WO2025129345A1 - Encapsulated mushroom extract powder materials using lyophilization - Google Patents

Abstract

Encapsulated mushroom powders and their method of manufacture using lyophilization are described. Liquid mushroom extract is combined with mushroom protein powder as an encapsulating agent and subjected to lyophilization, and milled or ground into a flowable powder. The method provides exclusively mushroom-derived encapsulated mushroom powders that are high in bioactive compounds with good flow properties. A process to make a concentrated mushroom extract is also described, wherein ground, dried mushrooms are extracted using a first extraction in ethanol followed by a second extraction in water. Various extraction times and temperatures are provided, and sonication may also be employed. The resulting mushroom extracts comprise beta-glucan, ergosterol and proteins.

Description

Encapsulated Mushroom Extract Powder Materials Using Lyophilization

This application is a continuation-in-part of US provisional patent application serial number 63/611,831 filed on December 19, 2023, the disclosure of which is hereby incorporated by reference.

Field of the Invention

[0001] The invention relates to encapsulated lyophilized powder forms of highly concentrated mushroom extracts in which the encapsulating agents (food-grade mushroom protein powder) help to protect the bioactive components of the mushroom extract from oxidants while preventing brittle crystals and enhancing product flow and solubility properties.

Background of the Invention

[0002] Mushrooms are one of the most diverse organisms on earth that have played a crucial role in human welfare since ancient times.¹ They have been universally used as both food and medicine by various civilizations due to their delicious taste, flavour, dietary qualities, and numerous medicinal properties including the anticancer, antihypertensive, antiinflammatory, antimicrobial, antiviral, antioxidant, hypoglycemic, hypolipidemic, and immunomodulatory activities.¹'³ These medicinal properties are conferred by a wide range of bioactive molecules they contain, such as polysaccharides, terpenes, statins, bioactive peptides, phenolic compounds, and carotenoids. Among the myriad of bioactive molecules present in mushroom species, researchers and marketers primarily emphasize the nutraceutical properties of a select few molecules, notably -glucan, ergosterols, and proteins. [0003] [3-glucan, a water-soluble glucan, is an important component of the cell walls of most fungi and many plants. It is a predominant non-starch polysaccharide consisting of linear chains of -D-glucose linked by P-(l,3)-, (1,4)-, and/or (l,6)-D-glycosidic linkage. It can contain over 25,000 D-glucose units in either branched or unbranched forms.^5,6 P-glucan stimulates the immune system in the digestive tract and produces pro-inflammatory metabolites that activate immune cells such as T cells.⁷ It has also been shown to lower tumor incidence, tumor volume, and the total number of tumor nodules.8 Other health benefits of P- glucans include maintaining serum cholesterol and blood glucose levels, as well as properties that help prevent cardiovascular diseases and hypertension.^{6, 9> 10} [0004] Ergosterol serves as a provitamin of ergocalciferol, which possesses antioxidant, anti-inflammatory, and anti-cancer. Studies have shown that extracts from ergosterol- enriched fungi can effectively reduce cholesterol absorption and inhibit its biosynthesis within the human body.¹¹ Furthermore, the conversion of ergosterol to vitamin D2, which is commonly used in the treatment of various medical conditions such as different types of cancer, multiple sclerosis (MS), dementia, seasonal affective disorder (SAD), and psoriasis, adds another beneficial aspect to the consumption of mushroom tinctures.¹²

[0005] Mushroom proteins, comprising approximately 19% to 37% of their dry weight, typically possess a complete essential amino acid profile. These proteins not only fulfil dietary requirements but also offer certain economic advantages when compared to animal and plant sources.¹³ The Food and Agriculture Organization (FAO) recommends edible mushrooms as a food source to meet the protein requirements of developing countries, particularly in populations heavily dependent on cereal crops.¹⁴

[0006] Fresh mushrooms typically contain about 85-90% moisture, 3% protein, 4% carbohydrates, 0.3-0.4% fats, and 1% minerals and vitamins.⁴ Needless to say, dried mushroom bodies have higher concentrations of bioactive molecules, such as -glucan, proteins, ergosterol, ergothioneine, flavonoids, and chitin, with actual beneficial biological properties. To further concentrate these beneficial molecules, mushroom extracts are prepared and provided to consumers as tinctures.

[0007] Besides their higher concentration of bioactive molecules, compared to fresh or dried mushrooms, tinctures have a longer shelflife, are easier to administer, and are absorbed more quickly. Optimizing extraction parameters, such as time, temperature, and the application of ultrasound waves, can enhance the bioactive compound content in tinctures while simultaneously reducing energy consumption and processing time. However, for the purpose of concentrating the bioactive molecules, extending the product’s shelflife, and enabling versatile applications — be it for sale as capsules or as a supplement for coffee, soup or spaghetti sauce — the mushroom tinctures could be dried and subsequently made into powder form.

[0008] Mushroom powder samples, retaining the majority of bioactive compounds present in mushroom tinctures, can be prepared using two distinct methods: spray drying and lyophilization. In the latter, known as freeze drying, water and other food-grade extraction solvents, such as ethanol, are sublimed from frozen mushroom tinctures under low-pressure conditions.

