Encapsulated mushroom extract powder materials using a spray drier

Abstract

Disclosed is a dry, flowable composition comprising a mushroom extract that was made by a process comprising: (a) spray drying said extract mixed with an oligosaccharide to make a first powder, and (b) mixing said first powder with a protein powder to make a flowable second powder. Preferably, the mushroom extract contains β-glucan, eergosterol, and mushroom proteins.

Description

Encapsulated Mushroom Extract Powder Materials Using a Spray DrierThis application is a continuation-in-part of US provisional patent application serial number 63 / 611,831, the disclosure of which is hereby incorporated by referenceField of the Invention

[0001] The invention relates to encapsulated, spray-dried powder forms of highly concentrated mushroom extracts in which the encapsulating agents (food-grade oligosaccharide and mushroom or other similar protein powder) help to protect the bioactive components of the mushroom extract from oxidants while 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.1They 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.1'3These 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 (3-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 P-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, 6P-glucan stimulates the immune system in the digestive tract and produces pro-inflammatory metabolites that activate immune cells such as T cells.7It has also been shown to lower tumor incidence, tumor volume, and the total number of tumor nodules.8Other 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 properties. Studies have shown that extracts from ergosterol-enriched fungi can effectively reduce cholesterol absorption and inhibit its biosynthesis within the human body.11Furthermore, 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.13The 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.4Needless 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 shelf life, 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 shelf life, 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 former, liquid material is atomised into droplets and rapidly dried to produce a fine powder. Typically, due to their biocompatibility, stability, high glass transition temperature, and cost-effectiveness, sugar substances (such as maltodextrin) are utilized as encapsulating agents. To date, such processes have not produced satisfactory powders having both flowability and sufficient concentration of the desired extract.Summary of the Invention

[0009] It is an object of the invention to provide a dry, flowable, powder containing a mushroom extract and a process for making that powder by spray drying.

[0010] It is further an object of the invention to provide a flowable mushroom extract in a size suitable for delivery by nasal spray.

[0011] In accordance with these and other objects of the invention that will become apparent from the description herein, a dry, flowable composition according to the invention comprises a mushroom extract that was made by a process comprising: (a) spray drying said extract mixed with an oligosaccharide to make a first powder, and (b) mixing said first powder with a protein powder to make a flowable second powder. Preferably, the mushroom extract contains -glucan, eergosterol, and mushroom proteins.

[0012] Further, the process for making the extract comprises contacting mushroom bodies with water or ethanol under extraction conditions. Preferably, the mushroom bodies are contacted with ethanol before they are contacted with water.

[0013] Preferably, the extraction conditions generally include a temperature of less than 300 °C. When the mushroom powder includes powdered Lion’s Mane or Reishi, the extraction conditions comprise a temperature of 45 °C.

[0014] Preferably, the extraction conditions include an extraction time within the range of 20-140 minutes and more preferably the time is within the range of 50-140 minutes. When the mushroom powder comprises powdered Lion’s Mane or Reishi, the extraction time is preferably within the range of 50-100 minutes and more preferably, when the powder comprises Lion’s Mane, the extraction is performed for a time within the range of 60-100 minutes. When the powder includes powdered Reishi, the extraction time is most preferably within the range of 30-80 minutes.

[0015] The process of the present invention provides a dry, flowable mushroom extract powder with high concentrations of -glucan, ergosterol, and mushroom proteins in a size suitable for administration by nasal spray and similar delivery mechanisms.Brief Description of the Drawings

[0016] 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.

[0017] Figure 2 shows the first derivative curves for the tests of Figure 1.

[0018] Figure 3 is a chart showing protein concentration v. extraction time.

[0019] Figure 4 is a chart showing -glucan concentration v. extraction time.

[0020] Figure 5 shows representative Scanning Electron Microscope (SEM) images of spray-dried mushroom extracts made from mushroom extract tinctures.

[0021] Figure 6 shows a portion of the SEM image in Figure 5 at a higher magnification.Detailed Description

[0022] 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.

