method for the preparation of a mycelial coffee product and mycelial coffee product prepared by said

专利摘要:
MICELLIED COFFEE PRODUCTS AND METHODS TO PRODUCE THEM. The present invention relates to a method for preparing a coffee product. This method comprises providing the green coffee beans and sterilizing the green coffee beans to provide prepared green coffee beans, and a step of inoculating the green coffee beans prepared with a prepared fungal component and cultivating the inoculum to prepare the coffee product. mycelia. The methods of the present invention result in prepared green coffee beans and mycelia coffee products that have reduced levels of undesirable taste components, such as chlorogenic acids, and higher levels of mycelia products, such as beta glucans and polysaccharides, compared to broad beans. green coffee initials.
公开号:BR112015023652B1
申请号:R112015023652-9
申请日:2014-03-15
公开日:2020-12-01
发明作者:Brooks John Kelly；James Patrick Langan
申请人:Mycotechnology, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] Provisional Order No. U.S. 61 / 802,256, filed March 15, 2013 and entitled "Myceliated Agricultural Substrates for Production of Nutraceutical Formulations and Functional Foods"; U.S. Patent Application 13 / 844,685, filed March 15, 2013 and entitled "A Method for Myceliating Coffee (amended)"; U.S. Patent Application 13 / 859,719, filed April 9, 2013 and entitled "Method of Myceliation of Agricultural Substrates for Producing Functional Foods and Nutraceuticals"; U.S. Patent Application 13 / 874,832, filed May 1, 2013 and entitled "Extract of a Myceliated Agricultural Substrate and it's Use as a Nutraceutical Composition"; Provisional application in U.S. 61 / 844,498, filed July 10, 2013 and entitled "Method for Manufacturing Coffee by Fermentation"; Provisional Application in U.S. 61 / 396,863, filed July 23, 2013 and entitled "Coffee Decafeination Process Including Fungal Components"; Provisional Application in U.S. 61 / 866,371, filed August 15, 2013 and entitled "Cocao Beans Including Metaboloites of 2-Methoxy-3- Isopropylpyrazine and Method for Metabolizing 2-Methoxy-3- Isopropylpyrazine in Cocao Beans"; Provisional Application in U.S. 61 / 867,501, filed August 19, 2013 and entitled "Cocao Beans including Methyl Pyrazines Produced by Fermenation, and a Method of Increasing Methyl Pyrazines in Cocao"; U.S. Patent Application 14 / 020,512, filed September 6, 2013 and entitled "Improved Method for Myceliating Raw Coffee Beans Including Removal of Chlorogenic Acids"; U.S. Patent Application 14 / 020,781, filed September 6, 2013 and entitled "Method for Myceliating Raw Coffee Beans Including Removal of Chlorogenic Acids"; Provisional Order No. 61 / 878,037, filed on September 15, 2013 and entitled "Myceliating Green Coffee Beans to Lessen the Tooth-Staining Impact of Coffee Melanoidins" and Provisional Order No. 61 / 896,097, filed on October 27, 2013 and entitled "Cocao Myceliation in an Aqueous Solution"; all of which are incorporated in their entirety into this document as a reference. TECHNICAL FIELD
[0002] The methods and products refer to the use of fungal strains to improve the taste on agricultural substrates, in particular coffee. BACKGROUND
[0003] The coffee was mixed or infused with fungal components like dried mushroom powders or extracts. These mixtures have the benefit of providing additional nutritional value derived from polysaccharides. However, this method of mixing fungal components does little to improve the taste of coffee.
[0004] U.S. Patent Publication 20100239711 A1 to Pei-Jung Let et al. describes a method of making coffee by solid state fermentation using fungal mycelium. It is a natural process. Untreated green coffee is deposited in a dust-free container, the coffee is inoculated with a fungal strain, performed to perform a sterile operation to implant fungi in the coffee beans, and a fungal fermentation process is started. Anthrodia Camphorate, a fungal strain native to Taiwan, is used. The process lasts between 15 to 60 days. The entire disclosure of U.S. Patent Publication 20100239711 is incorporated herein by reference in its entirety.
[0005] Many strains of fungi can coexist with chlorogenic acids, but the presence of chlorogenic acids reduces the ability of these fungal strains to optimally metabolize various substrates, including green coffee beans.
[0006] A portion of the coffee market is represented by decaffeinated coffee. Current sets of caffeine extraction procedures include pre-humidifying the beans to a moisture content of 40% or more, then extraction with a water-immiscible organic solvent, and after decaffeination, the residual solvent is removed from the broad beans (desolventization), usually by steam pickling.
[0007] A disadvantage to these caffeine removal processes is that there is some flavor removal of precursor flavor and aroma compounds, especially water-insoluble waxes, during decaffeination of green beans.
[0008] A need remains in the art for improved coffee products that have reduced levels of undesirable taste components and / or higher levels of flavor and / or health-promoting components compared to green coffee beans, and for methods of obtaining these. products. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 is a flow chart of a method of creating a mycelial agricultural product according to the present invention.
[00010] Figure 2 is a flow chart of a method of creating an extract of mycelial agricultural product for human consumption to effect neuroregeneration and neuroprotection in humans in accordance with the present invention.
[00011] Figure 3 is a flow chart of a method of removing caffeine from green coffee beans according to the current invention.
[00012] Figure 4 is a flow chart of a method of creating a mycelial coffee product.
[00013] Figure 5 is a flow chart of a method of creating a mycelial coffee product.
[00014] Figure 6 is a conveyor used in accordance with the present invention.
[00015] Figure 7 is a conveyor used in accordance with the present invention to deliver containers to an autoclave.
[00016] Figures 8 (A), 8 (B), 8 (C) show autoclavable bags that have a rolled surface, a stack of autoclavable bags and a rolled autoclavable material. SUMMARY OF THE INVENTION
[00017] In one embodiment, the present invention provides a method for preparing a mycelial coffee product. This method includes the step of providing prepared green coffee beans, which includes providing green coffee beans and sterilizing green coffee beans or other agricultural substrate to provide prepared green coffee beans. The method also includes the step of providing a prepared fungal component. The method also comprises inoculating the green coffee beans prepared with the prepared fungal component and cultivating the inoculum to prepare the mycelial coffee product.
[00018] In one embodiment, the method includes reducing the amount of unwanted taste components. The reduction of an amount of undesirable flavor components optionally includes at least one or two aqueous extractions of green coffee beans. An undesirable taste component includes chlorogenic acid.
[00019] In one embodiment, the method includes a step of hydrating green coffee beans, optionally to about 60%. The methods of the present invention also include where the prepared fungal component is G. lucidum, optionally, strain of G. lucidum 806, C. sinensis and / or T. melanosporum. The methods of the invention include screening various strains of fungi and selecting a strain that has an enhanced ability to grow in, metabolize or use green coffee beans and / or selecting a strain that has enhanced ability to remove one or more components of undesirable taste of green coffee beans and / or enhanced caffeine removal from green coffee beans.
[00020] In another embodiment, the prepared fungal component is kept in an undefined medium that comprises a watery green coffee bean extract and an energy source. The maintenance of the fungus strain causes an adaptation of the fungi resulting in an increase in the ability of the fungi to proliferate, metabolize or use green coffee beans.
[00021] The methods of the invention also include where the culture step is a fermentation step performed under semianaerobic conditions, optionally for about 7 days.
[00022] The methods of the present invention result in mycelial coffee products that have reduced levels of undesirable taste components, such as colloid acids, and increased levels of mycelial products, such as beta glucans and polysaccharides, in relation to green coffee beans.
[00023] Green coffee beans can come from Coffea Arabica or Coffea robusta.
[00024] The present invention includes a mycelial coffee product and green coffee beans prepared using the methods of the invention and a mycelial coffee product that has reduced levels of undesirable taste components and increased levels of mycelial products in relation to broad beans of green coffee.
[00025] The methods also include a method for reducing the levels of undesirable taste components in green coffee beans and a method for increasing the levels of mycelial products in green coffee beans as described in this document. DETAILED DESCRIPTION OF THE INVENTION
[00026] In one embodiment, the present invention provides a method for preparing a mycelial coffee product. This method includes the step of supplying prepared green coffee beans, which includes providing green coffee beans and sterilizing green coffee beans to provide prepared green coffee beans. The method also includes the step of providing a prepared fungal component. The method also comprises inoculating the prepared green coffee beans with the prepared fungal component and cultivating the prepared green coffee beans and the prepared fungal component to allow mycelia to produce the mycelia coffee product. These steps can be performed in any order.
[00027] In one embodiment, prepared green coffee beans are provided, including the stage of supplying green coffee beans. Coffee refers to the genus Coffea, which is a genus of flowering plants whose seeds, called coffee beans, are used to make coffee. The same is a member of the Rubiaceae family. Coffee beans can be selected from one of several varieties of coffee that are different cultivars derived through reproduction or natural selection of coffee plants. Coffee beans of the same variety grow in different places and may have different characteristics such as flavor (the flavor criteria include terms like "citrus type" or "earthy"), caffeine content, touch or taste, and acidity. The green coffee beans useful for the present invention can be any coffee species, including Coffea arabica and Coffea robusta (also known as Coffea conephora); additional coffee species useful for the present invention include Coffea benghalensis or Bengal coffee; Coffea congensis or Congo coffee; Coffea liberica or Liberian coffee; Coffea stenophylla or Sierra Leone coffee; Coffea excelsia, another Liberian coffee; Coffea bonnieri; Coffea gallienii and Coffea mogeneti. The invention includes any varietals or strains of the species listed above. For example, many varieties of the Arabica type are named by the country or region in which they are predominantly found or in which they originate. Some of the exemplary Arabica coffee varieties include Typica, Bourbon, Caturra, Catuai, Mundo Nova and Blue Mountain. Also included in the invention are and coffee-derived species including any genetically modified (GMO) strains or cultivars and also any variety of (non-GMO) heirloom coffee strains or cultivars.
[00028] A green coffee bean refers to a raw unroasted coffee bean. In general, the raw fruit of the coffee plant is called a coffee cherry. To prepare the green coffee bean, in general, the coffee cherry has the fruit or pulp removed and the seed, or fruit, is then subjected to drying. The fruit and pulp can be removed from the green coffee bean using various methods known in the art and include a wet process in which the fruit / pulp is removed from the coffee bean before drying, and a dry process in which all cherries are dried before mechanical peeling, separation, classification and packaging. The dried coffee beans useful for the present process may include dried coffee beans that are fresh or may undergo an aging process. Dried coffee beans can be stored in sackcloth bags, lined sackcloth bags or in vacuum-sealed containers before entering the present process.
[00029] In some embodiments, the green coffee bean is not subjected to drying before being used in the processes of the present invention. In this modality, after the green coffee bean is harvested, the green coffee bean has the pulp removed by any process known in the art and then can be used in the present invention without additional treatment of green coffee bean, such as drying. In this modality, the hydration and / or washing step as described below is not necessary or is avoided through the use of green coffee beans not subjected to drying. HYDRATION AND WASH STEP
[00030] In one embodiment, green coffee beans are prepared for use in the present methods of the invention, resulting in a prepared green coffee bean.
[00031] In some modalities, green coffee beans are prepared by a hydration stage of green coffee beans. Hydration is particularly useful where green bean beans have been subjected to drying. Hydration ensures that green coffee beans have an ideal moisture content for the growing process (mycelia). Hydration can be accomplished by a number of methods known in the art.