Summary of the Invention

[0009] In accordance with the disclosure herein, the invention relates to a dry, flowable composition that comprises an encapsulated mushroom extract and powder that was made by a process comprising: lyophilizing a mushroom extract tincture with a mushroom protein powder and milling or grinding the lyophilized composition to make a flowable powder.

[0010] Preferably, the lypholizing process comprises: (a) mixing mushroom protein powder with liquid mushroom extract in a first weight ratio to make a first mixture, (b) subjecting the first mixture to high-shear mixing to make a second mixture, (c) rapidly freezing the second mixture, (d) placing the frozen second mixture into a lyophilizer until dried, and (e) milling or grinding the dried second mixture into a powder.

[0011] The composition and its process of manufacture provide a particulate product that is high in bioactive components with good flow properties. Encapsulating mushroom tinctures with proteins also prevents the formation of a brittle crystal with very low flowability when the pure extract is lyophilized. Therefore, mixing the extract with small dosages of mushroom protein powder benefits the flowability of the final powder product.

Brief Description of the Drawings

[0012] Figure 1 shows the weight loss curves for Lion’s Mane, Reishi, and Cordyceps mushrooms using thermogravimetric analysis (TGA) recorded in the temperature range from room temperature to 600 °C.

[0013] Figure 2 shows the first derivative curves for the tests of Figure 1.

[0014] Figure 3 is a chart showing protein concentration v. extraction time.

[0015] Figure 4 is a chart showing -glucan concentration v. extraction time.

Detailed Description of the Invention

[0016] Mushrooms, as macro fungi, offer diverse benefits, encompassing nutrition, medicine, and economics. Mushroom tinctures have gained popularity over fresh mushroom consumption due to their higher concentration of bioactive molecules, reduced digestive distress, and convenience and accessibility. Nevertheless, mushroom powder holds an advantage over liquid extracts. Not only does it extend the product’s shelflife, but also it could contain certain compounds such as antioxidants, stabilizers, and solubilizers.

[0017] The present invention is applicable for mushrooms that contain -glucan and ergosterol. Especially preferred are mushrooms that contain psilocybin.

[0018] A 3x3 full-factorial Design of Experiment (DoE) was employed to optimize extraction parameters, including extraction temperature and time as well as the application of ultrasonic waves, for maximizing the concentration of bioactive compounds in extracts from mushrooms. A non-exhaustive list of mushrooms of interest includes: Ganoderma lingzhi (Reishi), Hericium erinaceus (Lion’s Mane), Cordyceps militaris (Cordyceps), Inonotus obliquus (Chaga), Trametes versicolor (Turkey tail), Grifola frondosa (Maitake), Lentinula edodes (Shiitake) and Fomitopsis officinalis (Agarikon). The results for the first three mushrooms are presented as representative models, but we reserve the application of this technology to all mushroom species and their mixtures.

[0019] Accordingly, the invention provides a dry, flowable composition and its method of manufacture that comprise an encapsulated mushroom extract made by a process comprising lyophilizing highly concentrated mushroom extracts, a “tincture,” with a mushroom protein powder (MPP) to make a flowable second powder.

[0020] The resulting product shows low cohesiveness and good flowability at a particle size suitable for administration by nasal spray.

[0021] Moreover, to create powder substance rich in only mushroom-derived compounds, lyophilization with MPP was used for the present invention. This added protein component serves to shield the tincture’s bioactive molecules, such as -glucan, from oxidants and environmental stressors by encapsulating them.

[0022] Encapsulating mushroom tinctures with proteins also prevents the formation of a brittle crystal with very low flowability when the pure extract is lyophilized. Therefore, mixing the extract with small dosages of MPP benefits the flowability of the final powder product.

[0023] To produce exclusively mushroom-derived powder samples, lyophilization was employed alongside Shiitake mushroom protein as the encapsulating agent. This approach, along with its methodology and the characterisation values for the resulting samples, is explained below. [0024] While industry and markets have focused on obtaining high -glucan containing extracts and tinctures from mushrooms, these occupy larger volumes and are difficult to package in concentrated formats.

[0025] A way out of this problem might be to pack lyophilized encapsulated mushroom P-glucan inside gelatine or agar-agar capsules, or simply package them as food sachets. This formulation makes it possible to prepare a finely-ground, entirely mushroom-derived powder composition exhibiting decent flowability and solubility suitable for human consumption using a lyophilizer, concentrated mushroom extracts/tinctures, and mushroom-derived proteins.

[0026] Maceration

[0027] Mushroom extracts can be obtained by immersing chopped fresh or ground dried mushroom bodies in water or ethanol for a specific duration at certain temperatures.

