Natural extraction of cannabis-based plant compounds from cannabis plants by yeast fermentation

Abstract

A fermentation process for the extraction of cannabis-based plant compounds is described. According to the method, wine or beer yeast is used for fermenting large fried dough twists to obtain the cannabis sativa vinasse marinade rich in cannabis sativa-based plant compounds.

Description

Background Art Technical Field

[0001] The present disclosure relates to the natural extraction of cannabis-based plant compounds (e.g., cannabinoids, terpenes, and phenolic compounds) from fresh and dried cannabis plants by yeast fermentation.

[0002] Description of the Prior Art

[0003] The cannabis plant (including marijuana and hemp) contains varying amounts of cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), and tetrahydrocannabinol (THC), which are known to have beneficial psychoactive and medicinal effects. Other active components such as terpenoids and flavonoids are known to synergistically contribute to these beneficial effects in addition to providing flavor. The complex synergy between all the different components of cannabis is also known as the "entourage" effect.

[0004] Cannabinoid extracts are typically obtained by various extraction methods, including supercritical CO2, alcohol extraction, and butane or propane extraction, etc. However, some volatile or reactive compounds (including flavor terpenes) may degrade and be lost (e.g., by evaporation) or chemically change (e.g., degrade) to different substances under extraction conditions.

[0005] However, there is a need to develop natural botanical preparations, particularly whole plant preparations, that do not involve heating and do not involve harsh industrial chemical extraction methods. Summary of the invention

[0006] Disclosed herein is a natural extraction method that captures all of the active plant compounds present in whole plant cannabis flowers, thereby providing certain beneficial effects of the whole plant or involving harsh industrial chemical extraction methods. Specifically, the method involves progressively fermenting cannabis (e.g., whole plant cannabis flowers) such that one or more cannabinoids or other plant compounds are extracted from the cannabis and enriched in a "cannabis lees," which is a naturally occurring suspension culture medium containing fermentation byproducts such as dead yeast cells.

[0007] Thus, one embodiment provides a method for extracting cannabis-based plant compounds, the method comprising: providing a yeast starter comprising yeast (Saccharomyces), sugar and water; fermenting the yeast starter for a first period of time sufficient to provide a first fermentation medium, thereby the yeast proliferates; adding cannabis to the first fermentation medium to provide a second fermentation medium; and fermenting the second fermentation medium for a period of time to provide a cannabis brine comprising cannabis-based plant compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Exemplary embodiments are shown in the accompanying drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0009] Figure 1 A flow chart providing an overview of a fermentation process according to one embodiment is depicted.

[0010] Figure 2 A container of fermented cannabis flower is shown with brine forming at the bottom of the container. DETAILED DESCRIPTION

[0011] Disclosed herein is a method of combining fermentation and extraction to provide extracted cannabis-based plant compounds. The method provides an entourage effect with synergistic beneficial effects to consumers of the resulting fermented beverage by extracting a range of plant compounds such as cannabinoids, flavonoids and terpenes that are naturally present in a given cannabis plant, thereby retaining the entourage effect of ingesting the whole plant.

[0012] Advantageously, the fermentation process produces ethanol, which is used as an extraction medium to extract plant compounds from the cannabis plant (preferably the whole flower). In addition, as the fermentation proceeds, cannabis lees are formed. Cannabis lees are a suspension of byproducts of the fermentation process, which include complex natural molecules (e.g., proteins, fatty acids, polysaccharides, enzymes, etc.) from dead or decomposed yeast cells. It was surprisingly found that cannabis lees have significant solubilizing and emulsifying capabilities. The plant compounds extracted with ethanol are enriched and co-suspended in the cannabis lees, which can be easily separated from the rest of the fermentation broth.

[0013] like Figure 1 As shown, the natural extraction process (100) starts with a first fermentation step, in which yeast (e.g., Saccharomyeces cerevisiae) is rehydrated and activated with water and sugar to form a yeast starter (102). The first fermentation step (104) causes the yeast cells to proliferate and initially produce ethanol, resulting in a first fermentation medium (106). Typically, the first fermentation step is carried out aerobically to produce a robust yeast starter. Ethanol is also produced as a fermentation product. Typically, the ethanol content is less than 5% v / v of the first fermentation medium, or more typically less than 3% v / v. In some embodiments, the initial ethanol content is 0.1% v / v to 5% v / v, or more typically 1% v / v to 3% v / v.