[00032] Hydration can be carried out by an aqueous medium. The aqueous medium includes water and, optionally, additional excipients. Water can be deionized, tap, distilled or mineralized. Other excipients can be added to the water, such as buffers to maintain a certain pH, sodium chloride, citric acid and / or ascorbic acid. The pH can be neutral or adjusted. The temperature of the aqueous medium can be room temperature or high in temperature to accelerate the hydration process.
[00033] Hydration can be carried out by allowing the green coffee beans to be soaked in the aqueous medium for any appropriate length of time, ranging from a few seconds or less to an entire night. The immersion step for hydration and / or the aqueous extraction step can take less than one second, at least five seconds, at least ten seconds, at least thirty seconds, at least one minute, at least five minutes, at least ten minutes, at least twenty minutes, at least thirty minutes, at least forty minutes, at least fifty minutes, at least one hour, at least one and a half hours, at least two hours, at least two and a half hours, at least three hours at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours or at least fifteen hours, at least eighteen hours, at least twenty four hours, at least thirty-six hours, or at least forty-eight hours. However, the time for the hydration step must be selected in view of the fact that the green coffee beans are not sterile and are not immersed for a long time, which can encourage the growth of unwanted organisms.
[00034] Green coffee beans can be hydrated at any temperature that provides effective hydration. In one embodiment, the temperature of the aqueous component is room temperature. The hydration temperature must be selected in view of the fact that at high temperatures, the components of desirable taste can be changed.
[00035] The moisture content of hydrated green coffee beans is optionally between about 20% and about 80% moisture content, between 40% and 70% moisture content. In one embodiment, the moisture content is at least about 30%, at least about 50%, or at least about 60%.
[00036] The hydration step can take place in a container. In one embodiment, the container is a drum, such as a 0.24 m2 (55 gallon) drum. In this modality, the volumetric ratio of 1: 1 (equal volumes) between green coffee beans: aqueous component was allowed to rest for 5 to 24 hours. For example, the 0.24 m2 (55 gallon) drum is half loaded with green coffee beans and an aqueous medium is added to fully load the drum. In this mode, green coffee beans can absorb the entire aqueous component. In another mode, cocoa beans or other agricultural substrate, kept in a container, can be loaded with water to completely submerge the beans, in one mode, without significant excess.
[00037] In another modality, green coffee beans that have been degummed but have not yet been dried, which have, on average, a moisture content of 60%, which can be used. This method avoids the hydration step.
[00038] In one embodiment, the step to supply prepared coffee beans optionally includes a step to remove unwanted taste components by washing or rinsing the green coffee beans. The wash or rinse can be the aqueous medium, as described above. In one embodiment, green coffee beans are optionally washed or rinsed before, during, or after the optional hydration step. The washing, drying and / or rinsing of green coffee beans can be carried out by any method known in the art. Green coffee beans can be washed once, at least twice, at least three times, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least fifty times or more. In one embodiment, the washing step is performed twice. The washing or rinsing step may include optional soaking times, as described in this document.
[00039] In one embodiment, the green coffee beans are washed by a method for filling in a container that holds the green coffee beans with water, allowing the water to be immersed from 10 seconds to 4 hours, draining the water and repeating the beans as many times as desired, or to raise the beans to the desired mixing level. The washing and rinsing step can also be carried out until the green coffee beans have had a certain amount of undesirable taste component removed.
[00040] The broad beans of green coffee can be washed at any temperature that allows the efficient extraction of components of unwanted taste; in one embodiment, the temperature of the aqueous medium temperature is ambient. The washing temperature must be selected taking into account the fact that components with a pleasant taste can be altered, destroyed and / or extracted at elevated temperatures.
[00041] In another embodiment, the aqueous medium or excessive component is removed and / or separated and / or drained from the hydrated coffee beans after the hydration stage. This step can also be referred to as an aqueous extraction step. This step can be done to remove unwanted taste components.
[00042] Most components of coffee include caffeine, minerals, tannic acid, cellulose, water, fat, protein and fiber. Coffee contains methylxanthine, such as caffeine, theophylline, and theobromine, flavonoids, phenols, phenolic acids, volatile alkaloids, non-volatile alkaloids. Coffee also contains some components of unwanted taste. These components will contribute to the perception of a bitter and / or sour taste for coffee. These tastes are usually attenuated by the addition of sugar or cream to mask the bitterness of the components. The bitterness and / or reduced bitterness can be noticed in more premium, more expensive coffee varieties, such as Arabica coffee.
[00043] The hydration step, aqueous extraction step, washing and / or rinsing step can optionally reduce and / or remove unwanted taste components from green coffee beans and can be carried out as described in this document until the amount of undesirable taste component has been removed from the green coffee beans.
[00044] Green coffee beans (and roasted conventional coffee beans) are known to contain several components that contribute to an acrid and bitter taste. These flavor components are considered to be undesirable in coffee. One of these components is chlorogenic acid which is an ester of caffeic acid and quinic acid and can be toxic as an acrylic acid derivative. During roasting, coffee smoke is toxic to the respiratory system. Chlorogenic acids contribute to a bitter and / or sour taste in coffee and coffee products, and can be chlorogenic acid for humans, mammals, and various strains of fungus.
[00045] Chlorogenic acid has a green color and its presence in the green coffee beans contributes to the green color of the beans. Optionally, even when the steps are taken to remove chlorogenic acid while creating the prepared green coffee beans, at least a certain amount of chlorogenic acid is left in the prepared green coffee beans, as it contributes to the characteristic flavor of coffee. In some modalities, at least some chlorogenic acid is retained in the green coffee beans. Chlorogenic acid has been reported to carry certain health benefits, particularly in its ability to mediate insulin levels and thus assist in fat metabolism. It has also been identified as a powerful antioxidant. Chlorogenic acid is converted to a lactone after roasting as a result of dehydration in the chinic acid portion. Lactone can be incorporated into macromolecules known as melanoidins, molecules in which the mass is above 25,000 kD and are the result of several and subsequent Maillard reactions (any of the carbonyl or hydroxyl groups in the lactone would serve as nucleophiles in this reaction), making the same less bioavailable than in pure form. In addition, extensive roasting (ie dark roasting) will degrade the lactone in the hydroxylated phenylindanes. Therefore, the roasted mycelia product may not contain chlorogenic acid, instead it may contain lactone. The biofunctionalities of chlorogenic acid lactone are much less understood. Chlorogenic acid has also been implicated as an important flavor in the taste profile of coffee.
[00046] If quantitative removal of chlorogenic acid from green coffee beans is desired, green coffee beans can be autoclaved in excess water; for example, 0.45 kg (1 lb) of green coffee beans can be mixed in 3 l of water and autoclaved in a liquid cycle for 30 to 80 minutes, resulting in white beans. However, quantitative removal of chlorogenic acid is not preferred.
[00047] Without being limited by theory, the inventors believe that the chlorogenic acid of green coffee beans can reduce the proliferation of at least some of the fungal strains of the present invention and may additionally interfere with some or all of the processes of the present invention. through the interference or reduction of fungus proliferation and / or the ability of the fungus to metabolize, or mycelia the green coffee beans. When chlorogenic acids are not removed from green coffee beans, the highest mixing component (eg 60%) of green coffee beans is preferred. The removal of at least part of the chlorogenic acid allows the stage of culture and mycelia to occur at a lower moisture content, such as 30% and higher. The inventors attribute this to 20% of the cellulose content of coffee.
[00048] In some modalities, as optionally measured by the intensity and / or presence of the green color of the green coffee beans, the step of aqueous extraction, washing and / or rinsing is carried out until about 5% of the chlorogenic acids are removed; in other modalities, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65 %, up to 70%, up to 75%, up to 80%, up to 85%, up to 90% or up to 95% of the chlorogenic acids are removed in the processes of the present invention. In some embodiments, about 25% to about 80% of the chlorogenic acids are removed. In one embodiment, about 45 to 50% of the chlorogenic acids are removed.
[00049] Other components of undesirable taste that contribute to a bitter taste in coffee include quinic acid, 5-hydroxymethylfurfural, 2-methylfuran, furfuryl alcohol, trigonelline, caffeic acid, citric acid, malic acid, lactic acid, pyruvic acid, acetic acid , pyrazine, thiazole, quinolone, phenylpyridine, caffeine, among others. Robusta coffee contains higher levels of both caffeine and chlorogenic acids, which leads to greater bitterness and astringency in Robusta coffee. Additional unwanted taste components may include one or more of theophylline, theobromine, paraxanthin, liberin, methyliberine, trigonelline (N-methylnicotinate); hydrophobic amino acids such as isoleucine, leucine, valine, tyrosine, phenylalanine, gamma-aminobutyric acid; diceethopiperazines such as cyclo (proline-proline), cyclo (prolinaleucine), and cyclo (proline-isoleucine); acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid; nonanoic acid; decanoic acid and derivatives of such fatty acids; valeric-3-methyl, acetaldehyde, propanal, butanal, pentanal acid; carboxylic acid-5-hydroxytryptamides with an amide bond to fatty acids (C6 to C24 unsaturated); triglyceride linoleic acid, palmitic acid and related esters; diterpenes that include cafestol, kahweol, 16-O-methyl-cafestol, cafestal and kahweal; chlorogenic acids; polyphenols and chlorogenic acids such as ferulic acid and dimethoxycinnamic acid-3,4, which are connected by an ester bond to the hydroxyl groups of quinic acid. Other components with an unpleasant and / or bitter taste include chlorogenic acid lactones and lactone breakdown products such as phenylindanes. One or more of the compounds named above can be reduced and / or removed by the methods of the invention.
[00050] In some embodiments, the determination of the extent of removal of at least one component of undesirable taste is determined by the appearance, taste and / or chemical composition (by methods known in the art) of the mycelial coffee product. Alternatively, the appearance or chemical composition of green coffee beans can be determined by known methods. This determination can be quantitative, for example, the chemical composition of the mycelia coffee product can be measured by test methods or determined qualitatively by the taste test by well-versed people.
[00051] In a modality, up to 5% of an or, but among the components of undesirable taste are removed; in other modalities, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65 %, up to 70%, up to 75%, up to 80%, up to 85%, up to 90% or up to 95% of one or more of the undesirable flavor components are removed in the processes of the present invention. In one embodiment, one or more of the undesirable flavor components are removed quantitatively.
[00052] The removal of components of undesirable taste may increase the value of low quality coffee and / or make it more drinkable. Mycelial coffee products produced by this method can be used to mix with less expensive coffee beans which leads to a lower cost product that has improved taste properties. The amount of sugar, milk and milk substitutes to be added to coffee can be reduced. The present methods lead to the enhanced taste profile of mycelia coffee products due to the perception that mycelia coffee products provide a richer, softer and / or sweet coffee with less bitter, unpleasant and / or acidic tastes. Green coffee beans and mycelia coffee beans that have been subjected to hydration and / or aqueous extraction steps as described above will have reduced amounts of chlorogenic acid. Mycelial coffee products of the present invention will demonstrate enhanced taste. In one embodiment, the enhanced taste results from the removal and / or reduction of compounds with a bitter taste such as chlorogenic acid in mycelium coffee beans.
[00053] In one embodiment, the reduction of desirable flavor components such as volatile oils is minimized by the processes of the present invention. When processing green beans from Robusta coffee, the technique teaches how to steam, extract or steam distill the beans before roasting, which can remove many desirable volatile oils from Robusta coffee beans. The processes of the present invention prevent the step of steaming the Robusta coffee beans and thereby help to preserve the desirable volatile oils that contribute to the flavor of the coffee.