However, to determine the optimal extraction time and temperature for maximizing the concentration of target bioactive molecules, a 3-by-3 full-factorial Design of Experiment (3² DoE) was devised for each mushroom species. The specific DoE temperature and time levels for each mushroom were determined respectively based on its thermal profile (using thermogravimetric analysis - TGA) and the duration required to extract bioactive compounds from its unique cell wall structure (running a time-course extraction study explained below). [0028] The extraction protocol begins by mixing a defined amount of ground, dried mushrooms (x g) with five times its mass in solvent volume (5x mL) in an amber centrifuge tube. This mixture (1:5 solid-to-solvent ratio) is then heated in a water bath with adjustable heat (0 - 80 °C) to a predefined temperature for a specified period before being filtered through four layers of cheesecloth (Grade 90). The filtrate is then centrifuged at 300 rpm for 3 minutes, and the supernatant is stored in the dark at 4 °C in an amber vial. The remaining mushroom residue undergoes a second extraction with fresh solvent (different from the first extraction solvent) under identical conditions, and the resulting extract is also stored in the dark in an amber vial at 4 °C.

[0029] For sonication-assisted extraction, the same procedure is followed using a 2.0 L Digital Ultrasonic Cleaner operating at 40 kHz with adjustable heating settings. To ensure reliability and consistency, both water bath and sonication-assisted extractions were performed in quadruplicate. [0030] To conduct a time-course study on the effects of using water and ethanol as the first or second extraction solvent, two sets of extractions were tested: one starting with water, then ethanol, and the other with ethanol, then water. The extraction conditions were uniformly maintained at 45 °C for 45 min in a water bath to ensure consistency in comparison. Upon completion of the extraction processes, the extracts were quantitatively analyzed to determine their concentrations of -glucan, ergosterol, and protein. To ensure reliability and consistency, all extractions were conducted in quadruplicates. All methods used for extraction and quantification of bioactives were validated for efficiency, repeatability and accuracy.

[0031] Thermogravimetric analysis (TGA) was performed as follows: Approximately 2 mg of each mushroom sample was loaded into pre-weighed TGA aluminum pans and placed on the instrument's sample platform. Each sample was then subjected to a temperature ramp from room temperature to 600 °C at a constant rate of 10 °C/min, with continuous weight monitoring. The weight loss (%) for each sample was plotted against the furnace temperature to generate the respective TGA curve. Additionally, the first derivative of the sample weight with respect to temperature was calculated to produce the first derivative curve for each TGA plot. To reduce noise, the curves were smoothed by averaging 20 adjacent points.

[0032] To determine how the order of extraction solvents, specifically HPLC-grade water and food-grade ethanol (with polarity indices of 10.2 and 4.3, respectively), impacts the yield of targeted bioactive molecules in the resulting extract, an extraction procedure was conducted at 35 °C for 40 min in a water bath to ensure consistency in comparison for each solvent order.

[0033] The results (presented in Table 1) show that -glucan and protein content, with a few exceptions, exhibited significant variance between the two solvent orders. Ergosterol analysis suggests that its extraction is more sensitive to the choice of solvent, with ethanol as the initial solvent generally enhancing its yield, especially in the case of Lion’s Mane and Cordyceps. This can be attributed to the higher affinity of ergosterol, being an alcohol, for ethanol. The results signify that using ethanol as the first extraction solvent yields higher concentrations of bioactive molecules in the extracts compared to when it is used as the second extraction solvent. Table 1. Comparing the bioactive compounds of the Reishi, Lion’s Mane, and Cordyceps mushroom extracts obtained using water and ethanol, respectively as the first and second extraction solvents (WE) and vice versa (EW).

[0034] To determine the thermal profile of each mushroom species and, more importantly, to establish the maximum temperature that could be used during the extraction process without altering the chemical composition of the mushroom cells, TGA measurements were conducted. As shown in Figure 1, Lion’s Mane and Reishi respectively lost 5 and 10% of their weight when heated to 100 °C, likely due to the evaporation of entrapped water or the degradation of small volatile molecules, whereas Cordyceps was barely affected, indicating its lower content of humidity and volatiles, or a more thermally stable cell wall structure. The first derivative of each thermogram was plotted to pinpoint the exact temperature at which each physical or chemical transition occurred. This analysis revealed that Lion’s Mane and Reishi begin to degrade small volatiles at approximately 45 °C, while Cordyceps remains thermally stable up to 300 °C, Figure 2.

[0035] To systematically investigate the optimal extraction time range, a time-course extraction was conducted on the three mushrooms under study. P-glucan and protein concentrations were measured every 20 minutes during a l-to-10 solid-to-solvent water- assisted extraction. As expected, a gradual increase in the concentration of extracted proteins was observed for all three mushrooms until reaching a plateau, Figure 3. For Reishi and Lion’s Mane, protein concentrations plateaued at 60 minutes, suggesting an equilibrium between soluble proteins and those remaining in the solid matrix, possibly due to water's limited capacity to extract additional proteins from the mushroom cell walls. Interestingly, Cordyceps required 140 minutes to reach saturation, potentially reflecting differences in its cell wall structure or the solubility of its protein constituents. Additionally, -glucan concentrations showed a significant increase in all three mushrooms’ extracts until peaking, followed by a sharp decrease to very low levels, Figure 4. This pattern could be explained by the continued extraction of -glucans until saturation, followed by a rapid decrease probably due to the activation of -glucanase enzymes, microbial activity, or oxidative degradation. Monitoring the pH of the extraction slurry throughout the process (pH = 5.4) suggests not only the optimal pH of the extract for -glucanase activity but also the potential for acidic hydrolysis of -glucans.