[0014] Cannabis flower (108) is then added to the first fermentation medium (106) to provide a second fermentation medium (110). During the second fermentation step, ethanol is continuously produced, which provides an extraction medium. During fermentation, the ethanol content may be as high as 8%, more typically up to 5%. The cannabis (including fresh, dried or weathered flower, or decarboxylated flower) is fermented (112) under first aerobic and then anaerobic conditions for a sufficient period of time to allow for the natural extraction of a comprehensive broad spectrum of cannabis-based plant compounds (116).

[0015] Optionally, additional yeast (Saccharomyces cerevisiae) (114) can be added to the second fermentation medium, which can have a starting ethanol content of 1% to 3%. The additional brewer's yeast enhances alcoholic fermentation and increases the ethanol content, for example, to 4.5% to 8% (v / v). Higher ethanol content increases the efficiency of extracting terpenoids and cannabinoids from the cannabis.

[0016] As the second fermentation step proceeds, cannabis brine is formed and collected at the bottom of the container. The brine typically present in winemaking is a sediment of dead yeast cells and other particles that settle to the bottom of the container at the end of fermentation. Similarly, as used herein, "cannabis brine" refers to cannabis fermentation byproducts, such as dead or inactivated yeast cells and other fine particles or precipitates. Specifically, brine is composed of dead yeast cells and cellular components from decomposed yeast cells, including proteins (e.g., mannoproteins), polysaccharides (glucans), phenolic compounds, enzymes, fatty acids (e.g., phospholipids), and amino acids.

[0017] Thus, the cannabis brine is a heterogeneous suspension with solubilizing and emulsifying capabilities, which is rich in extracted cannabis plant compounds. In some embodiments, the extracted plant compounds can be further enzymatically converted to provide more active or more bioavailable compounds.

[0018] Thus, the cannabis fermentation produces a complex mixture of active cannabis-based plant compounds (e.g., cannabinoids, terpenes, and phenolic compounds) that are extracted from the cannabis flower, thereby retaining the entourage effect of ingesting the whole plant. Additionally, the second fermentation produces Saccharomyces cerevisiae enzymes that cause enzymatic hydrolysis of the cannabis-based plant compounds extracted from the cannabis flower to make these plant compounds more bioavailable. Specifically, the second fermentation decarboxylates approximately 50% of the inactive cannabinoids into bioactive cannabinoids (e.g., inactive CBD-a was found to be decarboxylated into bioactive CBD).

[0019] These and other aspects of the disclosure are described in more detail herein.

[0020] First fermentation step - yeast initiation culture

[0021] As used herein, a yeast starter refers to actively multiplying yeast cells in water with sugar as a food source for the yeast culture.

[0022] Suitable yeasts are wine yeast or beer yeast, including, for example, the genus Saccharomyces and any species thereof (e.g., S. cerevisiae; S. bayanus; S. boulardii; S. pastorianus; S. kudriavzevii; S. uvarum; S. eubayanus).

[0023] In a preferred embodiment, Saccharomyces cerevisiae is used. Saccharomyces cerevisiae is the most commonly used yeast species in food and beverage fermentation. Saccharomyces cerevisiae is also known as beer yeast or baker's yeast, and all strains thereof ferment sugars (as substrates) to produce ethanol and carbon dioxide.

[0024] Any type of sugar can be used, including, for example, sucrose. Typically, the amount of sugar is about 10 to 30 times the amount of yeast (by weight). More typically, the amount of sugar is about 15 to 20 times the amount of yeast (by weight).

[0025] Thus, a yeast starter culture is initiated by rehydration of Saccharomyces cerevisiae in filtered warm water (e.g., about 72° F. to 75° F.) The weight ratio of yeast to water may be in the range of 1% w / v to 5% w / v, more typically in the range of 1% w / v to 3% w / v.

[0026] The initial culture is the first stage of the yeast life cycle, which involves the growth of the yeast population. Once the yeast cell culture reaches a critical mass, alcohol fermentation is performed. The yeast starter is preferably placed in a dark environment at ambient room temperature (e.g., about 68°F to 78°F) for 24 to 48 hours to aerobically increase the yeast population exponentially with the potential beneficial effect of inhibiting the growth of harmful bacteria and mold.

[0027] During the first fermentation step, it is possible to initially produce ethanol. Typically, the amount of ethanol at this stage is about 0.1% v / v to 5% v / v. More typically, the ethanol content is about 0.1% v / v to 4% v / v, or even more typically 0.5% v / v to 4% v / v, 1% v / v to 4% v / v, 1% v / v to 3% v / v, 1% v / v to 2% v / v, etc.