[0023] Suitable mushroom species include Ganoderma lingzhi (Reishi), Hericium erinaceus (Lion’s Man), Cordyceps militaris (Cordyceps), Inonotus obliquus (Chaga), Trametes versicolor (Turkey tail), Grifola frondosa (Maitake), Lentinulci edodes (Shiitake) and Fomitopsis officinalis (Agarikon). The results for the first three mushrooms are presented as representative models, but the application of this invention is applicable to all mushroom species and their mixtures.

[0024] Accordingly, the invention provides a dry, flowable composition and its method of manufacture that comprise an encapsulated mushroom extract made by a process comprising: spray drying a highly concentrated mushroom extract with an oligosaccharide and a protein source to make a flowable powder. In a preferred embodiment, this protein source is mushroom protein powder (MPP).

[0025] The resulting product shows low cohesiveness and good flowability at a particle size that is suitable for administration by nasal spray, capsule, tablet, transdermal patch, and similar delivery systems. It is particularly preferred that the mushroom powder extracts according to the invention (or corresponding pharmaceutical compositions) are administered orally, sublingually, or nasally (e.g., as a nasal spray or as nose drops). Suitable dosage forms for oral administration include, e.g., coated or uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders or granules for reconstitution, dispersible powders or granules, medicated gums, chewing tablets, or effervescent tablets.

[0026] In various embodiments, the agents are formulated as a dosage form (e.g., tablet, capsule, gel, lollipop), parenteral, intraspinal infusion, inhalation, nasal spray, transdermal patch, iontophoresis transport, absorbing gel, liquid, liquid tannate, suppositories, injection,I V drip, other formulation, or a combination thereof to treat subjects. The mushroom extracts according to the invention or pharmaceutical compositions are preferably administered orally, or by infusion, spray, gel, or inhalation.

[0027] The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human (e.g., a male human or a female human) or a non- human mammal. Most preferably, the subject / patient to be treated in accordance with the invention is a human.

[0028] Encapsulating or binding mushroom tinctures with proteins (such as, but not limited to, whey protein or mushroom protein) prevents the formation of a viscous paste with no flowability when the pure extract is spray dried. Therefore, mixing the extract with dosages of protein powder benefits the flowability of the final powder product. Furthermore, this added protein component serves to shield the tincture’s bioactive molecules, such as -glucan, from oxidants and environmental stressors by encapsulating them. To create powder substance rich in mushroom-derived compounds, in a preferred embodiment, mushroom protein powder (MPP) was used in the examples presented below.

[0029] In accordance with the present invention, mushroom powders are prepared by combining an oligosaccharide before the spray drying process. Suitable oligosaccharides include at least one member selected from the group consisting of maltodextrin, tapioca, potato, com, rice, wheat, carboxymethyl starch, carboxymethyl chitosan, chitosan oligosaccharide, hydroxy propyl methyl starch, hydroxy propyl cellulose, ethyl cellulose, methyl cellulose, and octenyl succinic anhydride modified starch. This formulation makes it possible to prepare a finely-ground, mushroom-derived powder composition exhibiting decent flowability and solubility suitable for human consumption using a spray drier, concentrated mushroom extract / tincture, and proteins.

[0030] The spray-dried samples used in the examples of this application were formulated using maltodextrin, although other sugar-based materials can be used. Maltodextrin, a preferred food-grade oligosaccharide, helps the encapsulation process, increases the solubility of the resulting powder, and contributes to the texture and mouthfeel of the product.

[0031] To yield highly water-soluble spray-dried mushroom powder, maltodextrin was combined with either MPP or whey protein. This approach, along with its methodology and the characterisation values for the resulting samples, is explained below.

[0032] While industry and market have focused on obtaining high -glucan containing extracts and tinctures from mushrooms, these occupy larger volumes and are difficult topackage in concentrated formats. A way out of this problem might be to pack spray-dried encapsulated mushroom -glucan inside gelatine or agar-agar capsules, or simply package them as food sachets. However, spray drying the mushroom extracts without additives does not result in dry, flowable powders.Maceration

[0033] 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 maximising the concentration of target bioactive molecules, a 3-by-3 full-factorial Design of Experiment (32DoE) 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).