[0036] Based on the results obtained for each mushroom species, the extraction conditions for Reishi and Lion’s Mane were set at 35, 45, and 55 °C for duration levels of 30, 45, and 60 min (Table 2). For Cordyceps, extractions were conducted at 40, 60, and 80 °C for 40, 60, and 90 min to compare the outcomes (Table 3).

Table 2. Extraction parameters selected for full-factorial DoE runs on Reishi and Lion’s Mane mushrooms.

Table 3. Extraction parameters selected for full-factorial DoE runs on Cordyceps mushrooms.

[0037] Statistical Analysis

[0038] All extractions were performed in quadruplicate. To minimise the effect of human error, one outlier from each set, identified using Microsoft Excel (Microsoft Corporation, 2023), was excluded. The results for each extraction condition were then expressed as the mean of the three closest values ± their standard deviation. A Student’s t-test was conducted to assess significant differences between extraction conditions, with p-values less than 0.05 considered significant. The bar charts and graphs presented in this article and the supplementary information were plotted using OriginPro 8.5.0 SRI (OriginLab Corporation). [0039] Optimum Extraction Conditions

[0040] The content of bioactive molecules, mainly -glucan and ergosterol, in dried mushroom powders significantly influences the nutraceutical and marketing value of the end product. This is greatly influenced by several factors, including the cultivation conditions affecting the fungi's bioactive molecule content, extraction yield, and the amounts of additives used to aid the drying process of the extract. Therefore, to produce mushroom extract powders rich in bioactive molecules while maintaining decent flowability and solubility, it is essential to use extracts from well-cultivated mushrooms with high bioactive molecule content, combined with minimal additives. Equally important is choosing the right extraction parameters to maximise bioactive content and ensure the quality of the final product. To measure the -glucan and ergosterol contents in the final products, the Megazyme -Glucan Assay Kit (Y east and Mushroom) and high-performance liquid chromatography (HPLC) are recommended. The methodology for both is explained below. [0041] For the present invention, the preferred extraction conditions include (a) a temperature of less than 300° C, a duration within the range of 20-140 minutes, a solvent that comprises a C1-C4 alcohol (preferably ethanol) or water, and virtually any mushroom species but is preferably selected from Lion’s Mane, Reishi, and Cordyceps. Preferred extraction conditions include a temperature of 45° C when said mushroom powder comprises powdered Lion’s Mane or Reishi; a time within the range of 20-140 minutes (preferably within a range of SO- O minutes, 50-100 minutes when the MPP comprises powdered Lion’s Mane or Reishi, or a time within the range of 60-100 minutes when said powder comprises powdered Lion’s Mane or 30-80 minutes when said powder comprises powdered Reishi). The most preferred conditions are presented in Table 4, supra.

[0042] The actual encapsulation and powder formation process comprises: (a) mixing mushroom protein powder with liquid mushroom extract in a first weight ratio to make a first mixture, (b) subjecting the first mixture to high-shear mixing to make a second mixture, (c) rapidly freezing the second mixture, (d) placing the frozen second mixture into a lyophilizer until dried, and (e) milling or grinding the dried second mixture into a powder.

[0043] Megazyme B-Glucan Assay Kit (Yeast and Mushroom)

[0044] The Megazyme [3-Glucan Assay Kit (Y east and Mushroom), was employed according to the instruction provided by the company to measure the concentration of [3- glucan in mushroom extract powders. This kit assesses total and a-glucans, respectively, by completely hydrolysing glucans and enzymatically digesting a-glucans into glucose. The absorbance is then read at 510 nm using a spectrophotometer to calculate the concentration of total and a-glucans. Lastly, the [3-glucan concentration is determined by subtracting the a- glucan content from that of the total glucans in the extract sample.

[0045] Ergosterol Extraction Procedure

[0046] Approximately 500 mg of samples were weighed into 15 mL falcon tubes and 5 mL HPLC grade ethanol added. In the case where there was insufficient sample, approximately 100 mg of samples are used with 1 mL ethanol for the extraction process to ensure concentration of target analytes are above the limit of detection or quantification. Samples are vortexed for 10 seconds and placed in an ultrasonic bath for 15 mins. After sonication, tubes are placed in a centrifuge and spun at 400 g for 5 mins and the clear supernatants collected for HPLC analysis.

[0047] Ganoderic Acid A (GAA) Extraction Procedure

[0048] Approximately 500 mg of MPP sample was extracted using 5 mL ethanol. After addition of ethanol, sample mixture was vortexed for 10 seconds and placed in an ultrasonic bath for 15 min. The sample was transferred to a centrifuge and spun for 5 mins at 400 g. The ethanol supernatant (1 mL) was transferred to HPLC vials for data acquisition.

