DESC:EARLIEST PRIORITY DATE:
This Application claims priority from a Provisional patent application filed in India having Patent Application No. 202241014674, filed on April 17, 2022, and titled “A PROCESS FOR DERIVING MUSHROOM EXTRACT”
FIELD OF INVENTION
[0001] Embodiments of a present disclosure relate to plant-based extraction technique and more particularly to a process for extraction of a plurality of bioactive molecules from any herbal, plant material, or mycological raw material comprising spices, roots, leaves, seeds or stems from plants, and mycelium, spores, fruiting bodies or sclerotia from fungi.

BACKGROUND
[0002] Edible and medicinal or functional mushrooms are an important constituent of our daily diet due to their rich nutrition and beneficial properties for human health. Most edible mushrooms are rich in nutrients such as polysaccharides, protein, vitamins, mineral elements, and various functional factors. These substances are essential for human and long-term consumption provides a beneficial effect on improvement of human health.
[0003] Mushroom cell walls are made of chitin, a natural fibrous substance generally considered to be indigestible in the human gut. Hence, mushrooms need to be extracted to make nutrients bioavailable.
[0004] Mushroom extracts have gained popularity as remedies for a variety of ailments including seasonal allergies, common cold, insomnia, inflammation, and cancer. Tinctures, powders, and pills with single mushroom extract or as a combination of numerous mushroom extracts are available in the market.
[0005] Traditional methods of mushroom extraction depend on use of boiling water (hot water extraction (HWE)) or a mix of ethanol and water (ethanol extraction). These methods are biased in their affinity towards desired bioactive molecules. The HWE preferentially extracts polysaccharides and other water-soluble components with a lower triterpene and phenolic content. The ethanol extraction contains a higher proportion of triterpenes and phenolics with minimal polysaccharide extraction as a result of ethanol induced precipitation of polysaccharides.

[0006] WO2016118034A2 relates to a process for extraction of active biological compounds from plants by simultaneous application of ultrasounds and microwaves in a laminar continuous flow regime of extraction, under conditions of a controlled flow regime.
[0007] KR101825104B1 relates to a method for producing ganoderma lucidum an extract obtained by extracting ganoderma lucidum with ethanol and then concentrating under reduced pressure to dryness, immersing the powder in water containing an emulsifier, and then treating with ultrasonic waves.
[0008] Although the methods, reported heretofore, are relatively simple and cost-effective, the disadvantages are also present with longer operating cycle and higher consumption of ethanol. However, the high temperature required in the traditional water extraction method breaks down the active polysaccharides structure in edible mushroom.
[0009] Traditionally, acid-base extraction (ABE) method is also used for extraction of mushroom. However, the disadvantage of this method is that it produces some side-products and/or result in degradation of compounds.
[0010] Therefore, there is a need for an efficient extraction method with high extraction rate.

SUMMARY
[0011] In accordance with an embodiment of the present invention, a process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof. The process comprises grinding and mixing of the at least one of plant material, mycological raw material, and combinations thereof to obtain a finely grounded material. The finely grounded material is extracted with an organic solvent to obtain an organic extract. The organic extract is subjected to ultrasonic sound waves of a predefined frequency applied at a predefined amplitude power. The organic extract is concentrated under vacuum evaporation and the finely grounded material is recovered. A recovered finely grounded material is extracted with water to obtain an aqueous extract. The organic and aqueous extract comprises at least one of plant material, mycological raw material, and combinations thereof and the organic solvent or water in a ratio of 1:30. The aqueous extract is subjected to ultrasonic sound waves of the predefined frequency applied at the predefined amplitude power. The aqueous extract is filtered and mixed together with the concentrated organic extract to obtain a mixed extract. The mixed extract is dried to obtain the plurality of bioactive molecules.
[0012] To further clarify the advantages and features of the present invention, a more particular description of the invention will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the invention and are therefore not to be considered limiting in scope. The invention will be described and explained with additional specificity and detail with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS
The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which:
[0013] FIG. 1 is a flowchart representing steps involved in a process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof, in accordance with an embodiment of the present invention.
[0014] Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, in terms of the method steps, chemical compounds, and parameters used herein may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

DETAILED DESCRIPTION
[0015] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.