[00054] In an optional step, the coffee beans can be decaffeinated by conventional processes before, subsequently or in addition to the methods of the present invention. HEAT TREATMENT
[00055] The methods of the present invention optionally further comprise a method of heat treatment such as pasteurization and / or sterilization of green coffee beans. In one embodiment, green coffee beans are sterilized to provide prepared green coffee beans. This step can be achieved by any method known in the art. For example, this step can be performed under atmospheric pressure or under increased pressure. This step can also be called "pre-processing". This step is performed to reduce or remove undesirable fungal or microbial contaminants in green coffee beans.
[00056] Methods for pasteurization and / or sterilization can be performed as known in the art. As an example of pasteurization, green coffee beans can be subjected to dry heat treatment with atmospheric pressure from 62 ° C (145 ° F) to 87 ° C (190 ° F) for 30 to 90 minutes, alternatively from 60 ° C (140 ° F) to 98 ° C (210 ° F) for 20 to 100 minutes.
[00057] The sterilization of green coffee beans can be performed as known in the art. For example, green coffee beans can be sterilized by heating under pressure to 0.1 MPa (15 pounds / inch2) from 121 to 122 ° C for 20 to 100 minutes, such as 90 minutes. Other examples of suitable sterilization conditions include sterilization from 108 to 109 ° C to 0.03 Mpa (5 pounds / inch2), from 115 to 116 ° C to 0.07 Mpa (10 pounds / inch2), for 20 to 90 minutes . In another mode, the steam is superheated from 121 to 123 ° C (250 to 255 ° F). Pressures can range from 0.03 to 0.17 Mpa (5 to 25 pounds per square inch). Green coffee beans can be sterilized in a container. The container can optionally be the same as the container used for the aqueous extraction and / or hydration step. The container can be optionally sealed and the green coffee beans can be sterilized by applying heat to the outside of the container. In one embodiment, heating is provided by applying steam to the outside of the container for a period of time sufficient to allow the contents to be sterilized.
[00058] The sealed container of some modalities can provide some advantages. For example, sealing the container minimizes the outflow of flavor components and aromatic components from the green coffee beans, which can be perceived by the lack of coffee aroma from water vapor from the pressure cooker or autoclave. during the sterilization process. The seal also prevents the aromatic and flavor components soluble in water from escaping from the green coffee beans directly into the water vapor, hot air or heated water.
[00059] Suitable containers include containers known in the art for mushroom cultivation. Optionally, the containers have a section to exchange air or gases, but do not allow the passage of any other component. Such sections are known in the art and include filter strips. In one embodiment, the container is a drum, for example, a 0.24 m2 (55 gallon) drum.
[00060] In some embodiments, the containers of the present invention can be glass, stainless steel, polyethylene or high density polypropylene bags, thermoset. Fermenters and bioreactors can also be used as containers for the present invention. In some embodiments, the containers have a means for exchanging gas that prevent the passage of contaminants, such as zones or filter valves.
[00061] In one embodiment, the container is a bag, for example, a polypropylene bag capable of autoclaving with filter strips, a high density polyethylene bag capable of autoclaving with filter zones and a bag of gamma-irradiated polyethylene with filter zones.
[00062] An additional advantage of the bags described above is that, when sealed, they conform to the shape of green coffee beans when pressurized during the sterilization stage. Conformation of the bags to the shape of green coffee beans inhibits the movement of green coffee beans in relation to each other, which prevents or minimizes the degradation of the surface of the beans. This conformation of the bag to the shape of green coffee beans also improves heat exchange, since the lack of air prevents the air insulation of heating green coffee beans. Scholarships can be any size. In one embodiment, the bags are stretched or flattened to speed up the heating process, for example, the length can be three times the diameter of the bag. This dimension can also facilitate the beneficial stacking of the bags or positioning of the bags for sterilization.
[00063] The size of the bags to be used can be chosen according to the volume or quantity of green coffee beans to be treated by the methods of the present invention. Exemplary amounts of green coffee beans for use per bag include 0.45 kg (1 lb) of green coffee beans to 68.04 kg (150 lbs) of green coffee beans, although greater or lesser amounts of green coffee beans contemplated. For example, quantities of 1 gram or 4,535.93 kg (10,000 lb) of green coffee beans can be treated by the method.
[00064] In another modality, the bags are flattened, with a thickness of 1/10 or less of the sum of the peripheral edges of each bag. The bags can be round in shape, with a circumference that defines the peripheral edges of each bag. Alternatively, the bags can be rectangular so that the sum of the sides defines the peripheral edges of each bag. The bags can be combined so that a series of rectangular bags can be easily handled in a production environment. All bags have breathable plasters (filter strips) that provide an approach to an anaerobic environment. In yet another modality, the bags are flattened to prevent a layer of broad beans from being less than three broad beans in thickness. Therefore, the heat quickly penetrates the flattened bags for the broad beans to effect sterilization or pasteurization. In this modality, due to pressurization, the bag will conform to the shape of the green coffee beans, and this will produce a pebbled surface on the outer surface of each bag when pressure is applied. The pebble bag surface forms interstitial spaces that allow heat to penetrate between the bags that are stacked to speed up the sterilization or pasteurization process. The pebbled surface of the bags also induces turbulent fluid flow along the surface of the bag to improve the heat exchange for the green coffee beans.
[00065] In another embodiment, green coffee beans are vacuum packed in the bags to eliminate air that could remove the volatile flavor or aromatic components from the bags.
[00066] In another modality, the bags are replaced by sheets of material that can be actuated by autoclave, such as BPA-free plastic. A base sheet is dispensed continuously along the top of a conveyor, the green coffee beans are then placed on the dispensed base sheet. A second top sheet is overlaid with green coffee beans and sealed to the base sheet. A vacuum is applied between the top and bottom sheets to evacuate air, then the sheets are sealed at predetermined distances to form sections. Each section maintains a predetermined volume of green coffee beans. The sections are transported through an autoclave, or oven, to carry out the pasteurization or sterilization process. The heat can be applied in a pressurized or non-pressurized environment in the form of water vapor, hot water under pressure, hot air in turbulent flow or laminar over the leaves or other heated fluid. In a variation of this modality, the sections containing the green coffee beans are laminated and placed in an autoclave for pressurization or sterilization. A lamination can contain several sections.
[00067] Since green coffee beans cause a pebbled surface on the outside of the leaves, there is interstitial space on the outside surface of the leaves to accelerate the pasteurization or sterilization process allowing the heated fluid to readily penetrate between the leaves. The pebbled leaf surface also induces turbulent fluid flow which further improves the heat exchange for green coffee beans. The pebbled surface inhibits the relative movement between the beans to ensure that the green coffee beans do not crack, break or rub. Fungal component
[00068] The fungal component to be used with the present invention can be a fungus from the phylum Basidiomycotina of Eumycota, including any fungus that belongs to Polyporaceae and Hericiaceae, in which the fungi selected from Eumycota's Basidiomycotina include Eumycota, including by the hair least one selected from Basidiomycotina and Ascomycotina, including the strains: Hericium erinaceus, Pleurotus ostreatus, Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotus djamor, Tramete versicolor, Lentinula edodes, Armillariella mellea, Moschina, Agaricus blazei, leafy Grifola, Pholiota nameko, cylindracea Agrocybe, ornatipes Boletus, Ganoderma lucidum, Ganoderma applanatum, marmoreus Hypsizygus, Morchella hortensis, Phellinus linteus, Headset Auricularia, fuciformis Tremella, obliquus Inonotus, fometarius Famines, sulfureus Laetiporus, nobillismus Bridgeoporus, Cordyceps sinensis , Military cordyceps is, Xylaria nigripes, Tuber melanosporum, Polyporus umbellatus. The fungi used in this invention for clean mycelium of green coffee beans may also include Eumycota Ascomycotina, including Clavicipitaceae, in which fungi selected from Eumycota Ascomycotina include Clavicipitaceae such as Cordyceps sinensis and Cordyceps militarus; and Xylariaceae, such as Xylaria nigripes. Combinations of the strains identified above are also contemplated. In some embodiments, the present invention uses Ganoderma lucidum or Cordyceps sinensis.
[00069] In general, the invention does not contemplate the use of the following fungi: Rhizopus chinensis, R. oligosporus, Aspergillus flavusoryzae, A tamari, A. niger, A. nidulans, A.oyae, Fusarium venenatum, F. graminearum, Saccharomyces cerevisiae, S. exiguus, S. pombe, Saccharomycopisis (Candida) lipolytica, Candida utilis, C. krusei or C. tropicalis, Pichia saitoi, Kluyveromyces fragilis, Endomycopsis fibuliger, Ascomycete Chaetomium, Zygosaccharomyces rouxii, Geucorumum racchi, camemberti, P. notatum, P. griseofulvuum, P. grisea, P. chrysogenum, P. roqueforti, P. nalgiovense, Neurospora intermedia, Amylomyces rouxii, Endomycopsis burtonii, Psycilocibin, Monascus purpureus, Debaryomyces hansenii, Ashbya gossypi niveum, T. inflatum, Streptomyces, Neocosmospora, Stachybotrys, Beauveria, Cephalosporium acremonium, C. acremonium, Gibberella fujikuroi, Fusidium coccineum, Monascus ruber, Claviceps fusiformis, C. paspali, C. pu rpurea, Aminita muscaria or A. phalloides.
[00070] The fungal components useful in the present invention can be prepared by methods as described herein. For example, in one embodiment, a pure strain of fungus is used. In some embodiments, the pure fungus strain may be able to proliferate effectively in and / or mycelia the coffee beans prepared to prepare mycelia coffee products. Any strain of fungus identified herein that has the capacity to proliferate effectively in and / or mycelia the prepared coffee beans can be used for the methods of the present invention.
[00071] It was surprisingly concluded by the inventors of the present invention that some fungal strains have a marked and / or increased ability to proliferate in, metabolize or otherwise use and / or modify green coffee beans and / or remove one or more components of undesirable taste of green coffee beans and / or better tolerate the presence of green coffee beans (or extract) in media. In one embodiment, the taste component is chlorogenic acid. In another modality, the fungal component reduces or removes caffeine from green coffee beans.
[00072] Therefore, the methods of the invention have as an optional additional step, a method for selecting a fungal component that has a marked and / or increased ability to proliferate in, metabolize or otherwise use and / or modify green coffee beans and / or removing one or more components of undesirable taste from and / or removing caffeine and / or better tolerating the presence of green coffee beans (or extracts) in media. This method comprises screening several strains of a desired fungal species to select a suitable fungal component (strain) that exhibits the marked and / or increased ability to proliferate in, metabolize or otherwise use and / or modify green and / or broad bean beans remove one or more components of undesirable taste and / or caffeine from green coffee beans and / or have a better ability to tolerate the presence of green coffee beans and use that selected strain (s) in the methods of the invention .
[00073] In one embodiment, a suitable strain will generate a distinctly dark ring of coffee matter around the perimeter of the colony, showing that the strain actively rejects and excludes certain aspects of coffee as a food source. The dark color of the ring, without sticking to any theory, shows that chlorogenic acid is rejected as a food source and is removed / volatilized from the strain.
[00074] In one embodiment, a pure strain of any commercially available Ganoderma lucidum is used as the fungal component. Although all strains of Ganoderma lucidum are effective for the present invention, it has been surprisingly concluded that some selected strains have the enhanced capabilities useful for the present invention as described herein. Such a useful strain for the fungal component of the present invention is strain 806 of Ganoderma lucidum, (Alice Chen; Buffalo, NY; 4/94) commercially available from The Pennsylvania State University Mushroom Culture Collection, available from to the College of Agriculture Sciences, Department of Plant Pathology and Environmental Microbiology, 117 Buckhout Laboratory, The Pennsylvania State University, University Park, Pennsylvania, USA 16802).