[0028] Second Fermentation Step - Add Cannabis

[0029] After the first fermentation step is performed for 24 to 48 hours, cannabis flowers (e.g., dried weathered and / or fresh cannabis flowers) are added to the yeast starter to prepare a second fermentation medium. The amount of cannabis flowers (dry weight) can be 0.5 to 5 times (by weight), or more typically 1 to 3 times, the amount of yeast added in the first fermentation step.

[0030] After 3 to 5 days of aerobic fermentation, an airlock can be placed on the container containing the yeast starter and cannabis flower. The cannabis is then fermented aerobically with the yeast in a dark environment at ambient room temperature (e.g., about 68° F. to 78° F.) for 2 to 3 months.

[0031] The second fermentation medium is further fermented anaerobically under conditions and duration that are optimal for distributing the cannabis extract into the fermentation broth. As used herein, the cannabis extract can be any compound derived from the added cannabis plant material. Typically, cannabis flowers (including fresh, weathered or decarboxylated flowers) are added because they are rich in active plant compounds such as cannabinoids and terpenes.

[0032] After a fermentation period of 2 to 3 months, cannabis-based compounds from the cannabis are extracted and allowed to accumulate at the bottom of the container, forming a tan opaque layer. This tan opaque layer represents the cannabis brine (the brine co-suspended with cannabinoid compounds and other cannabis precipitates). The brine consists of dead yeast cells containing proteins and molecules such as mannoproteins, polysaccharides, fatty acids and amino acids. Without wishing to be bound by theory, it is believed that the fatty acids and proteins in the brine have significant solubilizing and emulsifying abilities, making it possible to extract and enrich lipophilic compounds such as CBD or THC.

[0033] A.Marijuana

[0034] Cannabis is a genus of flowering plants in the family Cannabaceae. Unless otherwise specified, cannabis refers broadly to any cultivated strain within the genus, including cannabis and industrial hemp.

[0035] Drug cannabis is a broad classification of many strains of cannabis that contain more than 0.3% (dry weight) of tetrahydrocannabinol (THC), the main psychoactive ingredient. Drug cannabis (or medical marijuana) is consumed primarily for its cannabinoid components, including THC and CBD, in an attempt to treat a disease or improve symptoms. Marijuana is used to reduce nausea and vomiting during chemotherapy, improve appetite in HIV / AIDS patients, and treat chronic pain and muscle spasms. The potential for cannabinoids to affect stroke is in preliminary research. Although the evidence is inconclusive, drug cannabis has been tried to treat depression, anxiety, attention deficit hyperactivity disorder, Tourette syndrome, post-traumatic stress disorder, and psychosis.

[0036] Certain strains of the cannabis plant are called industrial hemp, but the term is generally used to refer only to varieties of cannabis cultivated for non-medicinal uses (i.e., having a THC content of 0.3% or less by dry weight). Industrial hemp has long been used for medicinal purposes for its hemp fiber, hemp seeds and their oil, and hemp leaves. Industrial hemp products are made from hemp plants that have been selected for their high fiber production.

[0037] Of the more than 100 cannabinoids identified in the cannabis plant, phytocannabinoids primarily include cannabidiol (CBD), tetrahydrocannabinol (THC), cannabigerol (CBG). Although structurally different, cannabinoids all act on cannabinoid receptors (e.g., CB1 and CB2 receptors), which are located throughout the body and are involved in appetite, pain perception, mood, and memory.

[0038] Tetrahydrocannabinol, or THC, is the main psychoactive cannabinoid component of marijuana. THC acts on CB1 receptors, primarily in the brain, and provides the "high" psychoactive effects that users experience. THC is also used to treat conditions such as pain, muscle spasms, glaucoma, insomnia, loss of appetite, nausea, and anxiety.

[0039] In the cannabis plant, THC exists primarily as non-psychoactive tetrahydrocannabinolic acid (THC-a). THC-a can be converted to THC by decarboxylation, which is usually performed by heating. However, the fermentation process described herein simultaneously causes an enzymatic reaction that decarboxylates THC-a to produce THC.

[0040] "Cannabidiol" or "CBD" is a non-psychoactive cannabinoid. When combined with THC, CBD exhibits some modulatory properties to reduce adverse THC effects. CBD is also known to have its own therapeutic properties, particularly for the treatment of epilepsy and more recently autism. Due to CBD's antidepressant and neuroprotective effects, it may also benefit those who experience nausea, inflammation, and anxiety.