[0016] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more components, compounds, and ingredients preceded by "comprises... a" does not, without more constraints, preclude the existence of other components or compounds or ingredients or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.
[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.
[0018] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
[0019] Embodiments of the present invention relates to a process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof. The process mainly involves extraction using ultrasonic sound waves. The process enables extraction of terpenoids, flavonoids, polyphenols, isoflavones, carotenoids, phytosterols, proteins, polysaccharides, peptides, lipopeptids, lipids, lipoproteins, glycoproteins, proteoglycans, glycosides, alkaloids, saponins, and combination thereof without use of external heating and therefore prevents breakdown of heat labile compounds.
[0020] As used herein the term “ultrasonic sound waves” refers to vibrations of frequencies greater than the upper limit of the audible range for humans i.e., greater than about 20 kilohertz.
[0021] In an embodiment, the process for deriving at least one of plant material, mycological raw material, and combinations thereof is provided. The process optionally required heating.
[0022] FIG. 1 is a flowchart representing steps involved in the process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof, in accordance with an embodiment of the present invention.
[0023] In an embodiment, the process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof begins with grinding and mixing the at least one of plant material, mycological raw material, and combinations thereof to obtain a finely grounded material, at step 102. The plant material is selected from one of fruits, flowers, seeds, stems, roots, barks, leaves, and combinations thereof, and the mycological raw material comprises one of spores, fruiting bodies, mycelium, sclerotia, and combination thereof. The mycological raw material comprises dried medicinal and edible mushrooms. The mycological raw material is selected from one of a genus from a group consisting of Pleurotus, Hericium, Grifola, Lentinus, Lentinula, Cordyceps, Trametes, Inonotus, Psilocybe, Ganoderma, Fomes, Fomitopsis, Wolfiporia, Amanita, Agaricus, Tremella, Auricularia, Lignosus, Flammulina, Coprinellus, Schizophyllum, Morchella, Agrocybe, Macrocyb, Polyporales, Agaricales, and combinations thereof.
[0024] In an embodiment, the finely grounded material is extracted with an organic solvent to obtain an organic extract, at step 104. The organic extract comprises the at least one of plant material, mycological raw material, and combinations thereof and the organic solvent in a ratio of 1:30. The organic extract is subjected to ultrasonic sound waves of a predefined frequency applied at a predefined amplitude power, at step 106.
[0025] In an embodiment, the organic solvent is selected from one of a group of organic solvents consisting of acetone, ethyl acetate, hexane, heptane, dichloromethane, methanol, ethanol, tetrahydrofuran, acetonitrile, dimethylformamide, toluene, dimethyl sulfoxide, and combinations thereof. In one embodiment, water is used as the solvent. In another exemplary embodiment, ethanol is used as the solvent. The predefined frequency of 15-20kHz is applied at the predefined amplitude power of 300-500W. The organic extract or the aqueous extract is subjected to ultrasonic sound waves for the predefined duration of 1-90 minutes. The organic extract or the aqueous extract is subjected to ultrasonic sound waves for a predefined duration with a predefined on or off pulse. The predefined duration is 8-10 minutes with the predefined on or off pulse of 5-8 seconds.
[0026] In an embodiment, the organic extract is concentrated under vacuum evaporation and the finely grounded material is recovered, at step 108. The recovered finely grounded material is extracted with water to obtain an aqueous extract, at step 110. The aqueous extract comprises the at least one of plant material, mycological raw material, and combinations thereof and the water in a ratio of 1:30. The aqueous extract is subjected to ultrasonic sound waves of the predefined frequency applied at the predefined amplitude power, at step 112. The aqueous extract is filtered and mixed together with the concentrated organic extract to obtain a mixed extract, at step 114. The mixed extract is dried to obtain the plurality of bioactive molecules, at step 116.
[0027] The ultrasonic sound waves penetrate through matrix of the mushrooms breaking them open and releasing the compounds of interest. A phenomenon of ultrasonic cavitation enables to break open the cells and extracellular matrix of the mushroom. This releases the native molecules trapped within the matrix into surrounding liquid. Since this is a physical method of extraction, the extraction is not inherently selective to the nature of molecules being extracted.