[0049] HPLC Analysis for Ganoderic Acid A

[0050] HPLC analysis was performed using an Agilent 1100 HPLC instrument with a quaternary pump and diode array detector with a Zorbax SB C18 column. The mobile phase constituted of 0.03% aqueous phosphoric acid (A) and acetonitrile (B) following a gradient flow of 31% A for 4 minutes, then 45% A for 15 mins, a further 100% A for 3 mins, and an equilibration time of 1 min with 31% A. A total runtime of 21 mins was applied per analysis with the column conditioned at 35 °C and a flow rate of 0.7 mL/min. Detection of GAA was at 252 nm and samples were run alongside standard dilutions of known concentrations in mg/mL. Data generated from HPLC analysis was processed using a calibration curve generated from standards run alongside samples.

[0051] HPLC Analysis for Ergosterol

[0052] Using an Agilent 1260 HPLC instrument fitted with a quaternary pump and diode array detector (DAD), ethanol extracts from mushroom powder samples are analyzed for detection and quantification of ergosterol. Analysis was performed using an Agilent Zorbax SB Cl 8 column, methanol/0.1% formic acid as the mobile phase at a flow rate of 1 mL/min with detection of ergosterol at 282 nm. Sample injection is maintained at 5 pL and total run time per sample acquisition was 11 mins. Standard dilutions of ergosterol (0.918 mg/mL - 0.002 mg/mL) are run alongside samples. Peak areas from the standard dilutions run are used to generate a calibration curve and further determination of target analytes concentration in samples based on their peak areas.

[0053] Our DoE results demonstrated that, in the case of [3-glucans and proteins, both extraction time and temperature exert independent and non-linear effects on the concentration of bioactive molecules extracted from Reishi, Lion’s Mane, and Cordyceps mushrooms.

Contrary to what was noted by Oscar Benito-Roman et al. (Benito-Roman, 6., et al., Dissolution of (l-3),(l-4)-f-Glucans in Pressurized Hot Water: Quantitative Assessment of the Degradation and the Effective Extraction, International Journal of Carbohydrate Chemistry, 2016. 2016(1): p. 2189837), we observed that the solubility of [3-glucans in water and ethanol does not always directly correlate with temperature. The solubility of these polysaccharides depends on factors such as the molecules to which they are bound, their 1,3- /1,6-linkage ratio, and their molecular weight. Additionally, the complexity of each mushroom’s cell wall influences the extraction behaviour of its cell wall -glucans. Prolonged exposure of mushroom slurry to high temperatures may increase the risk of enzymatic or microbial degradation of bioactive molecules such as -glucans and proteins. The extraction of ergosterol, vitamin D2, and ganoderic acid A showed an almost direct dependency on both extraction time and temperature, with a gradual increase in their concentration as these parameters were increased. Furthermore, while ultrasonic waves are often thought to facilitate the extraction process, they do not always increase the yield of all bioactive molecules. For instance, in the case of Lion’s Mane, a regular water bath led to higher yields of -glucans and ergosterol compared to using a sonicator.

[0054] Table 4 presents the optimal extraction conditions, specifically extraction time, temperature, and method, determined for Lion’s Mane, Reishi, and Cordyceps mushrooms.

Table 4. Optimal extraction conditions, such as extraction time, temperature, and method, determined for Lion’s Mane, Reishi, and Cordyceps mushrooms.

[0055] To further concentrate the 1:5 extracts to generate approximately 1 : 1 extracts (equal mass of total solvent and solid mushroom mass used in the extraction) solvent reduction by rotary evaporation was employed. Initially, adhering to the optimal extraction protocols specific to each mushroom variety, five batches of 1:5 mushroom extracts were prepared, each comprising of both ethanol and water-assisted fractions. The extracts derived from the same solvent were then combined resulting in two groups: one containing all water- assisted extracts and the other containing all ethanol-assisted extracts. The volume of these combined extracts was initially measured before being transferred to a rotary evaporator. The pump pressure settings for the ethanol and water-assisted extracts were adjusted to 175 and 60 mbar, respectively, while the water bath temperature was set at 40 °C. The objective was to achieve extracts with a 1 : 1 ratio by carefully reducing the volume of each extract to one- fifth of its original volume, as monitored using a graduated cylinder. Fresh solvent was added to make up for any solvent loss below the targeted level.

[0056] Lyophilization [0057] The resulting extract is subsequently combined with MPP to encapsulate the extract’s bioactive molecules within protein. The quantity of MPP used varies based on the desired -glucan content, as well as the desired flowability and solubility of the resulting powder. To study the impact of MPP% on properties of the final powder, different weight ratios of MPP to highly concentrated liquid extract were measured and mixed. This mixture was then diluted with 5-10 mL of distilled water before being subjected to high-shear mixing using a rotor-stator mixer at 10,000 rpm for 3 mins (1 min on, 1 min rest, x 3). The resulting mixture was rapidly frozen using liquid nitrogen and subsequently placed in a lyophilizer for 48 h to be completely dried. The dried samples were finely ground using an automated ball mill (a mortar could be used alternatively). The resulting powder can be stored in sealed containers at room temperature.

[0058] To assess the flowability of these MPP-encapsulated mushroom powders, a set of physical tests was conducted, encompassing loose bulk density (LBD), tapped density (TD), Hausner’s ratio (HR), angle of repose (0), and Carr’s index (CI). The methodology for each test is explained below.