[0041] In industrial hemp plants, CBD is primarily present as cannabidiolic acid ("CBD-a"), the carboxylic acid form of CBD. Like THC-a, CBD-a can also be converted to CBD by decarboxylation via the enzymatic reactions that occur during fermentation as described herein.

[0042] Plant terpenes are naturally occurring hydrocarbons with different structures. Although terpenes are present in small amounts (less than 5% by weight) of the plant mass, they impart a powerful and desirable flavor to plant extracts. It is believed that terpenes may also modulate the pharmacological effects of cannabinoids.

[0043] Phytocannabinoids and terpenes accumulate in the secretory cavity of glandular hairs and are primarily found in cannabis flowers (typically flowers of female plants). Therefore, the methods disclosed herein preferably utilize cannabis flowers, including fresh or weathered (dried) flowers. During the second fermentation step, cannabinoids and terpenes are extracted.

[0044] The cannabis flower can be weathered (dried) by methods known in the art for use. Alternatively, decarboxylated cannabis flowers can be used. For example, heating or baking dried cannabis flowers at about 100° C. to 120° C. for 60 to 90 minutes can effectively convert CBD-a and THC-a into CBD and THC, respectively.

[0045] B. Fermentation Conditions

[0046] The second fermentation is carried out aerobically for 2 to 3 days and then anaerobically for 2 to 3 months. During this period, the metabolites produced by the yeast form an alcohol extract that selectively separates phytocannabinoids and terpenes from the cannabis flower. In particular, many cannabinoids have one or two phenolic moieties that can be protonated under alcoholic conditions.

[0047] The second fermentation produces Saccharomyces cerevisiae enzymes that cause enzymatic hydrolysis of the cannabis-based plant compounds extracted from the cannabis flower to make these hydrophobic plant compounds more bioavailable. The yeast enzymatic hydrolysis allows micelles to be formed from the hydrolyzed lipid plant compounds, thereby facilitating the bioaccessibility of the cannabis-based hydrophobic plant compounds.

[0048] During the second anaerobic fermentation, ethanol is continuously produced by the yeast. Although the second fermentation medium starts with an ethanol content of 1% to 3% (v / v), when additional wine / beer yeast is added, the ethanol can be as high as 5% to 8% (v / v) during the second fermentation step. The increase in the amount of ethanol promotes the extraction of various active components of the cannabis flower.

[0049] In more specific embodiments, cannabis flowers (fresh, weathered or decarboxylated) are added at 2 g / liter to 10 g / liter of the second fermentation medium. The fermentation is typically carried out under anaerobic conditions at ambient room temperature (e.g., about 68°F to 78°F) for a sufficient period of time (e.g., 2 to 3 months).

[0050] Enrichment and isolation of extracted cannabis-based plant compounds

[0051] The enzymatic hydrolysis of the yeast cells at the end of the fermentation results in a similar enzymatic hydrolysis of the nutrient-rich cannabis plant cells, which then releases many cannabinoid plant compounds and emulsifies them to increase bioavailability. The dead yeast cells and the cannabis-based plant compound precipitate will settle to the bottom of the final fermentation broth to form a cannabis brine (brine mixed with cannabinoid compounds). The cannabis brine contains the cannabis-based plant compounds of the whole cannabis plant to retain the entourage effect. The cannabis brine can be analyzed by methods known in the art to determine the amount of terpenes and cannabinoids present.

[0052] In other embodiments, cannabis flower containing a significant percentage (10% to 20%) of THC rather than low-THC cannabis flower may also be used to ferment and produce THC-rich cannabis brine.

[0053] Example

[0054] The present disclosure is further illustrated by the following examples. The following examples are non-limiting and merely represent various aspects of the present disclosure.

[0055] Example 1

[0056] Fermentation of industrial hemp flowers

[0057] Cannabis flowers are fermented according to the two-step process disclosed herein.

[0058] The yeast starter culture is initiated by rehydrating 11.5 g of dried Saccharomyces cerevisiae (brew yeast) with 500 ml of filtered warm water (e.g., 72°F to 75°F) and 220 g of sugar in a conical flask (1000 ml) or similar container. Once the yeast reaches a critical mass, alcohol fermentation is performed. The yeast starter is placed in a dark environment at ambient room temperature (e.g., about 68°F to 78°F) for 24 to 48 hours to increase the yeast population through aerobic fermentation.