[00075] This strain was surprisingly determined by the present inventors to more effectively proliferate, metabolize or otherwise use and / or modify green coffee beans and / or tolerate green coffee beans and / or remove a or more components of undesirable taste of green coffee beans, including chlorogenic acid. In another modality, this strain can remove and / or reduce the amount of caffeine in green coffee beans. Therefore, in one embodiment, the fungal component is strain 806 of Ganoderma lucidum Alice Chen; Buffalo, NY; 4/94. These selected strain (s) were (were) deposited with ATCC, as described below in this document.
[00076] In one embodiment, a pure strain of any commercially available Cordyceps sinensis is used as the fungal component. Although all strains of Cordyceps sinensis are effective for the present invention, it has been surprisingly concluded that some selected strains have the enhanced capabilities useful for the present invention as described in the present document. Such a useful strain for the fungal component of the present invention is Cordyceps sinensis (Cepa 1009 Caterpillar Fungus; Colorado Corp, 1/2014) commercially available from The Pennsylvania State University Mushroom Culture Collection, available from the College of Agriculture Sciences, Department of Plant Pathology and Environmental Microbiology, 117 Buckhout Laboratory, The Pennsylvania State University, University Park, Pennsylvania, USA 16802). These selected strain (s) were (were) deposited with ATCC, as described below in this document.
[00077] This strain was surprisingly determined by the present inventors to more effectively proliferate, metabolize or otherwise use and / or modify green coffee beans and / or remove one or more components of undesirable taste from the beans of green coffee, including chlorogenic acid, and / or better tolerate the presence of green coffee beans (or extract) in media. In another modality, this strain can remove and / or reduce the amount of caffeine in green coffee beans. Therefore, in one modality, the fungal component is Cordyceps sinensis (Cepa 1009 Caterpillar Fungus; Colorado Corp, 1/2014). These selected strain (s) were (were) deposited with ATCC, as described below in this document.
[00078] Similarly, the strains selected for H. erinaceus, T. versicolor, L. edodes, T. matsutake, F. velutipes, A. blazei, G. frondosa, P. nameko, L. officinalis, M. hortensis, M angusticeps, A. auricula, T. fuciformis, I. obliquus, F. fomentarius, L. sulfureus, for example, (or for any species of fungi mentioned in this document) were obtained by screening several strains of each species to select an appropriate fungal component (strain) that exhibits the marked and / or increased ability to proliferate in, metabolize or otherwise use and / or modify green coffee beans and / or remove one or more components of undesirable taste and / or caffeine from green coffee beans and / or has a better ability to tolerate the presence of green coffee beans, and to use that selected strain (s) in the methods of the invention. Therefore, in some modalities, the selected strain (s) of H. erinaceus, T. versicolor, L. edodes, T. matsutake, F. velutipes, A. blazei, G. frondosa, P. nameko , L. officinalis, M. hortensis, M. angusticeps, A. auricula, T. fuciformis, I. obliquus, F. fomentarius, L. sulfureus is (are) used in the processes of the present invention. These selected strain (s) were (were) deposited with ATCC, as described below in this document.
[00079] In one embodiment, a pure strain of Tuber melanosporum is commercially obtained. In another embodiment, at least one strain is generated from wild truffle mushroom (s) and used in the present invention as the fungal component. Although antibiotics can be used in the culture, strains of more vigorous proliferation have been found in cases where the culture medium lacked antibiotics. For example, a pure strain that comprises a maternal strain to the original truffle mushroom can be generated by cultivation on undefined agar that comprises oak leaves and oak branches. A pure strain comprising a parent strain of the original truffle mushroom can be generated by cultivation on undefined agar that comprises oak leaves and oak branches. A pure strain comprising a strain comprising a cultivar derived from non-sexual genetic recombination or anastomosis of both types of maternal and paternal mating can be generated by cultivation on indefinite agar comprising oak leaves and oak branches. In some embodiments, appropriate isolates (strains) for use include isolates that result in a tasty and pleasant aroma / taste in mycelia coffee, reminiscent of flowers (in some embodiments, achieved by maternal strains to a truffle mushroom as described in this document) or isolates (strains) that result in a tasty and pleasant aroma / taste, reminiscent of truffles, (in some modalities, reached by a cultivar derived from non-sexual genetic recombination or anastomosis of both types of maternal and paternal mating, as described in this document). These selected strain (s) were (were) deposited with ATCC, as described in this document.
[00080] In one embodiment, the methods of the invention include screening various strains of a T. melanosporum, created as described above, to select a suitable fungal component (strain) that exhibits the marked and / or increased ability to proliferate in, metabolize or otherwise use and / or modify green coffee beans and / or remove one or more components of undesirable taste and / or caffeine from green coffee beans and / or have a better ability to tolerate the presence of green coffee beans and use that (s) strain (s) selected in the methods of the invention. These strain (s) were (were) deposited with ATCC as described in this document.
[00081] All strains referenced in this document are deposited with the ATCC at 10801 University Boulevard, Manassas, Virginia 20110-2209 USA under the provisions of the Budapest Treaty. The deposit will be irrevocably and without restriction or condition available to the public upon the issuance of a patent and will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure Purposes. These deposits were made only as a convenience to those skilled in the art and are not an admission that a deposit is required under 35 U.S.C. §112. However, it should be understood that the availability of a deposit does not constitute a license to practice the present invention in derogation from the patent rights granted by government action. The deposit will be kept without restriction in the ATCC Deposit, which is a public deposit, for a period of 30 years or 5 years after the most recent application or for the enforceable life of the patent, whichever is longer, and will be replaced if the same become ever feasible during that period. MAINTENANCE AND ADAPTATION OF THE Fungal Component
[00082] The fungal components useful in the present invention can be prepared by the methods as described herein. For example, in one embodiment, the fungal component is optionally proliferated, maintained and / or propagated in an undefined medium that comprises coffee bean extract prior to use for inoculating green coffee beans. In one embodiment, the fungal component is maintained indefinitely in the undefined medium which comprises green coffee bean extract in solid, floating and submerged morphologies. Without sticking to the theory, the inventors believe that maintaining the fungal component in indefinite media that comprise green coffee beans plays an important role in the health and long-term availability of the fungal component. It is believed that the perpetual and subtle changes made from batch to batch of agar and liquid media when using the undefined medium comprising green coffee beans effectively prevent the phenomenon of undesirable genetic drift that will occur over time for the fungal components when they are maintained in identical (defined) media.
[00083] The undefined medium that comprises green coffee bean extract can be made by several methods. In one embodiment, the undefined medium comprises aqueous green coffee extract. Optionally, additional energy sources can be added. The materials are optionally organic and at least RO filtered water. It has surprisingly been revealed by the inventors that the medium can comprise aqueous green coffee extract without any additional added excipients, as well as an additional energy source to proliferate the fungi of the present invention.
[00084] The solid media comprises the indefinite medium which comprises extract of green coffee bean. In one embodiment, the undefined medium comprising means of green coffee bean extract agar to proliferate fungi for the eventual purpose of mycelializing sterile green coffee beans may comprise the green coffee bean filtrate. In one embodiment, 0.05 to 45.36 kg (0.1 to 100 lb) of green coffee beans are in 0.1 to 100 l of water, respectively, for 1 minute to 12 hours. The filtrate was collected through 1 to 3 filtrations of the mixture and 14 to 17 g / l of agar were added. This base solution was then mixed with the undefined aqueous vegetable extract filtrate (of any type, but ideally organic), such as malt extract, yeast extract, potato. In one embodiment, the vegetable is potato. The aqueous potato mixture can be prepared by softening 1 to 300 g of potato dough in boiling or pressurized water, mashed and the filtrate was collected through 1 to 3 filtrations. Optionally, fruit juice with no added sugar can also be added to the base green coffee agar media. In one embodiment, the medium comprises 0.1 to 10% by weight of malt extract, 0.1 to 10% by weight of undefined vegetable extract with the essence of green coffee bean, 0.1 to 10% by weight of extract yeast, 0.1 to 10% by weight of peptone, 0.1 to 10% by weight of glucose, 20 to 80% by weight of water and 1 to 90% by weight of whole green coffee beans or fava extract of green coffee.
[00085] As a non-limiting example of the media, for example, 0.91 kg (2 lb) of green coffee beans, or pulverized or whole, can be mixed with 0.95 l (0.25 gallon) of water at room temperature. The mixture can be mixed. The mixture is then allowed to be extracted for 20 minutes with stirring, then filtered three times through fine mesh. Separately, about 5 organic potatoes will be placed in 10 l of water and autoclaved for 20 minutes to soften the potatoes. The potatoes are then sprayed with a potato masher and then filtered through fine mesh three times. 1 l of unsweetened commercial fruit juice can be added. These solutions are combined and autoclaved.
[00086] Once prepared, the medium can be sterilized by any method known in the art. Once the medium cooled, it was poured into Petri dishes and fungal cultures were propagated from plate to plate in sterile operation, as is known in the art. Inclined surfaces for vials and test tubes can be prepared by this method. Petri dishes can be inoculated with floating or submerged liquid tissue culture and mycelial substrate.
[00087] The liquid medium is the indefinite medium that comprises green coffee bean extract. Green coffee bean extract and undefined vegetable extract were prepared as described for the solid medium, except that no agar was added. In the case of preparing to make a floating culture, 1 to 10 tablespoons of flour were added to the mixture, in one embodiment, about 1 tablespoon per 10-15 l of culture. The medium can be sterilized by methods known in the art. Once cooled, the vessel can be inoculated in sterile operation with a colonized section Petri dish, from other liquid tissue cultures or from samples of the mycelial substrate.
[00088] In one embodiment, the fungal component for inoculation into green coffee beans may be prepared by submerged liquid tissue culture using the undefined medium comprising liquid medium of green coffee bean extract as defined in this document and stirred on a shaking table. In one embodiment, the agitation rate is 50 to 240 rpm or 85 to 95 RPM and incubated for 4 to 90 days. In one embodiment, the incubation temperature is 30.56 to 31.67 ° C (87 to 89 ° F).
[00089] In one embodiment, the fungal component is trained and / or adapted and / or maintained in its ability to proliferate, metabolize or otherwise use and / or effectively modify green coffee beans. In one embodiment, the fungal component is selected and / or trained and / or adapted and / or maintained in its ability to remove or reduce one or more components of undesirable taste from green coffee beans or to remove or reduce the amount of caffeine. Methods for determining whether an undesirable taste and / or caffeine component has been reduced or removed have been disclosed herein and also found in the art.
[00090] In one embodiment, the trained and / or adapted and / or maintained fungal component is prepared from wild disinfected wild fungi. Such fungi with changed, improved and adapted properties, as described in this document, in relation to the initiation strains, both selected and unselected, were developed by these methods. These adapted strains were deposited with the ATCC, as described in another paragraph in this document. In one embodiment, the fungal component trained and / or adapted and / or maintained is prepared together with Ganoderma lucidum. In one embodiment, the fungal component trained and / or adapted and / or maintained is prepared with the strain Ganoderma lucidum 806 Alice Chen; Buffalo, NI; 4/94. In another modality, the fungal component trained and / or adapted and / or maintained is prepared together with Cordyceps sinensis (Caterpillar Fungi of Cepa 1009; Colorado Corp, 1/2014). In one embodiment, the fungal component trained and / or adapted and / or maintained is prepared together with H. erinaceus, T. versicolor, L. edodes, T. matsutake, F. velutipes, A. blazei, G. frondosa, P. nameko, L. officinalis, M. hortensis, M. angusticeps, A. auricula, T. fuciformis, I. obliquus, F. fomentarius, L. sulfureus. In one embodiment, the fungal component trained and / or adapted and / or maintained is prepared together with the pure strain of Tuber melanosporum, obtained by culturing a mushroom truffled by the methods described in this document. Those fungi that have changed, improved and adapted properties as described in this document, in relation to the initiation strains, were deposited with ATCC, as described in this document.