[0034] The extraction protocol begins by mixing a defined amount of ground, dried mushrooms (x g) with five times its mass in solvent volume (5% 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.

[0035] 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.

[0036] 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 analysed to determine their concentrations of -glucan, ergosterol, protein and ganoderic acid A (for Reishi mushrooms). To ensure reliability and consistency, all extractions were conducted inquadruplicates. All methods used for extraction and quantification of bioactives were validated for efficiency, repeatability and accuracy.

[0037] Thermogravimetric analysis (TGA) was performed as follows: Approximately 2 mg of each mushroom sample was loaded into pre-weighed TGA aluminium pans (TA Instruments) 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.

[0038] 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 mins in a water bath to ensure consistency in comparison for each solvent order.

[0039] 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).

[0040] Preferably, the extraction conditions generally include a temperature of less than 300 °C. When the mushroom powder includes powdered Lion’s Mane or Reishi, the extraction conditions comprise a temperature of 45° C.

[0041] Preferably, the extraction conditions include an extraction time within the range of 20-140 minutes and more preferably the time is within the range of 50-140 minutes. When the mushroom powder comprises powdered Lion’s Mane or Reishi, the extraction time is preferably within the range of 50-100 minutes and more preferably, when the powder comprises Lion’s Mane, the extraction is performed for a time within the range of 60-100 minutes. When the powder includes powdered Reishi, the extraction time is most preferably within the range of 30-80 minutes.

[0042] To determine the thermal profilse 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. See, Figure 2.

[0043] To systematically investigate the optimal extraction time range, a time-course extraction was conducted on the three mushrooms under study. -glucan and protein concentrations were measured every 20 mins 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 2. For Reishi and Lion’s Mane, protein concentrations plateaued at 60 mins, 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 mins 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 3. 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 f>- glucanase activity but also the potential for acidic hydrolysis of [3-glucans.

[0044] 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 mins (Table 2). For Cordyceps, extractions were conducted at 40, 60, and 80 °C for 40, 60, and 90 mins 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.Statistical Analysis

[0045] The run numbers in the DoE were randomized to enhance reliability and accuracy by reducing the impact of uncontrolled factors, such as environmental variability or instrument drift, which could otherwise introduce bias. 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).Optimum Extraction Conditions

[0046] 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 (Yeast and Mushroom) and high-performance liquid chromatography (HPLC) are recommended. The methodology for both is explained below.Megazyme B-Glucan Assay Kit (Yeast and Mushroom)

[0047] The Megazyme [3-Glucan Assay Kit (Y east and Mushroom), acquired from Neogen in Michigan, United States, 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.High-Performance Liquid ChromatographyErgosterol Extraction

[0048] Approximately 500 mg of samples are weighed into 15 mL falcon tubes and 5 mL HPLC grade ethanol added. In the case where samples are not enough, approximately 100 mg of samples are used with 1 mL ethanol for the extraction process. Samples are vortexed for 10 seconds and place in an ultrasonic bath for 15 mins. After sonication, tubes are placed in a centrifuge and spun at 400 g for 5 mins and clear supernatants collected for HPLC analysis.HPLC analysis

[0049] Using an Agilent 1260 HPLC instrument fitted with a quaternary pump and diode array detector (DAD), ethanol extracts from mushroom powder samples are analysed for detection and quantification of ergosterol. Analysis is performed using an Agilent Zorbax SB C18 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 minutes. Standard dilutions of ergosterol (0.918 mg / mL - 0.002 mg / mL) were run alongside samples. Peak areas from the standard dilutions run are (were) used to generate a calibration curve and further determination of target analytes concentration in samples based on their peak areas.

[0050] 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., 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 l,3- / l,6-linkage ratio, and their molecular weight. Additionally, the complexity ofeach 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.

[0051] 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.

[0052] 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 via rotary evaporation was employed. This process involves concentrating mushroom extracts by volume reduction through rotary evaporation. 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 approximate 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.Spray drying

[0053] The resulting extract is subsequently combined with maltodextrin and mushroom protein powder (MPP) or whey protein to encapsulate the extract’s bioactive molecules within protein molecules. 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 minutes (1 minute on, 1 minute rest x 3). The resulting mixture was rapidly introduced to the spray drier. 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.