[0059] The loose bulk density (LBD) of powder refers to the overall density of the particles, encompassing the spaces or voids between each particle. In order to measure the LBD of a powdered sample, precisely 2 g of the sample is weighed before being poured into a 10 mL graduated cylinder. The volume it occupies is recorded as bulk volume (V0). LBD is calculated as the ratio of mass to volume according to Equation 1.

[0060] Equation 1:

LED > Weight of the sample powder (g) W Volume before tapping (mL) Vo

[0061] The tapped density (TD) of a powder signifies the ratio of the powder's mass to the volume it occupies after undergoing tapping for a specified duration. It demonstrates the powder's density under conditions of random packing. Determining the TD of the mushroom powder involved tapping the glass cylinder (previously used) 20 times from a 2 cm height onto a soft surface (layers of paper towel) until a consistent volume (Vi) was achieved. The TD was calculated by dividing the mass by the final volume using Equation 2.

[0062] Equation 2:

> > Weight of the sample powder (g) W

Tapped volume of the packing (mL) Vi [0063] The Hausner ratio (HR), assessing compressibility and particle interaction, is derived by dividing tapped density by loose bulk density (as per Equation 3). This ratio gauges powder flowability by analyzing volume alterations caused by tapping the bulk powder.

[0064] Equation 3: > Tapped density > TD Loose bulk density LBD

[0065] Carr's index (CI) quantifies the compressibility and flowability of a powder and is calculated as a percentage using Equation 4.

[0066] Equation 4: 100

[0067] The angle of repose (0) characterizes interparticulate friction among individual particles, reflecting the flow properties of powders and similar materials. To measure the angle of repose for the powdered tinctures, the fixed powder funnel method was employed. Using a plastic funnel and clean graph paper, the procedure involved positioning the funnel at a height (h) of 2 cm above the paper surface and allowing 2 g of the powder to form a heap upon it. The resulting heap's radius (r) was measured by outlining its circular shape on the paper using a marker. Determining the end-to-end diameter involved measuring perpendicular lengths, computing their average, and using Equation 5 to calculate the angle of repose.

[0068] Equation 5:

[0069] The series of tests explained above was applied to lyophilized Reishi, Lion’s

Mane, and Cordyceps highly concentrated samples (solid-to-solvent ratio of 1: 1) mixed with different percentages of a Shiitake mushroom protein (0 - 100%).

[0070] Additionally, to compare the flowability of the resulting mushroom powders with some commercially available counterparts, the same parameters were tested for Reishi, Lion’s Mane and Cordyceps samples obtained from the Canadian market. [0071] Table 5 displays the flowability properties of powder samples obtained through the lyophilization of highly-concentrated Lion’s Mane extracts mixed 0, 2, 5, 10, 20, 50, and 100% MPP.

Table 5. Flowability values, namely loose bulk density, tapped density, Hausner ratio, Carr’s index and angle of repose, calculated for Lion’s Mane mushroom powder samples containing 0, 2, 5, 10, 20, 50, and 100% of mushroom protein powder.

[0072] The flowability properties of mushroom powders consisting of highly- concentrated Reishi mushroom extracts encapsulated with 0, 2, 5, 10, 20, 50, and 100% Shiitake MPP are tabulated in Table 6.

Table 6. The calculated values for loose bulk density, tapped density, Hausner ratio, Carr’s index and angle of repose of highly-concentrated Reishi mushroom powder samples containing 0, 2, 5, 10, 20, 50, and 100% of mushroom protein powder.

[0073] Table 7 shows the flowability properties of powder samples obtained through the lyophilization of highly-concentrated Cordyceps extracts mixed 0, 2, 5, 10, 20, 50, and 100%. Table 7. Flowability values, namely loose bulk density, tapped density, Hausner ratio, Carr’s index and angle of repose, calculated for Cordyceps mushroom powder samples containing 0, 2, 5, 10, 20, 50, and 100% of mushroom protein powder.

[0074] The water solubility index (WSI) serves as an indicator of the total soluble solids, reflecting a powder's ability to dissolve in water. This parameter holds significance, particularly for products aimed at quick and complete dissolution in water, such as instant beverages or food additives. High-water solubility index values are essential for achieving swift dissolution. Moreover, enhanced solubility not only influences desired mouthfeel and appearance but also ensures optimal absorption and utilization in the body.

[0075] To determine the water solubility index (WSI), 0.2 g of the powder (SI, g) is measured into a centrifuge tube containing 10 mL of distilled water at ambient temperature. This mixture undergoes incubation in a water bath set at specific temperatures (e.g. room temperature and 80 °C) for 30 mins, followed by centrifugation at 3000 rpm for 15 mins. The resulting supernatant is meticulously collected in a pre-weighed evaporating dish (S2, g) and dried overnight at 105 °C. After drying, the dish with residue is reweighed (S3, g; for ensuring sample dryness, another check of S3 could be conducted after an additional night in the oven). By acquiring values for SI, S2, and S3, the WSI can be calculated using Equation 6.

[0076] Equation 6:

[0077] The water solubility index of lyophilized mushroom samples (Table 8) along with that of commercial mushroom powders/capsules (Table 10) was calculated using the method explained above. Table 8. Water solubility index of mushroom powders containing 0, 2, 5, 10, 20, 50, and 100% MPP.