[00091] The training and / or adaptation and / or maintenance step, as described in this document, can be optionally conducted in an undefined medium that comprises liquid media or solid media of green coffee bean extract, as defined in this document. In one embodiment, fungi can be grown for 4 to 90 days at any temperature known in the art to grow fungi, for example, 30.56 ° C to 31.67 ° C (87 to 89 ° F). The reinoculation of the fungal component grown in fresh media, as described in this document, can be performed at an appropriate time, as determined by a person skilled in the art depending on the rate of proliferation, proliferation cycle, and appearance of the fungal component. The proliferation cycle and the reinoculation of the fungal component in fresh media, in some modalities, are carried out more than once, more than twice, more than three times, more than four times, more than five times, more than ten times, more than fifteen times, more than twenty times, more than twenty five times, more than thirty times, more than forty times, more than fifty times, more than seventy-five times, or a hundred times or more. The fungal component, through these methods, for example, can better recognize green coffee beans or any particular component of green coffee beans as an energy source, better tolerate the presence of green coffee bean extract in the media (as measured by an increased proliferation rate, for example), better remove components of undesirable taste, or better remove caffeine. In one embodiment, the undesirable taste component to be removed and / or reduced is chlorogenic acid.
[00092] Therefore, the methods of the invention have as an optional additional step, a method for preparing a trained and / or adapted and / or maintained fungal component that comprises a fungal component that has an enhanced and / or increased ability to proliferate, metabolize or, otherwise, use and / or modify green coffee beans and / or remove one or more components of undesirable taste from green coffee beans, and / or remove caffeine, and use the trained and / or adapted fungal component and / or maintained in the present invention. The methods of the invention further comprise the use of any one of the trained fungal component (s), adapted and / or maintained (s) as described herein, in the methods of the present invention. PREPARATION OF A Fungal Component For Inoculation Of Green Coffee Beans
[00093] In one embodiment, methods for preparing the fungal component to inoculate the prepared green coffee beans include scaling a fungal component as defined herein in a liquid culture. Such a fungal component, which is reliable for inoculating prepared coffee beans is called a "prepared fungal component".
[00094] In one embodiment, the prepared fungal component is in solid culture. In another embodiment, the prepared fungal component is in a liquid culture. In another embodiment, the fungal component prepared is a mixture of liquid and solid culture. Liquid culture can be achieved by any means known in the art and includes the use of a bioreactor. For example, when using a bioreactor to prepare the fungal component, the bioreactor can be prepared by diluting the undefined medium which comprises liquid media of green coffee bean extract to 1,000x with filtered / RO water. The bioreactor jacket can be vaporized in a way to sterilize the media or, alternatively, the media can be sterilized by injecting water vapor into the vessel.
[00095] The means for use in preparing a fungal component for use to inoculate the prepared green coffee beans can be any means known in the art, or can be done by the methods disclosed herein. The media may additionally comprise trace elements and organic substances, such as, water, nucleic acids, and minerals. The media can be diluted up to 1,000x with filtered / RO water. The dilution can be 1x, about 2x, about 3x, about 4x, about 5x, about 6x, about 7x, about 8x, about 9x, about 10x, about 10x, about 15x, about 20x, about 25x, about 30x, about 35x, about 40x, about 45x, about 50x, about 55x, about 60x, about 65x, about 70x, about 80x, about 90x, about 100x, about 150x, about 200x, about 250x, about 300x, about 350x, about 400x, about 450x, about 500x, about 550x, about 600x, about 600x, about 650x, about 700x, about 750x, about 800x, about 850x, about 900x, about 950x or about 1,000x. In some embodiments, the dilution is about 5x to about 100x. For a 100 l bioreactor, the media can be diluted about 10x, for example.
[00096] In one embodiment, to inoculate the reactor, the media can be pumped from another reactor through a sterile line with an internal line pump, or by creating positive pressure by spraying air into the reactor with a compressor of air that passes through the air through the 0.2 / 0.5 micron internal line capsule filters, then through a check valve with a specific crack pressure, for example 0.01 MPa at 0, 02 MPa (2 to 3 psi).
[00097] Methods for inoculating the bioreactor to prepare the fungal component include inoculating the bioreactor with a colonized section excised from Petri Dish and / or a sample of mycelial agriculture using a sterile procedure, or pouring liquid tissue culture floating or submerged in the bioreactor through the nozzle.
[00098] Optionally, the fungal component can be stirred during culture by methods known in the art. For example, in a bioreactor, agitation can be carried out by a combination of sprayed air and a motorized paddle that allows both a turbulent environment and a mechanical shear force. The inventors, without limitation, found that the combination is superior to the execution of the method, either individually, since sprayed air creates most of the turbulence in the upper half of the crop, at the same time that it affects the bottom less, which can be kept agitated by a motorized paddle, although the paddle does not have to be driven it has a high RPM as used normally in the art. The combination creates the appropriate spherical sizes of small hyphae without damaging the mycelium.
[00099] Liquid fermentation agitation and rotation techniques are known in the art and include mechanical shear which includes using magnetic stir bars, stainless steel impulses, sterile high pressure ambient air injection, injection in high pressure from sterile media, and / or the use of shaking tables. Higher agitation and rotation rates, in combination with air and media injection, produce small spheres of mycelium.
[000100] The fungal component can be proliferated until it is ready for inoculation of the prepared green coffee beans, as determined by a person skilled in the art. In some modalities, the fungal component can be proliferated for 48 hours before use to inoculate the green coffee beans. The determination of whether the fungal cultures that comprise the fungal component are suitable for inoculation of the prepared green coffee beans can be made by a person skilled in the art. For example, in one modality, fungal culture, when in liquid media, is suitable for inoculation while it is in the phase of exponential proliferation, both early and late. Senescent cultures and cultures in early proliferation stages with lower amounts of mycelium / ml can be used, but are not preferred. The prepared fungal component optionally appears well proliferated through the media, with visible mycelium that proliferates with each ml visible by microscope and by unaided vision.
[000101] In order to effect the most efficient myceliation of the most efficient coffee, the fungal component defined sizes of hyphal spheres that allows the cultivation of hyphae in three dimensions around the spherical conglomeration of the fungal strain culture. In one embodiment, the size of the hyphal sphere is less than 10 mm in diameter, less than 2 mm in diameter, less than 1 mm in diameter, less than 100 microns in diameter, less than 10 microns in diameter, less than 5 microns in diameter, less than 2 microns in diameter, or less than 1 micron in diameter. In another embodiment, the spherical spherical conglomeration has a size range of 5 microns to 1 millimeter in diameter, or, a size range of 10 to 50 microns in diameter.
[000102] These methods result in a fungal component prepared for inoculation of prepared green coffee beans. INOCULATION AND MYCELLATION OF PREPARED GREEN COFFEE BEADS
[000103] The prepared green coffee beans are inoculated with the prepared fungal component. The fungal component prepared to be used can be any fungal component as defined in the present invention. The inoculation of the fungal component prepared in the prepared green coffee beans can be carried out by any method known in the art. This stage can be referred to in a variety of ways as the culture stage, the fermentation stage, and / or the mycelium stage.
[000104] Mycelia can occur in a container as described in this document. In one embodiment, mycelia occurs in a 0.24 m2 (55 gallon) drum as described in this document. In this modality, the 0.24 m2 (55 gallon) drums have a lid that contains two doors, and one door can be used as an inoculation door, while the other can be used to spread filtered air to the bottom of the culture, and simultaneously serve as a gateway. In some embodiments, the inoculation port is a quick-disconnect socket, which is attached to a quick-disconnect plug at the end of a harvest line during inoculation. Optionally, green coffee beans prepared in a plurality of drums can be myceliated in a cycle by means of a sterile collection tube connected to the bioreactor harvest line, with the infrastructure included to target any single drum or all together at once . In one embodiment, a system for consistently volumetric inoculant dispensing by culture is used.
[000105] In one embodiment, the culture can be injected pneumatically into a container that includes the prepared green coffee beans. The mixture can optionally be injected into bags to optimize mycelial growth. In another embodiment, the prepared green coffee beans are inoculated by pouring the culture into the container that keeps the coffee beans sterilized either manually or through a valve built into the fermentor or bioreactor, from any variety of liquid tissue culture.
[000106] In one embodiment, the prepared green coffee beans are cooled to a temperature between 26.6 to 37.7 ° C (80 to 100 ° F) before inoculation with the prepared fungal component. Cooling can be completed by refrigeration or at room temperature. The step of mycelializing the prepared green coffee beans can occur for between 4 and 90 days, for between about seven and twenty-one days and, in one modality, for about seven days, and at any temperature, for example, at 30.5 to 31.6 ° C (87 to 89 ° F). The multiplication of fungal mycelia by fermentation is carried out by efficiently controlling ambient light, such as by a control model of 40% lighting and 60% darkness, and also controlling sterile air flow and temperature at 30 at 31.1 ° C (86 to 88) or 30.5 to 31.6 ° C (87 to 89 ° F), or between 12 and 35 ° C, or between 24 ° C to 32 ° C.
[000107] The relative humidity of this culture, mycelium and / or fermentation stage is between 20% and 80%, in some modalities, around 60%.
[000108] The step of mycelializing the prepared green coffee beans is preferably completed in an anaerobic or semi-anaerobic environment. Methods known in the art can be used to induce and / or maintain optional anaerobic metabolic activity of the fungal component prepared as described by the Pasteur Effect. In an alternative embodiment, the prepared green coffee beans are removed from the leaves and deposited in large stainless steel vats in a sterile environment. The vats regulate the levels of oxygen and temperature, and allow optional anaerobic activity and mycelial growth in the prepared green coffee beans. Optional anaerobic activity metabolizes more cellulose per unit time, which means that the coffee substrate is consumed at a faster rate than in an aerobic environment. In some cases, mycelial growth is nine times faster than in an aerobic environment (that is, nine times more cellulose molecules are metabolized to ATP). Another benefit is that the anaerobic environment inhibits the growth of the fruiting body. An anaerobic environment also guarantees a reduction in unwanted bacterial growth, and another unwanted microbial growth.
[000109] The expansion of the fungus mycelia is monitored by microscope, and growth programs documented by photography.
[000110] The longer the incubation period, the greater the production of the dry weight of mycelium and the greater the flavor intensification of the prepared green coffee. Some strains will form primordial tissues and fruit bodies in 30 days (Hericium erinaceus is particularly prone to fruiting while in culture, as is Ganoderma lucidum and Flamulina velutipes). In some modalities, the harvesting of mycelial coffee beans is carried out before the body tissues of cultured fruits. However, a prolonged incubation period for a specific fungus does not guarantee a high metabolite production or the accumulation of active material.