[0054] A powder matrix was used as wall material for encapsulating highly concentrated aqueous mushroom extract (see Table 5). For making the pre-spray system, all solids were poured into the mushroom extracts and subjected to high-shear mixing at 10,000 rpm for 3 min, with 1 min intervals between consecutive mixing cycles. This was then diluted with water. In a preferred embodiment the volume of water used was the same as the volume of the extract.

[0055] The powders were produced using a Buchi mini spray-drier B-290 in conjunction with a high-performance cyclone from Buchi Labortechnik AG. Nitrogen being inert in nature was preferred as a drying gas to avert any unwanted reactions that might occur in the presence of air as drying medium. The spray drying parameters were varied in accordance with the experimental data (Table 5). The feed flow rate is displayed in percentage (%) on the equipment and feed flow rate of 100% was experimentally determined to correspond to a flow rate of 39.6 g / min with reference to the pre-spray emulsion. An atomising airflow of 7.3-17.5 L / min corresponds to a setting from 30 to 50 mm; all experiments were conducted at 40 mm. The aspirator rates were set at 19.8-22 N m3 / h in all experiments; this corresponded to instrument setting of 90-100%. The powders were stored in clean, dry glass vials at room temperature in a steel cabinet.

[0056] Spray dryer conditions are summarized in Table 6, including inlet temperature, outlet temperature, aspiration, feed rate, time, and yield.Table 5. Components of the spray-drying system.Table 6. Spray drying variables and outcomes.Imaging the Spray-Dried Powder

[0057] To investigate the particle shape and size of the spray-dried powders, scanning electron microscopy (SEM) was used. This was performed as follows: about 4-5 mg of the powder was smeared onto double-sided carbon tape on an aluminium stub, and excess powder was removed by blowing compressed air onto the sample stub surface. SEM was then performed on a FEI Quanta 200 Environmental Scanning Electron Microscope (ESEM) with an ED AX Octane Plus SDD detector. Some sample SEM micrographs are attached as Figures 5 and 6.

[0058] The resulting powder particle sizes are mostly under 5 pm and are particularly well- suited for aerosolized drug inhalation and uptake through the respiratory tract such as with a nasal spray.

[0059] To assess the flowability of spray-dried protein-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.

[0060] 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 (Vo). LBD is calculated as the ratio of mass to volume according to Equation 1.Equation 1:

[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 ontoa 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.Equation 2:

[0062] The Hausner ratio (HR), assessing compressibility and particle interaction, is derived by dividing tapped density by loose bulk density. This ratio gauges powder flowability by analysing volume alterations caused by tapping the bulk powder. Hence, powder cohesiveness was assessed using the Hausner ratio (HR) using Equation 3.Equation 3:

[0063] Carr's index (CI) quantifies the compressibility and flowability of a powder and is calculated as a percentage from the loose bulk density and tapped density using Equation 4: Equation 4:

[0064] The angle of repose (9) 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) 2 cm above the paper surface and allowing 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 utilizing Equation 5 to calculate the angle of repose.Equation 5:

[0065] The series of tests explained above was applied to spray-dried Lion’s Mane and Reishi highly concentrated samples, and the results are presented in Table 7.

[0066] 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. The flowability properties of the spray-dried Lion’s Mane mushroom powder were examined, and the findings are presented in Table 7. These tests were performed in triplicate, and the numbers after ± indicate the standard deviation of the data from their average value.Table 5. Loose bulk density, tapped density, Hausner Ratio, Carr’s index and angle of repose for spray-dried Lion’s Mane and Reishi mushroom extract-derived powder samples.

[0067] To appraise the flowability of maltodextrin-assisted spray-dried mushroom powder samples, agent, loose bulk density, tapped density, Hausner Ratio, Carr’s index, and angle of repose were examined for five commercial mushroom powders / capsules. The findings from these evaluations are presented in Table 8.Table 6. Flowability values, including loose bulk density, tapped density, Hausner Ratio, Carr’s index, and angle of repose, calculated for five commercial mushroom powders / capsules

[0068] 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 utilzation in the body.

