherein the dosing of the media with sugar achieves a concentration of sugar in the media of about 20 g/L.

6. A method of any one of claims 1 to 5 wherein the sugar is provided to the media every 24 hours.

7. A method of any one of claims 1 to 6 wherein the sugar is first provided to the media after 24 hours from inoculation.

8. A method of any one of claims 1 to 7 wherein the sugar is glucose.

9. A method of any one of claims 1 to 8 wherein the agitation is high shear mixing.

10. A method of claim 9 wherein the fermentation is carried out at about 25 to about 350 C.

11. A method of any one of claims 1 to 10 wherein the fungi is a mushroom species.

12. A method of claim 11 wherein mushroom species may be selected from Inonotus obliquus, Trametes versicolor, (also known as Coriolus versicolor), Lentinula edodes, Hericium erinaceus, Cordyceps sinensis, Grifola frondosa, Schizophyllum commune, Flammulina velutipes, Pleurotus ostreatus, Agaricus bisporus, Auricularia auricula, Coprinus comatus, Phellinus linteus, or Laetiporus sulphureus.

13. A method of claim 12 wherein mushroom species is selected from Ganoderma lingzhi.

14. A method of any one of claims 1 to 13 wherein the mycelia comes from seed culture, and wherein the seed culture is first expanded.

15. A method of any one of claims 1 to 14 wherein the concentration of seed culture at the beginning of fermentation is about 2 to about 6% by volume.

16. A method of claim 14 or 15 wherein the seed culture is a combination of pre existing and expanded seed culture.

17. A method of any one of claims 1 to 16 wherein the inoculated media is incubated for up to 7 days.

18. A method of any one of claims 1 to 17 wherein the inoculated media is stirred.

19. A method of claim 18 wherein the inoculated media is stirred at about 80 to 160 RPM.

20. A method of any one of claims 1 to 19 wherein the media comprises: a) about 30 to 50 g/L glucose, b) about 2 to 6 g/L soy peptone, c) about 1 to 3 g/L KH2PO4, d) about 0.5 to 2 g/L MgSO4.7H20, or e) any combination of (a) to (d).

21. A method of any one of claims 1 to 20 wherein the method comprises the addition of insoluble ingredient.

22. A method of claim 21 comprising about 0.5 to about 4% w/v of insoluble ingredient.

23. A method of claim 21 or 22 wherein the insoluble ingredient is lotus ground rice bran.

24. A method of any one of claims 1 to 23 wherein the vessel used for fermentation is a bioreactor.

25. A method of any one of claims 1 to 24 wherein the method controls any one or more parameters selected from pH, dissolved oxygen, aeration, and shear force.

26. A method of any one of claims 1 to 25 wherein the mycelia is harvested before fruiting body formation.

27. A method of any one of claims 1 to 26 wherein the pH of the media is about 3 to 5.

28. A method of any one of claims 1 to 27 wherein the level of oxygen in the fermentation mixture may be about 20 to about 30%.

29. A method of any one of claims 1 to 28 comprising the addition of an anti-foam agent to the media.

30. A method of any one of claims 1 to 29 wherein the biomass is dried prior to extraction.

31. A method of any one of claims 1 to 30 wherein the method extracts at least 5% polysaccharide per dry weight of the biomass.

32. A isolate from a fungal biomass, the isolate comprising two or more polysaccharides having a molecular mass selected from

• about 1 to about 5 kDa,

• about10 to about20 kDa,and

• about100 to about 200 kDa.