[000111] The determination of when to coat the mycelia coffee product can be determined by a number of methods. Harvesting is usually carried out with a timer to optimize the taste profile of the mycelial coffee product according to the desired taste profile. For example, the essence profile of the mycelial culture can be used by the trained person to determine when the culture is ready. Determining the appearance of the culture can also be done by the trained person. In some modalities, harvesting can be done when the amount of mycelia in the culture is in the approximate amount of 2 to 3 fully grown Petri dishes (standard size) (for G. lucidum), or when the amount of the mycelia is in the approximate amounts 10 to 12 fully grown standard Petri dishes (for C. sinensis), per 3.6 kg (8 pounds) of coffee. Analytical methods of analysis that include high performance liquid chromatography (HPLC) can be used to measure total biomolecules in order to determine the ideal composition and culture conditions and the appropriate time (s) to collect the fungi.
[000112] In a non-limiting example of the present invention, about 3.6 kg (8 pounds) of green coffee beans prepared according to the processes of the invention, in a sealed container as described in this document, were inoculated with about 25 ml of fungal component prepared as described in this document. A decreased oxygen environment was obtained. Mycelia or culture was allowed to continue for seven days at 30.5 to 31.6 ° C (87 to 89 ° F), although as little as four days or as long as sixty or more days is also appropriate. The harvest was carried out when observers determined that an appropriate taste profile for the mycelial coffee product had been obtained. REDUCING CAFFEINE AND / OR COMPONENTS OF UNDESIRABLE TASTE DURING MYCELLATION
[000113] The culture or mycelia stage can also cause reduction and / or removal of components of undesirable taste as described in this document and / or caffeine. In some embodiments, determining the extent of removal of at least one component of undesirable taste is determined by the appearance, taste and / or chemical composition of the mycelium coffee product as is known in the art. This determination can be quantitative, for example, the chemical composition of the mycelial coffee product can be measured by methods of analysis for one or more of the components of undesirable taste by test methods as known in the art, or determined qualitatively by taste test by well-versed people.
[000114] In one embodiment, up to 5% of one or more of the unwanted taste components are removed; in other modalities, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65 %, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% of one or more of the unwanted flavor components are removed in the processes of the present invention. In one embodiment, one or more of the unwanted flavor components are removed quantitatively. The invention also relates to mycelial coffee products that have reduced levels of undesired taste components, as described herein.
[000115] In one embodiment, the unwanted taste component is chlorogenic acid and up to 5% of the chlorogenic acids are removed, in other embodiments, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95 % of chlorogenic acids are removed in the processes of the present invention. The invention also relates to mycelial coffee products that have reduced levels of chlorogenic acid, as described herein.
[000116] In one embodiment, caffeine is removed from coffee beans prepared during the culture or mycelia stage. In one modality, up to 5% of the caffeine is removed, in other modalities, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50 %, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% of the caffeine are removed in the processes of the present invention. The invention also relates to mycelial coffee products that have reduced levels of caffeine, as described in the present document.
[000117] The removal of components of unwanted taste may allow the value of weaker quality coffee to increase and / or make it more palatable. The mycelial coffee products produced by this method can be used to be mixed with less expensive coffee beans, leading to a lower cost product that has improved taste properties. The amount of sugar, milk and substitutions to add to coffee can be reduced. The present methods lead to the improved taste profile of mycelia coffee products due to the perception that mycelia coffee products offer a richer, softer and / or sweeter coffee, with less bitterness, roughness and / or acidic tastes. ADDITION OF FLAVOR AND / OR COMPONENTS BENEFICIAL TO HEALTH
[000118] The culture or mycelial processes of the present invention, in some embodiments, offer a mycelial coffee product with added flavor and / or health-beneficial components. For example, mycelial coffee products may contain exogenously added anti-tumor and immunomodulatory components that are beneficial to health.
[000119] Fungi are metabolically similar to animals, but structurally similar to plants in which they have a rigid cell wall formed, in general, by long molecular chains of sugar joined by beta (b-) bonds, in a way, difficult digestion and, to a lesser extent, easily digestible alpha (a-) bonds, together with membrane-bound proteins. In contrast, plant cell walls (such as coffee beans) are made up of cellulose polysaccharides whose glycosidic glucose bonds 1-> 4 are likewise difficult to digest for humans, but can be digested by fungi. The cell walls of fungi are basically composed of 1-> 3 glycosidic bonds, with 1-> 6 bonding side chains and, therefore, they can be decomposed by minimal processing, with the use of water, heat and mechanical treatment into molecules. smaller, easier to digest, and immunologically active polysaccharides of varying sizes of microparticles called beta glucans and glycoprotein-related compounds. The immune response to beta glucan depends on the structure of glucan a- or b, which has primary, secondary and tertiary chiral structures, which explain the differences in the immune response of a- and / or b-glucan profile of each fungus. Thus, mycelia coffee products added beneficial health components, including the molecules described above. Other beneficial health components present in mycelia coffee products can be components that have several properties, such as immunomodular, anti-aging, aphrodisiac, antitumor, antiviral, antibacterial and / or antifungal properties and include components such as a- and b-glucans, glycoproteins, proteins, ergosterols, sterols, triterpene and fatty acids, glucomannan, riboglucan, sterpuric acid, mannitol, ribitol, guanosine and adenosine.
[000120] Methyl pyrazines, which naturally develop in cocoa, are among the most important desired flavor compounds found in cocoa. In the presence of heat, such as roasted, they are produced through Maillard reactions, that is, between sugars and amino acids or peptides. Without sticking to the theory, it is believed that methyl pyrazines are also produced through fermentation and / or myceliation, which leads to increased and / or altered methyl pyrazines in mycelia coffee products and mycelia green coffee beans and other agricultural products.
[000121] Agaricus blazei can be used to add a single a- and glucans called glucomannan and riboglucan, which are antiviral, to the mycelial coffee product. Other polysaccharide extracts A. blazei may have anti-cancer effects and may be co-therapeutic with mycelial extract of the fungi listed in this document. Methods to optimize biomass and extracellular polysaccharide production were recorded. Therefore, myceliation with A. blazei and mycelial coffee products that contain flavor and / or beneficial components derived from A. blazei as described herein are also included in the present invention.
[000122] Cordyceps sinensis (C. sinensis) produces cordicépticoic acid, adenosine, D-mannitol and cordicepine-adenosine which are immunomodular and antiviral. C. Sinensis extracts have been shown to be anti-aging and aphrodisiac. Mycelial sterols isolated from C. sinensis have been shown to inhibit the proliferation of numerous cancer cell lines. Mycelial polysaccharide extracts C. sinensis have been shown to induce hypoglycemia. Therefore, myceliation with C. sinensis and mycelial coffee products that contain flavor and / or beneficial components derived from C. sinensis as described herein are also included in the present invention.
[000123] The mycelium Flammulina velutipes has been shown to have a polysaccharide profile that is immunomodular. The mycelium F. velutipes composes a unique ergosterol and amino acid profile, sterpuric acid, mannitol, ribitol and the nucleosides guanosine and adenosine, Enokipodins A-D extracted from the mycelium F. velutipes are broad spectrum microbicides. The proteins flamulin and velutin exhibit anti-HIV and anti-HPV activity. Therefore, myceliation with F. velutipes and mycelial coffee products that contain flavor and / or beneficial components derived from F. velutipes as described herein are also included in the present invention.
[000124] The polysaccharide profile of Ganoderma lucidum has been shown to be immunomodular in human cell lines and also in clinical studies. The extracts of the mycelium G. lucidum have anti-oxidative, anti-inflammatory and anti-mutagenic properties. G. lucidum extracts have been shown to be anti-aging and aphrodisiac. The G. lucidum triterpenoid profile was determined and demonstrated to be anti-hepatotoxic and hepatoprotective, anti-tumor, anti-angiogenic, anti-hypertensive, hypocholesterolemic, anti-histamine and anti-HIV. G. lucidum, in addition to producing polysaccharides and glycoproteins, also produces triterpenes such as ganoderic and lucidic acids, phenolic compounds and sterols that also have biological activities and therapeutic properties and are, in themselves, antioxidants, antitumor, antibacterials, anticancer, anti-inflammatory, antihistamines, hypotensive, sedative and meditative after oral consumption. Therefore, myceliation with G. lucidum and mycelial coffee products that contain taste and / or beneficial components derived from G. lucidum as described herein are also included in the present invention.
[000125] The polysaccharide profile Grifola frondosa proved to be immunomodular and antioxidative, G. frondosa produces ergosterols and an antioxidative profile of fatty acids. The antitumor effects of G. frondosa extracts in cancer cell lines in vitro have been investigated and shown to be promising as being hypoglycemic for diabetic patients. Therefore, myceliation with G. leafy and mycelial coffee products that contain flavor and / or beneficial components derived from G. leafy as described herein are also included in the present invention.
[000126] Mycelial and fruiting body extracts from Hericium erinaceus have been shown to be antimutagenic and immunomodulatory across multiple cell lines. H. erinaceus uniquely produces hericenones in fruit bodies and erinacins in the mycelium, structurally determined compounds that can pass through the barrier between the blood and the brain and promote secretion of Nerve Growth Factor (NGF) in certain regions of the brain. Erinacins have been shown to enhance NGF expression than hericenones. Therefore, myceliation with H. erinaceus and mycelial coffee products that contain flavor and / or health-promoting components derived from H. erinaceus as described herein are also included in the present invention.
[000127] Aspects of the Lentinula edodes' polysaccharide profile have been determined and shown as immunomodulators and antivirals. Lentinan and other metabolites have been studied because of their numerous health care benefits. In some countries, lentinan is classified as an "antineoplastic polysaccharide" and is available for clinical use. The addition of lentinan to standard cancer therapies has been shown to result in increased tumor necrosis and hepatocellular carcinoma and increased quality of life in patients with esophageal carcinoma. Therefore, myceliation with L. edodes and mycelial coffee products containing flavor and / or health-promoting components derived from L. edodes as described herein are also included in the present invention.
[000128] Extracts of Phellenis linteus have been shown to exhibit antitumor activity. Polysaccharide extracts of Polyporus umbellatus have been studied and shown to be anticancer, immunomodulatory, antimalarial and hepatoprotective. The mycelial polysaccharide extract of Inonotus obliquus demonstrated antitumor, hypoglycemic and antioxidant properties. The mycelium composition of Pleurotus ostreatus and fruit body were shown to be very similar, of which they differ only by the content of amino acids. The mycelial polysaccharide profile consists primarily of laminarin, the extract of which has been shown to be an immunomodulator. Lovastatin, isolated from the mycelial broth of P. ostreatus, exhibits anticarcinoma activity, inhibits the proliferation of bacteria and Fungus and lowers cholesterol. Trametes versicolor produces heteroglucans cin a- (1-4) - and b- (1-3) glycosidic bonds with fucose in PSK (Krestin) and rhamnose and arabinose in PSP, which have been shown to be anti-tumor and immunomodulatory. PSK, an approved drug, is an mycelial extract that exhibits immunomodulatory, antiviral and cholesterol-regulating properties. The mycelial polysaccharide extracts of Tremella fuciformis have been shown to be therapeutic for various circulatory disorders, to be neurologically healthy, anticarcinoma, anti-tumor and anti-aging.
[000129] Therefore, myceliation with Phellenis linteus, Polyporus umbellatus, Inonotus obliquus, Pleurotus ostreatus, Trametes versicolo and / or Tremella fuciformis (and any other fungal species described in this document) and mycelial coffee products that contain flavor and / or components health promoters derived from Phellenis linteus, Polyporus umbellatus, Inonotus obliquus, Pleurotus ostreatus, Trametes versicolo and / or Tremella fuciformis (or any other fungal species described herein) are also included in the present invention.