[0069] To determine the water solubility index (WSI), 0.2 grams 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.Equation 6:S3 - S2WSI% = — - — x 100SI

[0070] The water solubility index of spray-dried mushroom samples along with that of commercial mushroom powders / capsules was calculated using the method explained above and presented in Table 9.Table 9. Water solubility index for Lion’s Mane, Reishi, and mixed mushroom extract-derived powders with comparison to five commercial mushroom products.B-glucan Content

[0071] The [3-glucan content (g / lOOg) of spray-dried Lion’s Mane, Reishi and Cordyceps extracts, and commercial powders are displayed in Table 10.Table 10. P-glucan % of tinctures, spray-dried mushroom extracts and comparison to those of commercial mushroom powders.Ergosterol Content

[0072] The ergosterol content (mg / g) of spray-dried Lion’s Mane and Reishi extracts with comparison to commercial powders are shown in Table 11.Table 11. Concentration (mg / g) of ergosterols in mushroom powder samples.Flowability of Spray Dried Samples

[0073] The bulk and tapped densities provide a perspective from packing and the compaction profde of a material. Table 16 shows that the change of carrier agents affected the bulk density of mushrooms.

[0074] The tapped density value for each mushroom powder sample exceeds its bulk density due to denser packing conditions during tapping, a finding consistent 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.

[0075] 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 spray-dried samples with the corresponding flowability ranges in Table 12, an increase in the angle of repose leads to reduced flowability for spray-dried mushrooms. Flow properties of the samples ranged from excellent to passable, as per the criteria. Within this range, 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.

[0076] Hausner ratio (HR) serves as a common indicator for approximating powder flowability. HR values for spray-dried samples were used to classify the flow behaviour of dried mushroom tincture samples. Higher HR values denote increased powder cohesiveness and reduced flowability, Table 13. Hausner's ratio measures the material's ability to setle and help to assess the relative importance of interparticulate interactions as they influence flowability. Flow properties ranged from excellent to passable. Thus, powders exhibited less cohesive and more capable of flowing freely.Table 13. Characterisation of the powder flow type using Carr's index and Hausner ratio values.

[0077] Ahigher Carr’s Index (CI) indicates a larger difference between the tapped and bulk densities, which implies poorer flowability and lower compressibility; interpretation of these values is shown in Table 13. The mushroom powders are classified to have excellent to passable flowability and low to intermediate cohesiveness, Table 14. The higher the Carr’s index and Hausner ratio, the more cohesive the powder, and less able to flow freely.Table 14. Summary of mushroom powder data and comparison with commercial samples.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) Rono, J.; Niyokuri, A.; Izamuhaye, J. Relative performance of Oyster Mushroom (Pleurotus florida) on agro-industrial and agricultural substrate. 2013, 4.(4) Miah, M. N. B., A.; Shelly, N. J.; Bhattachaqya, 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). 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 / 10.1016 / j.jnutbio.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.Org / 10.1016 / j.physbeh.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 / 10.1016 / j.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 / ijfs.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: (a) mixing said extract with an oligosaccharide to make a first mixture, (b) mixing said first mixture with a protein powder to make second mixture, and (c) spraydrying the second mixture to make a flowable powder.

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

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

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

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

6. A process to make a concentrated extract from mushroom bodies by a process that comprises: concentrating the composition according to claim 3 by evaporating any ethanol or water by rotary evaporation.

7. A process according to claim 3 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 an extraction time of 20-140 minutes.

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

11. A process according to claim 10 wherein said an extraction 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 an extraction 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 an extraction 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 bound with a mushroom extract by a process that comprises: (a) mixing the protein powder with liquid mushroom extract, an oligosaccharide, and sodium chloride to make a first mixture, (b) subjecting the first mixture to high-shear mixing to make a second mixture, (c) diluting the second mixture with distilled water to make a third mixture, and (d) forming a dried powder by spray drying said third mixture.

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