[0059] After 24 to 48 hours, 10 g of cannabis flower (dried and weathered) is added to the yeast starter and fermented aerobically for 3 to 5 days. After 3 to 5 days, an airlock stopper is placed on a conical flask (1000 ml) or similar container. The cannabis is fermented aerobically with yeast in a dark environment at ambient room temperature (e.g., about 68°F to 78°F) for 2 to 3 months. The flask is shaken regularly to promote mixing during this period.

[0060] Figure 2The fermentation product obtained in a conical flask (200) after a second fermentation period of 2 to 3 months is shown. As shown, the remaining cannabis flower (210) floats on the top; the water / alcohol layer (220) is in the middle; and the brown opaque layer (230) settles to the bottom of the conical flask, which is the "cannabis brine". The cannabis brine is separated from the remaining fermentation liquid by pipetting, and its contents are quantitatively analyzed.

[0061] Table 1 shows the amount of CBD and CBD-a in fermented samples taken from cannabis brine marked at 1 month and 2 months. As shown in the figure, not only is CBD-a present in the cannabis brine with an increasing trend, but also CBD is formed due to decarboxylation.

[0062] The water / alcohol layer was also tested but showed no CBD or CBD-a. This suggests that the phytocannabinoids extracted by ethanol are ultimately concentrated in the cannabis brine, which is believed to be more solubilizing than ethanol.

[0063] As a control, CBD was also extracted directly (without fermentation) from industrial hemp flowers using water or methanol. As shown in Table 1, water extraction produced only a small amount of CBD-a but no CBD; while methanol (industrial chemical) extraction produced a larger amount of plant compounds from industrial hemp flowers.

[0064] Table 1

[0065]

[0066] ND = not detected; n / a = not analyzed; LOQ = limit of quantification; Total THC = (0.877 x THC-A) + Δ9-THC;

[0067] Total CBD = (0.877 x CBD-A) + CBD. *Δ8THC has lower precision and higher detection limit than other cannabinoids

[0068] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet are incorporated herein by reference in their entirety. If necessary, various aspects of the embodiments can be modified to provide additional embodiments using the concepts of various patents, applications, and disclosures.

[0069] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full range of equivalents to which these claims are entitled. Therefore, the claims are not limited by this disclosure.

[0070] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 304,518, filed on January 28, 2022, the entire contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for extracting cannabis-based plant compounds, the method comprising: providing a yeast starter comprising yeast, sugar, and water; allowing the yeast starter to ferment for a first period of time sufficient to provide a first fermentation medium, whereby the yeast proliferates; adding cannabis to the first fermentation medium to provide a second fermentation medium; and The second fermentation medium is fermented for a period of time to provide cannabis brine comprising cannabis-based plant compounds.

2. The method according to claim 1, wherein the yeast is Saccharomyces cerevisiae.

3. The method according to claim 1 or claim 2, wherein the amount of the sugar is 10 to 30 times the weight of the yeast, or preferably 15 to 20 times the weight of the yeast.

4. The process according to any one of the preceding claims, wherein the first fermentation medium has an ethanol content of 0.1% to 5% (v / v).

5. The method of any one of the preceding claims, wherein the first period of time is about 24 hours to 48 hours.

6. The method of any of the preceding claims, wherein the yeast starter is fermented at a temperature of about 72°F to 75°F.

7. The method of any one of the preceding claims, wherein the cannabis comprises flowers of industrial hemp or cannabis sativa.

8. The method according to any one of the preceding claims, wherein the dry weight of the cannabis is 0.5 to 5 times the amount of the yeast (by weight), preferably the dry weight of the cannabis is 1 to 3 times the amount of the yeast (by weight).

9. The process according to any one of the preceding claims, wherein the second fermentation medium is fermented aerobically for up to 5 days and subsequently anaerobically for at least 2 to 3 months.

10. A method according to any one of the preceding claims, wherein the cannabis is industrial hemp and the cannabis brine contains phytocannabinoids including CBD, CBG, CBD-a or mixtures thereof.

11. A method according to any one of the preceding claims, wherein the cannabis is narcotic cannabis and the cannabis brine contains phytocannabinoids including THC, THC-a or mixtures thereof.

12. The method of any one of the preceding claims, wherein the cannabis brine further comprises phospholipids.

13. The method of any one of the preceding claims, further comprising separating the cannabis brine.

14. A cannabis brine prepared by the method according to any one of claims 1 to 13.

15. A beverage comprising the cannabis brine according to claim 14.

16. A food material comprising the cannabis brine according to claim 14.

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