[0028] The resulting extract contains both hydrophobic and hydrophilic molecules thus reducing number of steps to two required in processing of at least one of plant material, mycological raw material, and combinations thereof by sequential extraction with organic solvent followed by water to obtain value added products. In an embodiment, the process for deriving the plurality of bioactive molecules from the at least one of plant material, mycological raw material, and combinations thereof is carried out at room temperature thereby bypassing a need for heating or boiling for deriving the at least one of plant material, mycological raw material, and combinations thereof. Thus, minimises heat induced denaturation of bioactive molecules and retains higher biological activity.
[0029] In another embodiment, the process for deriving the plurality of bioactive molecules from the at least one of plant material, mycological raw material, and combinations thereof is optionally carried out at a constantly maintained elevated temperature of 20 oC to 121 oC to increase polysaccharide yields.
[0030] The present invention provides the process for deriving at least one of plant material, mycological raw material, and combinations thereof. The process helps in eliminating dependencies on external solvent-based extraction for obtaining the plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof. The process provided by the present invention is simple and rapid, thereby making it possible to process large number of raw mushrooms in less amount of time.
[0031] The process of the present invention provides a significant improvement in yield and reduced processing time with ultrasonication and sequential extraction with organic solvent followed by water to obtain the value-added products. The processing time is reduced from several hours from 3-24 hrs to 30 minutes for ethanol extraction. Further, the processing time is reduced from several hours from 6-12 hrs to 30-90 min for polysaccharides. The increase in beta glucan yield is found to be around 30% and increase in phenolics yield is found to be around 80-100%.
EXAMPLES
[0032] In an exemplary embodiment, the mixed extract is obtained after ultrasound assisted dual extraction of the at least one of plant material, mycological raw material, and combinations thereof through sequential extraction with organic solvent followed by water. The mixed extract is subjected for spray drying and subjected for further evaluation to determine the quantity of the bioactive molecule present in the dried mixed extract. Also, the heat assisted mixed extract as a liquid formulation is subjected for further evaluation for determining the quantity of the bioactive molecule present in the heat assisted mixed extract. The liquid has 12% solids content and therefore the 100g weight used to represent the unit measure while quantifying the bioactive molecules is equivalent to 12g of dried extract. It is observed that the quantity of the phenol as total polyphenol in the mixed extract and the heat assisted mixed extract is found to be in a range of 28.2 mg/100gm to 503.3 mg/100gm. Further, the estimation of total glucan, alfa glucan, and beta glucan is carried out using an assay procedure for beta-glucan by megazyme kit for both the mixed extract and the heat assisted mixed extract. The quantity of the total glucan is found to be in a range of 1.70 g/100g to 15.77 g/100g, the alfa glucan is found to be in the range of 0.61 to 3.53 g/100g, and the beta glucan is found in the range of 1.09 to 12.24 g/100g in the mixed extract and the heat assisted mixed extract.
[0033] Tests are conducted with the help of FSSAI Approved & NABL Accredited Food, Water & Air Testing Laboratory to identify and ascertain efficiency of the present invention.
TEST 1:
PARTICULARS OF SAMPLE ANALYSED
Sample Quantity & Condition: Approx 50ml x 3 of sample in a client packaging is intact without any leaks or breaks.
Date of Start of Analysis: 18-Mar-2023
Date of End of Analysis: 28-Mar-2023
RESULTS OF ANALYSIS
Sr.No. Parameters Units Methods Results of Analysis
1 Phenol as total Polyphenol mg/100gm By Inhouse Method 28.2

TEST 2:
PARTICULARS OF SAMPLE ANALYSED
Sample Quantity & Condition: Approx 50ml x 3 of sample in a client packaging is intact without any leaks or breaks.
Date of Start of Analysis: 14-Mar-2023
Date of End of Analysis: 07-April-2023
RESULTS OF ANALYSIS
Sr.No. Parameters Units Methods Results of Analysis
1 Total Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 1.70
2 Alfa Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 0.61
3 Beta Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 1.09
TEST 3:
PARTICULARS OF SAMPLE ANALYSED
Sample Quantity & Condition: Approx 15 gm of sample in a client packaging is intact without any leaks or breaks.
Date of Start of Analysis: 18-Mar-2023
Date of End of Analysis: 28-Mar-2023
RESULTS OF ANALYSIS
Sr.No. Parameters Units Methods Results of Analysis
1 Phenol as total Polyphenol mg/100gm By Inhouse Method 28.2
TEST 4:
PARTICULARS OF SAMPLE ANALYSED
Sample Quantity & Condition: Approx 15 gm of sample in a client packaging is intact without any leaks or breaks.