[000130] The amounts of flavor components or health promoting components added by the fungal component as described in this document can be estimated by a person skilled in the art, and includes up to 1 ng of the component per unit of mycelial coffee product, or even 5 ng, up to 10 ng, up to 50 ng, up to 100 ng, up to 500 ng, up to 1 pg, up to 5 pg, up to 10 pg, up to 100 pg, up to 500 ug, up to 1 mg, up to 2 mg , up to 5 mg, up to 10 mg, up to 20 mg, up to 50 mg, up to 100 mg or up to 500 mg per unit mycelial coffee product. A unit of mycelial coffee product can be varied in different ways such as 1 g, 1 lb, 1 kg and the like. ADDITIONAL PROCESSING OF MICELLIED COFFEE PRODUCT
[000131] In some embodiments, once mycelialized completely, the myceliated coffee product is optionally rinsed after mycelialization. Rinsing can be performed to remove some or all parts of mycelia and / or other non-green coffee bean material.
[000132] In some embodiments, once myceliated completely, the mycelial coffee product is optionally dry. Drying can be carried out by means known in the art for drying green coffee beans. For example, mycelia coffee product can be spread on a dry surface to dry. In one embodiment, the mycelia coffee product is dried to about 11 to 13% moisture content.
[000133] Optionally, the dry or non-dry mycelial coffee product can be roasted and / or roasted by conventional methods known in the art. The optional toasting step is provided to disable the fungus, which may be desirable to reduce the risk of mycosis.
[000134] The mycelial coffee product can be extracted optionally to prepare an extract for use in food and / or beverage products. For example, 1,000 g of mycelia roasted coffee beans can be extracted completely with stirring; using 10 to 1,000 ml of pressurized water at 121 to 122 ° C as a buffer, which contains 0.01% to 10% citric acid and 0.01% to 10% ascorbic acid. The resulting aqueous extract can be further purified and concentrated using conventional methods. Mycelial coffee product extracts are given an extended shelf life by methods known in the art such as a formulation in 18% to 24% alcohol, 45% to 60% glycerol or the addition of 2.5 X volumes of honey or sugar similar to maple syrup or evaporated cane sugar.
[000135] Removing components of undesirable taste and / or caffeine, and / or adding mycelial products that increase flavor and / or promote health, will result in mycelial coffee products and may increase the value of unsatisfactory quality coffee and / or make them more drinkable. The mycelial coffee products produced by this method can be used to be mixed with less expensive coffee beans, which leads to a lower production cost with improved taste properties such as richer, softer, and / or sweet coffee with palates less bitter, sour and / or acidic. MODALITIES
[000136] Figure 1 shows a flow chart of a modality of a method of creating an extract of mycelial agricultural product (such as a mycelial coffee product) for human consumption. Step 10 provides an agricultural substrate, step 12 inoculates the substrate with a liquid medium that comprises a culture aliquot derived from liquid fermentation, step 14 allows the proliferation of mycelium and step 16 proliferates mycelium in the substrate, the step 18 includes boiling the substrate in water and separating the water-substrate mixture into aqueous and non-aqueous components after the proliferation of mycelium in the substrate reaches a desired stage, step 20 adds a mixture of small molecule components to the aqueous components by which the aqueous components mix in biologically available combinations with the small molecule components to facilitate the water solubility of the extract.
[000137] In one embodiment, step 24 uses a Basidiomycota fungus culture to allow step 12. In an alternative embodiment, step 26 uses Ascomycota fungus to allow step 12.
[000138] Figure 2 shows a method of creating an extract of mycelial agricultural product for human consumption, to effect neuroregeneration and neuroprotection in humans according to the present invention.
[000139] The method includes step 30 of supplying an agricultural substrate, step 32 of substrate inoculation by means of liquid comprising a culture aliquot derived from liquid fermentation, step 34 of allowing the proliferation of mycelium and proliferate mycelium on the substrate, step 36 of boiling the substrate in water and separating the mixture of water and substrate into aqueous and non-aqueous components. Step 36 occurs after the proliferation of mycelium in the substrate reaches a desired stage. Step 38 adds a mixture of small molecule components to the aqueous components to increase the water solubility of the extract to enhance passive absorption and uptake in humans.
[000140] Step 32 of inoculation includes step 40 of inoculating with a fungal component of the present invention.
[000141] Step 34 of allowing and proliferating mycelium on the substrate includes regulating temperature and humidity in a sterile environment. The mycelium then proliferates automatically. The mycelium is proliferated in a container that allows only a small amount of sterile air to enter the container. The volumes of sterile air are regulated by capping the container. This modulates the ambient oxygen in the container.
[000142] Step 34 proliferates the mycelium to a desired stage, for example, where the fruiting bodies begin to appear on the surface of the substrate.
[000143] Step 36 boils the substrate in water to separate the polysaccharides in the mycelium and other beneficial components to suspend in the water, which separates aqueous components from the residual substrate solids. The non-aqueous component includes the substrate solids. Separating the aqueous component from the non-aqueous component can be accomplished by filtration or siphoning. It can be appreciated that other methods known in the art can be used.
[000144] Step 38 adds a mixture of small molecule components to the aqueous component. This allows the aqueous components to be more bioavailable in vivo.
[000145] Figure 3 shows a method of the present invention 10. Method 10 includes step 12 of providing a fungal component in a liquid tissue culture medium, step 14 of adding a mixture that includes caffeine, such as undefined medium containing green coffee bean extract, liquid tissue culture medium to train the fungal component to consume caffeine, step 16 of pasteurization or sterilization of green coffee beans, step 18 of hydration of green coffee beans for moisture content of 10 to 80%, step 20 of inoculating green coffee beans with the fungal component, step 22 of providing ideal temperature, oxygen and moisture content to allow fungal proliferation and step 24 of drying and roasting of green coffee beans.
[000146] Figure 4 shows a method 48 of making coffee according to the present invention. Method 48 includes step 50 which supplies coffee beans in a container. Preferably, the container is an autoclavable bag.
[000147] Step 22 includes reducing oxygen levels to facilitate proliferation of facultative anaerobic mycelium in green coffee beans to maximize caffeine consumption rates by the fungal component mycelium. In step 50 the container is sealed, which allows only minimal leakage.
[000148] Step 52 moisturizes the green coffee beans to the ideal moisture content. In one embodiment, step 52 of hydrating the green coffee beans includes soaking the green coffee beans in water for two hours, and draining the green coffee beans for two hours before pasteurization sterilization. This is an important step due to the fact that the hydration of green coffee beans accelerates mycelial proliferation. The hydration stage of green coffee beans also removes unwanted residues and can remove some undesirable water-soluble components from green coffee beans. In an alternative embodiment, a fluid other than water is used. For example, water infused with an enzyme can eliminate unwanted components from green coffee beans before step 54 of sterilization. The pH of the water can be regulated to optimize the hydration step 52. Distilled water or mineralized water can be used for the hydration step 52.
[000149] Step 54 sterilizes the green coffee beans with water vapor and step 56 pressurizes the container. The pressurizing step 56 of the container is typically included with the sterilizing step 54. Consequently, the sterilizing step 54 is carried out with a pressure ranging from 0.35 to 1.75 kgf / cm2 (5 to 25 pounds per square inch) . Preferably, the pressure is 0.70 to 1.40 kgf / cm2 (10 to 20 pounds per square inch). In one embodiment, the pressure is 1.05 kgf / cm2 (15 pounds per square inch).
[000150] Sterilization under step 54 can be carried out under the conditions of 121 to 122 ° C at 1.05 kgf / cm2 (15 lb / in2; 115 to 116 ° C at 0.70 kgf / cm2 (10 lb / in) in2); and 108 to 109 ° C at 0.35 kgf / cm2 (5 lb / in2) for a period of 20 to 100 minutes, or pasteurization under the heat drying treatment conditions of 140 ° C to 210 ° C for a period of 20 to 100 minutes, these parameters can be varied.
[000151] Step 58 inoculates the pouches with a fungal component. In one embodiment, step 58 is performed with a liquid tissue culture that has fungal hypha sphere sizes from 5 to 100 microns in diameter to ensure consistent inoculation. In another embodiment, solid tissue culture is added to the green coffee beans and mixed with the green coffee beans in a consistent manner. In yet another embodiment, both liquid tissue culture and solid state tissue cultures are both added to the green coffee beans in the container to reduce the time required to propagate fungal mycelium in the sterilized coffee beans as described in step 60.
[000152] In one embodiment, step 58 is performed by cooling the container to 26.67 to 37.78 ° C (80 to 100 ° F) after steps 54 and 56 and injecting tissue culture of fungal liquid directly into the container . Step 60 propagates the fungal mycelium into the same container. The container can be shaken or rotated to ensure consistent distribution of the fungal liquid tissue culture. In an alternative embodiment, the green coffee beans are transferred from the containers to a vat for an inoculation step 58.
[000153] In another embodiment, step 58 is performed with a liquid tissue culture derived from liquid state fermentation. The culture of liquid tissue yields a pure culture of a fungal strain that comprises spherical conglomeration smaller than 2 millimeters in diameter, which allows hyphae to proliferate in three dimensions around the spherical conglomeration of the fungal strain culture. In a variation of this modality, liquid state fermentation yields a pure culture of a fungal strain that comprises spherical conglomeration ranging in size from 5 microns to 1 millimeter in diameter due to the shearing of hyphae structures during a process of agitation or stirring of the liquid medium . Stirring and / or rolling processes may include, but are not limited to, for example, mechanical shear using magnetic stir bars, stainless steel impellers, sterile high-pressure ambient air injection, sterile high-pressure injection and / or use of shaking tables. Higher rates of agitation and rolling, together with injections of medium and air, produce small mycelial spheres, aliquots which are used to inoculate solid agricultural substrate (s) for subsequent semianaerobic fermentation. Ideally, mycelial spherical conglomerations are less than 100 microns in diameter, and more preferably, within the range of 10 to 50 microns in diameter.
[000154] Step 58 of inoculating the pouches includes the use of a submerged or floating liquid culture, mycelium substrate such as grain or coffee, or petri dish culture to inoculate a solid state culture medium comprised of sterile or pasteurized green coffee beans. Special attention is paid to sub-varieties that exhibit a marked ability to ferment coffee. These strains can be selected and propagated by petri dish culture and liquid medium composed of a certain concentration of green coffee bean extract (as in, four to five green coffee beans are added to the preparation of tissue culture medium. liquid), in a submerged or floating liquid state and then used to inoculate sterilized green coffee beans at a faster rate than strains and sub-varieties that are not conditioned to ferment coffee.
[000155] Stage 60 of fungal mycelium propagation in sterile coffee beans is preferably performed in an anaerobic environment. Consequently, once the substrate (coffee beans) has been sterilized or pasteurized, the subsequent facultative anaerobic metabolic activity of the fungus as described by the Pasteur effect can be induced after step 58 of inoculation.
[000156] Figure 5 shows a method 10 of the present invention. Method 10 includes step 12 to supply the green coffee beans in a container, step 14 to prepare the green coffee beans for fungal myceliation by removing chlorogenic acids. In one embodiment, step 14 includes rinsing the beans in an aqueous solution.
[000157] Method 10 additionally includes step 16 of pasteurization or sterilization of green coffee beans, step 18 hydrates green coffee beans to a moisture content of 10% to 80%, step 20 inoculates the coffee beans green with a fungal component, step 22 regulates the temperature, oxygen and humidity in the container to myceliate the green coffee beans. This step 22 optimizes the mycelial rate. Step 24 rinses mycelia coffee beans to eliminate the accumulation on the surface of mycelial metabolites and other unwanted material caused by the mycelia step 22.