[0078] We have also applied this technology to mixtures of mushroom extracts. As a representative example, a 1:1 mixture of Lion’s Mane and Reishi extracts were encapsulated with varying amounts of MPP. Table 9 presents the water solubility indices for the mixed Lion’s Mane and Reishi extract encapsulated with 0, 10, 25 and 50% Shiitake protein powder. Table 9. Solubility of mixed Lion’s Mane and Reishi extract powders containing between 0 and 50% MPP.

[0079] The water solubility index for commercial mushroom powders/capsules were calculated and the results are shown in Table 10.

Table 10. Water solubility index for five commercial mushroom powders/capsules at room temperature and 80 °C.

[0080] Bioactive Molecules’ Profiles in Derived Powders

[0081] The [3-glucan content (in g/lOOg) and ergosterol content (in mg/g) of lyophilized Lion’s Mane, Reishi and Cordyceps extracts, their parent tinctures and comparison to commercial powders are displayed in Table 11. Table 11. -Glucan and ergosterol content of Lion’s Mane, Reishi, and Cordyceps mushrooms powders containing 0, 2, 5, 10, 20, 50 and 100% MPP with comparison to commercial products.

Discussion

Flowability of Lyophilized Samples

[0082] In the case of lyophilized extract samples, Lion’s mane with 0% MPP exhibits sticking and caking problems, leading to the formation of an agglomerate structure. An increase in the amount of MPP corresponds with a decrease in the loose bulk density (LBD) of Lion’s mane and Reishi mushroom powders, while there is no significant difference for Cordyceps. The slight reduction in the loose bulk density of the powder could be attributed to the potential presence of free protein particles filling the gaps between the larger protein-encapsulated extract particles.

[0083] The tapped density value for each mushroom powder sample exceeds its bulk density due to denser packing conditions during tapping, a finding consist with other published studies. The dried mushroom tinctures with a higher tapped density are beneficial to fill tablets or capsule products. Otherwise, it is recommended to use powders with low bulk and tapped densities for the formulation of supplementary foods with an even and packed texture.

[0084] The mushroom powder samples showed different flowability when tested for the angle of repose. Variations in the angle of repose could be related to differences in particle size and shape among the powder samples. Comparing the calculated angle of repose values for lyophilized samples with the corresponding flowability ranges in Table 12, all samples fall into the category of powders with “low cohesiveness, high flowability”. This categorisation suggests an anticipated enhancement in the granular bulk's flowability.

Table 12. The relationship between powder flowability and angle of repose.

[0085] Hausner ratio (HR) is a mathematical factor that used to serves as a common indicator for approximating powder flowability. HR values for lyophilized samples were used to classify the flow behaviour of dried mushroom tincture samples. Higher HR values denote increased powder cohesiveness and reduced flowability (Chinwan, et al, 2019). According to Table 3, higher values for HR were observed at 5% MPP in Lion's mane and 10% MPP in Reishi and Cordyceps powders. The higher the HR, the more cohesive the powder, and less able to flow freely. Table 13. Characterisation of the powder flow type using Carr's index and Hausner ratio values.

[0086] The impact of adding MPP to dried mushroom tincture samples is reflected in the CI values of the samples. The samples with the highest percentage of MPP exhibit “fair” flowability. Notably, a higher CI value indicates a larger difference between the tapped and bulk densities, which implies poorer flowability and more compressibility (more cohesive). So, the smaller the Carr’s index the better the flow properties.

[0087] Table 14 summarizes the data presented herein.

Table 14. Summary of the mushroom powder data.

References

(1) Martinez-Ibarra, E.; Gomez-Martin, M. B.; Armesto-Lopez, X. A. Climatic and

Socioeconomic Aspects of Mushrooms: The Case of Spain. Sustainability 2019, 11 (4), 1030.

(2) Kakon AJ, C. M. Nutritional and medicinal perspective of Hericium mushroom. Bangladesh Journal of Mushroom 2015, 9 (1), 67-75.

(3) Ng'etich; Nyamangyoku, O.; Rono, J.; Niyokuri, A.; Izamuhaye, J. Relative performance of Oyster Mushroom (Pleurotus florida) on agro-industrial and agricultural substrate. International journal of Agronomy and Plant Production 2013, 4, 109-116.

(4) Miah, M. N. B., A.; Shelly, N. J.; Bhattacharjya, D. K.; Paul, R. . K.; Kabir, M. H. Effect of Different Sawdust Substrates on the Growth, Yield and Proximate Composition of White Oyster Mushroom (Pleurotus Ostreatus). Bioresearch Communications (BRC) 2022, 3. 397- 410.

(5) Ahmad, A.; Kaleem, M. Chapter 11 - 0-Glucan as a Food Ingredient. In Biopolymers for Food Design, Grumezescu, A. M., Holban, A. M. Eds.; Academic Press, 2018; pp 351-381.

(6) Kaur, R.; Sharma, M.; Ji, D.; Xu, M.; Agyei, D. Structural Features, Modification, and Functionalities of Beta-Glucan. Fibers 2020, 8 (1), 1.