Date of Start of Analysis: 18-Mar-2023
Date of End of Analysis: 07-Apr-2023
RESULTS OF ANALYSIS
Sr.No. Parameters Units Methods Results of Analysis
1 Total Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 15.77
2 Alfa Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 3.53
3 Beta Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 12.24
3 Beta Glucan Estimation g/100g Assay Procedure For Beta-Glucan- By Megazyme Kit 1.09
[0034] In an embodiment, the process of the present invention enables the extraction of bioactive molecules comprising terpenoids, flavonoids, polyphenols, isoflavones, carotenoids, phytosterols, proteins, polysaccharides, peptides, lipopeptids, lipids, lipoproteins, glycoproteins, proteoglycans, glycosides, alkaloids, saponins, and combination thereof without use of external heating and therefore prevents breakdown of heat labile compounds. The process also reduces the number of extraction steps to two while decreasing the time of extraction to scale of a few minutes rather than hours. Therefore, the process is more efficient, has higher yield and economical when compared to currently used methods.
[0035] In an embodiment, the at least one of plant material, mycological raw material, and combinations thereof is subjected to selective fractionation to obtain the plurality of bioactive molecules. The selective fractionation is performed by altering the organic solvent and water to obtain differentiated extract of the plurality of bioactive molecules comprising one of terpenoids, flavonoids, polyphenols, isoflavones, carotenoids, phytosterols, proteins, polysaccharides, peptides, lipopeptids, lipids, lipoproteins, glycoproteins, proteoglycans, glycosides, alkaloids, saponins, and combination thereof.
[0036] The process of the present invention aids to obtain the plurality of bioactive molecules from any traditional plant material used in the practices of ayurveda, siddha, well known superfoods as well as nootropics and adaptogens. The plurality of bioactive molecules is extracted from a group of herbs consisting of Ashwagandha, Pepper, Physilum Husk, Lavender, Beetroot, Lemon Grass, Bacopa, Clitoria, Benincasa, Celastrus, Acorus, Nardostachys, Panax, Rhodiola, Ginkgo, Centella, Ocimum, Curcuma, Withania, Astragalus, Schisandra, Lycium, Convolvulus, Tinosporia, Asparagus, Moringa, Mucuna, and Embelica. The process of the present invention is faster than the current industry standard of double extracts with water and ethanol.
[0037] The derived plurality of bioactive molecules serves as a raw material for further processing. The derived plurality of bioactive molecules has application in nutritional supplements, nutraceuticals, medicinal tinctures. The derived plurality of bioactive molecules serves as the raw material for cosmetic formulations, topical formulations, antiseptics, wound healing matrices, food and beverage industry, health drinks, sports beverages, functional blends, nutrient bars, alcoholic and non-alcoholic beverages, baked items, fast food, and health food. The derived plurality of bioactive molecules is able to be used in pharmaceuticals such as ganoderic acids, glucans, lucidenic acids, other functional molecules with applications in longevity, treatment adjuncts, holistic medicine, immune support, stress relief, chemotherapy and radiation therapy; lifestyle enhancers such as adaptogens, nootropics, immune supplements, performance boosters, detoxifiers, and antioxidants.
[0038] While specific language has been used to describe the invention, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.
[0039] The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, order of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.
,CLAIMS:WE CLAIM:
1. A process for extracting a plurality of bioactive molecules from at least one of plant material, mycological raw material, and combinations thereof, said process comprising the steps of:
grinding and mixing the at least one of plant material, mycological raw material, and combinations thereof to obtain a finely grounded material,
wherein the plant material is selected from one of fruits, flowers, seeds, stems, roots, barks, leaves, and combinations thereof, and the mycological raw material comprises one of spores, fruiting bodies, mycelium, sclerotia, and combination thereof;
extracting the finely grounded material with an organic solvent to obtain an organic extract,
wherein the organic extract comprises the at least one of plant material, mycological raw material, and combinations thereof and the organic solvent in a ratio of 1:30;
subjecting the organic extract to ultrasonic sound waves of a predefined frequency applied at a predefined amplitude power;
concentrating the organic extract under vacuum evaporation and recovering the finely grounded material;
extracting a recovered finely grounded material with water to obtain an aqueous extract,
wherein the aqueous extract comprises the at least one of plant material, mycological raw material, and combinations thereof and the water in a ratio of 1:30;
subjecting the aqueous extract to ultrasonic sound waves of the predefined frequency applied at the predefined amplitude power;
filtering the aqueous extract and mixing the aqueous extract with the concentrated organic extract to obtain a mixed extract; and
drying the mixed extract to obtain the plurality of bioactive molecules.
2. The process as claimed in claim 1, wherein the mycological raw material comprises dried medicinal and edible mushrooms.
3. The process as claimed in claim 1, wherein the mycological raw material is selected from one of a genus from a group consisting of Pleurotus, Hericium, Grifola, Lentinus, Lentinula, Cordyceps, Trametes, Inonotus, Psilocybe, Ganoderma, Fomes, Fomitopsis, Wolfiporia, Amanita, Agaricus, Tremella, Auricularia, Lignosus, Flammulina, Coprinellus, Schizophyllum, Morchella, Agrocybe, Macrocyb, Polyporales, Agaricales, and combinations thereof.
4. The process as claimed in claim 1, wherein the plurality of bioactive molecules comprises one of terpenoids, flavonoids, polyphenols, isoflavones, carotenoids, phytosterols, proteins, polysaccharides, peptides, lipopeptids, lipids, lipoproteins, glycoproteins, proteoglycans, glycosides, alkaloids, saponins, and combinations thereof.
5. The process as claimed in claim 1, wherein the organic solvent is selected from one of a group of organic solvents consisting of acetone, ethyl acetate, hexane, heptane, dichloromethane, methanol, ethanol, tetrahydrofuran, acetonitrile, dimethylformamide, toluene, dimethyl sulfoxide, and combinations thereof.
6. The process as claimed in claim 1, wherein the predefined frequency of 15-20kHz is applied at the predefined amplitude power of 300-500W.
7. The process as claimed in claim 1, wherein the one of the organic extract and the aqueous extract is subjected to ultrasonic sound waves for the predefined duration of 1-90 minutes.
8. The process as claimed in claim 1, wherein the one of the organic extract and the aqueous extract is subjected to ultrasonic sound waves for the predefined duration with a predefined on or off pulse.
9. The process as claimed in claim 8, wherein the predefined duration is 8-10 minutes with the predefined on or off pulse of 5-8 seconds.
10. The process as claimed in claim 1, wherein the process for deriving the plurality of bioactive molecules from the at least one of plant material, mycological raw material, and combinations thereof is carried out at room temperature.
11. The process as claimed in claim 1, wherein the process for deriving the plurality of bioactive molecules from the at least one of plant material, mycological raw material, and combinations thereof is optionally carried out at a constantly maintained elevated temperature of ranging from 20 oC to 121 oC.
12. The process as claimed in claim 1, wherein the process further comprises subjecting the at least one of plant material, mycological raw material, and combinations thereof for a selective fractionation to obtain the plurality of bioactive molecules.
13. The process as claimed in claim 1, wherein the selective fractionation is performed by altering the organic solvent and water to obtain differentiated extract of the plurality of bioactive molecules comprising one of terpenoids, flavonoids, polyphenols, isoflavones, carotenoids, phytosterols, proteins, polysaccharides, peptides, lipopeptids, lipids, lipoproteins, glycoproteins, proteoglycans, glycosides, alkaloids, saponins, and combinations thereof.
14. The process as claimed in claim 1, wherein the process further comprises enriching the plurality of bioactive molecules with an extract of a group of herbs consisting of Ashwagandha, Pepper, Physilum Husk, Lavender, Beetroot, Lemon Grass, Bacopa, Clitoria, Benincasa, Celastrus, Acorus, Nardostachys, Panax, Rhodiola, Ginkgo, Centella, Ocimum, Curcuma, Withania, Astragalus, Schisandra, Lycium, Convolvulus, Tinosporia, Asparagus, Moringa, Mucuna, and Embelica.

Dated this 17th day of April 2023

Vidya Bhaskar Singh Nandiyal
Patent Agent (IN/PA-2912)
Agent for applicant