(7) de Graaff, P.; Govers, C.; Wichers, H. J.; Debets, R. Consumption of 0-glucans to spice up T cell treatment of tumors: a review. Expert Opinion on Biological Therapy 2018, 18 (10), 1023-1040. DOI: 10.1080/14712598.2018.1523392.

(8) Susanti, I.; Wibowo, H.; Siregar, N.; Pawiroharsono, S.; Sujatna, F. Antitumor Activity of Beta Glucan Extract from Oyster Mushroom (Pleurotus ostreatus Jacq. P. Kum) on DMBA- Induced Breast Cancer in vivo. International Journal of PharmTech Research 2018, 11, 190- 197. DOI: 10.20902/IJPTR.2018.11209.

(9) Jayachandran, M.; Chen, J.; Chung, S. S. M.; Xu, B. A critical review on the impacts of 0- glucans on gut microbiota and human health. The Journal of Nutritional Biochemistry 2018, 61, 101-110. DOI: https://doi.org/10.1016Zi.inutbio.2018.06.010.

(10) Volman, J. J.; Ramakers, J. D.; Plat, J. Dietary modulation of immune function by 0- glucans. Physiology & Behavior 2008, 94 (2), 276-284. DOI: https://doi.Or /10.1016/i.phvsbeh.2007.ll.045. (11) Correa, R. C. G; Peralta, R. M.; Bracht, A.; Ferreira, I. C. F. R. The emerging use of mycosterols in food industry along with the current trend of extended use of bioactive phytosterols. Trends in Food Science & Technology 2017, 67. 19-35. DOI: https://doi.org/10.1016Zi.tifs.2017.06.012.

(12) Hu, D.; Chen, W; Li, X.; Yue, T; Zhang, Z.; Feng, Z.; Li, C.; Bu, X.; Li, Q. X.; Hu, C. Y; et al. Ultraviolet Irradiation Increased the Concentration of Vitamin D2 and Decreased the Concentration of Ergosterol in Shiitake Mushroom (Lentinus edodes) and Oyster Mushroom (Pleurotus ostreatus) Powder in Ethanol Suspension. ACS Omega 2020, 5 (13), 7361-7368. DOI: 10.1021/acsomega.9b04321.

(13) Bach, F.; Helm, C. V; Bellettini, M. B.; Maciel, G. M.; Haminiuk, C. W. I. Edible mushrooms: a potential source of essential amino acids, glucans and minerals. International Journal of Food Science & Technology 2017, 52 (11), 2382-2392. DOI: https://doi.org/10.llll/iifs.13522.

(14) World Bank. World Development Reports. Oxford University Press, I., New York. 2004.

Claims

WHAT IS CLAIMED IS:

1. A dry, flowable composition that comprises a mushroom extract that was made by a process comprising: lyophilizing of a mushroom extract tincture with a mushroom protein powder to make a flowable powder.

2. A composition according to claim 1, wherein the mushroom extract contains P-glucan, eergosterol, and mushroom proteins.

3. A composition according to claim 2 made by a process that comprises: contacting mushroom bodies with water or ethanol under extraction conditions.

4. A composition according to claim 3 made by a process that comprises: contacting said mushroom bodies in ethanol before contact with water.

5. A composition according to claim 1 wherein the lyophilizing process comprises: (a) mixing the mushroom protein powder with liquid mushroom extract in a first weight ratio to make a first mixture, (b) subjecting the first mixture to high-shear mixing to make a second mixture, (c) rapidly freezing the second mixture, (d) placing the frozen second mixture into a lyophilizer until dried, and (e) milling or grinding the dried second mixture into a powder.

6. A process to make a concentrated extract from mushroom powder by a process that comprises contacting mushroom powder under extraction conditions with ethanol followed by water.

7. A process according to claim 6 wherein said extraction conditions comprise a temperature of less than 300° C.

8. A process according to claim 7 wherein said extraction conditions comprise a temperature of 45° C when said mushroom powder comprises powdered Lion’s Mane or Reishi.

9. A process according to claim 7 wherein said extraction conditions comprise a time of 20-140 minutes.

10. A process according to claim 9 wherein said time is within the range of 50-140 minutes.

11. A process according to claim 10 wherein said time is within the range of 50- 100 minutes when said powder comprises powdered Lion’s Mane or Reishi.

12. A process according to claim 9 wherein said time is within the range of 60-100 minutes when said powder comprises powdered Lion’s Mane.

13. A process according to claim 9 wherein said time is within the range of 30-80 minutes when said powder comprises powdered Reishi.

14. A process according to claim 9 wherein said extraction conditions occur within the time and temperatures listed in the table below:

15. A process to make a mushroom protein powder encapsulated in a mushroom extract by a process that comprises: (a) mixing mushroom protein powder with liquid mushroom extract in a first weight ratio to make a first mixture, (b) subjecting the first mixture to high-shear mixing to make a second mixture, (c) rapidly freezing the second mixture, (d) placing the frozen second mixture into a lyophilizer until dried, and (e) milling or grinding the dried second mixture into a powder.