[000158] Figure 6 shows a conveyor 10, a first blade 12 of autoclavable material, a second blade 14 of autoclavable material and a hopper 16 that distributes the coffee beans between blades 12 and 14. The first blade 12 extends from a cylinder 24 of autoclavable material and is deposited on the conveyor 10. The hopper 16 releases the coffee beans on the first blade 12. The second blade 14 is first laminated to a cylinder 26 and then deposited on the coffee beans and the first blade 12. The first blade 12 has edges 22a and 22b. The second blade 14 has edges 20a and 20b, which seal against the edges 22a and 22b of the first blade 12 to form a large autoclavable pouch, which can be laminated as shown in Figure 8c and designed to contain more than 68.04 kg (150 lbs) of coffee beans.
[000159] A vacuum can optionally be applied to extract air that is between the blades 24a and 24b, which optimizes the heat transfer to the coffee beans 18 when the coffee beans 18 are delivered to a sterilization chamber. The gap also inhibits the relative movement between the coffee beans 18. The conveyor 10 delivers the coffee beans 18 in a sterilization chamber.
[000160] Figure 7 shows the conveyor 10. The hopper 16 between the coffee beans for an autoclavable pouch 30. The autoclavable pouch 30 is then sealed and deposited on the conveyor 10. The conveyor 10 carries numerous autoclavable pouches 30, 32 , 34, 36, 38 and 40, for an autoclave 42. Autoclave 42 in one mode applies the heated fluid such as water vapor and pressure, to the autoclavable bags 30, 32, 34, 36, 38 and 40 to perform sterilization or pasteurization of coffee beans 18.
[000161] Figure 8a shows an autoclavable bag 30 filled with coffee beans. The autoclavable bag 30 is under pressure which causes an uneven surface 44. Alternatively, the autoclavable bag 30 is vacuum sealed to cause the uneven surface 44.
[000162] Figure 8b shows the autoclavable bags 30, 32, 34, 36 and 38 stacked for sterilization or pasteurization in an autoclave. The irregular surface 44 improves the heat transfer to the green coffee beans enclosed in the pouch 30. The autoclavable pouch 30 includes at least one respiratory patch 45 adhered to the irregular surface of the pouch 30. Preferably, the respiratory patch 45 includes air vents that have a diameter of 0.1 microns to allow a minimum amount of air to flow through pocket 30 and to ensure that the mycelium propagated within pocket 30 propagates anaerobically. It can be appreciated that respiratory plasters can have air vents of between 0.05 to 100 microns in diameter. Preferably, the respiratory plaster air outlets 45 have an air outlet diameter of 5 microns or less. More than a respiratory patch 45 can be adhered to the uneven surface of the pouch 30 and can be used in containers other than pouches according to the present invention.
[000163] Figure 8c shows a continuous bag 46 of autoclavable material that surrounds the coffee beans. Continuous pouch 46 is sealed to allow pouch 46 to be sterilized or pasteurized in an autoclave. In an alternative embodiment, pouch 46 is sealed in sections so that continuous pouch 46 has multiple sections, which are isolated from each other to inhibit the movement of air and coffee between the sections. The turbulent air flow during sterilization is shown with arrows that pass through the pouch 46 and around its surfaces.
[000164] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1
[000165] The pure and specific strains of the fungus obtained from the reference harvests were handled in sterile environments in plastic bags of 3.79 to 37.9 l (1 gal to 10 gal), 1 as much as 3.79 l ( 1 gal) in a glass jar or in petri dishes from 10 cm to 15 cm, using a medium based on vegetable or organic fruit that includes green coffee bean extracted with 1.5% agar (w / v), in order to monitor and ensure the general vigor and health of the strains.
[000166] The mycelium samples proliferated in a sterile air stream in a gentle environment for 2 to 4 weeks, then excised from the petri dishes and subsequently used for inoculation in liquid state fermentation using a plant-based medium and similar undefined fruit (but without agar), using ambient air, in 1 fourth part of 3.79 l (1gal) of glass jars. Some samples proliferated in agitated cultures and some proliferated in unstirred cultures in room air in stainless steel tanks designed for fermentation and / or commercial brewing.
[000167] Unstirred liquid fermentation formed a floating mass of hyphae that exhibited continuous proliferation at the interface of liquids and air. The mycelium of the agitated and / or rolling cultures grows very quickly as spheres of hyphae, which is hydrated, remained submerged and has the appearance of gelatinous microspheres in small diameter. The spheres of hydrated hyphae flattened by desiccation, in which they were used for the inoculation of petri dishes for propagation of strain and quality control.
[000168] It was found that the sphere diameter in liquid state fermentation must be inversely proportional to the volume and intensity of agitation. The hyphae shear became more effective at higher rolling and agitation intensity and once sheared, the hyphae formed new spheres of the smallest possible diameter, growing in size until they were sheared again. When used in the continuous liquid state fermentation, there was a constant ratio between the ball diameters and then a constant supply of balls in the order of microns was produced.
[000169] Thus, this example demonstrated that the mycelia sphere diameter was manipulated for more effective inoculation with the inoculation efficiency that is inversely proportional to the sphere diameter. EXAMPLE 2
[000170] Mycelium cultures from unsteady liquid state fermentation (proliferation period 2 to 4 weeks) formed a floating mass of hyphae, which were gently mixed with a sharp sterile culture device before being used for inoculation. Smooth mixing was achieved by mixing or low homogenization in a commercial mixer in small breaks at low speeds. Mixed liquid state culture aliquots were used to inoculate sterilized unprocessed vegetables and / or fruits, cereal grains and / or culinary seed or pasteurized culinary species, medicinal herbs, natural flavorings, tea mixtures, green vanilla beans, broad beans of green cocoa and green coffee beans. EXAMPLE 3
[000171] The substrates for mycelium (which contains both inoculated and substrate mycelium culture) in pots or bags were gently mixed every few days until they had commanded to the substrate and became somehow resistant to mixing or agitation , usually 2 to 4 weeks depending on the strain. The products were then in the form of soy meat. Mycelia green vanilla beans were cooked or roasted; mycelia green cocoa beans were roasted or roasted and mycelia green coffee beans were toasted or roasted. The mycelial grains presented in the form of soy meat or as an ingredient in food (s) including soups, chips, breads and meat substitutes, were produced safe to eat and bioavailable, cooking over low to medium heat, 62, 7 ° C (145 ° F) to 73.9 ° C (165 ° F), for 10 min to 60 min, at some point before consumption. Other cultures in pots or bags, such as herbs and spices were dried at 37.8 ° C (100 ° F) to 62.7 ° C (145 ° F) for 1 h to 24 h, packaged and used in a conventional manner.
[000172] The mycelial honey formulations were stirred for 10 min to 90 min at 37.8 ° C (100 ° F) to 51.7 ° C (125 ° F), then poured into small glass bottles. In addition, mycelialized agricultural products have been reformulated into added value products such as egg noodles, meat substitutes, especially flavors, cooking sauces, soup ingredients and the like. EXAMPLE 4
[000173] For a solid batch and liquid batch operation, pure cultures were aerobically proliferated and inoculated into large solid state and large industrial liquid state processors operated continuously and anaerobically for large scale fermentation. food products. After the medium cultures became completely white or a color representative of them for a particular species and had completely overproliferated and controlled the medium and were resistant to gentle mixing, the contents were collected, removed into plastic bags and refrigerated for quick use in 5 ° C (40 ° F) or frozen for long-term storage and subsequent use at -29 ° C (-20 ° F). The fermented medium was prepared in gastronomic human foods including: "soy meat style" meat substitutes, egg noodles, especially flavors, breads, strata and cooking sauces or used directly as a fresh ingredient in soup and / or recipes sautéed or packaged. EXAMPLE 5
[000174] Completely mycelialized agricultural substrates inoculating with pure cultures of fungal strains selected from A. blazei, C. sinensis, G. lucidum, H. erinaceus, G. frondosa, P. eryngii, P. ostreatus, P citrinopileatus, P. djamor, T. versicolor, L. edodes, F. velutipes, V. volvacea, H. marmoreus, P. nameko, T. melanosporum, M. hortensis, P. umbellatus and T. fuciformis were subjected to treatment hot for 1 hour to 24 hours before harvest for 1 min to 2 hours at 62.7 ° C (145 ° F) to 90.6 ° C (195 ° F) followed by recovery at room temperature for 45 min at 48 hours. This process showed a considerable decrease in RNA levels and was formulated in different nutraceutical compositions. EXAMPLE 6 SMALL BATTERY WORK
[000175] 22 kilograms (48 lbs.) Of coffee were divided into 48 equal portions in spherical clean room jars with lids built to allow gas division after an edge. These 22, 0.45 kilogram (48.1 lbs.) Coffee masses were soaked with% quarts of water for two hours. The water in the mixtures was filtered. The coffee pots were then subjected to 90 minutes of sterilization temperatures at 0.1 MPa (15 psi) and placed in a sterile laminar air flow to cool for 8 hours. Once cooled, the prepared green coffee beans were inoculated with half for whole colonies of fungi selected from one of the following: Ganoderma lucidum, Cordyceps sinensis, Tuber melanosporum, Hericium erinaceus, Agaricus blazei, Leafy grifola, Pleurotus ostreatus, Trametes versicolor, Laetiporus sulphureus, Flammulina velutipes, Lentinula edodes, Morchella angusticeps, Morchella crassipes, Morchella crecipes, Tremella fuciformis and Inonotus obliquus, causing three of each to proliferate on an undefined vegetable and a half of fruit juice agar containing coffee extract green as described in Example 8, with sterile tools and in sterile operation inside the lamination flow hood. The mycelial cultures for 7 to 21 days, with samples of each that are pulled out to dry and roast on the 7th, 14th and 21st days. The smell of the culture and taste of mycelia green coffee beans on the 7th day indicated that the cultures were complete, although longer periods of mycelia yielded greater cell mass. LARGE BATTERY WORK
[000176] 239.5 kilograms (528 lbs.) Of green coffee beans were drenched in two different procedures. In the first procedure, the broad beans were soaked three times, for 20 minutes, each soaking, in the second procedure, the broad beans were soaked for 20 minutes through a constant stream of filtered water. The broad beans were then packed in propylene bags with 0.2 micron breathing pieces, with the tops of the bags folded with rubber bands wrapped around the sides of the bags, so that the diffusion of current and gas can occur through the breathing piece and through the folded sides of the bags. The bags were sterilized using a liquid cycle at 0.15 MPa (22 psi) for 80 minutes and then allowed to cool for 8 hours. The bags were inoculated with fungi of the following species: Ganoderma lucidum, Cordyceps sinensis, Tuber melanosporum and Morchella angusticpes. The culture of Ganoderma lucidum was proliferated in a bioreactor, with 10 l of organic potato extract, 2 l of green coffee extract and 1 l of organic mango juice diluted in 100 liters with RO water. The bioreactor was sprayed with compressed air filtered through 0.2 micron hydrophobic capsule filters in-line and the reactor was maintained at 0.01 MPa to 0.02 MPa (2 to 3 psi) through the use of check valves in the air supply and ventilation lines with crack pressure rates from 0.01 MPa to 0.02 MPa (2 to 3 psi). The inoculant was promptly proliferated in 48 hours and was collected through a diaphragm valve located at the bottom of the reactor, which leads to a harvest line that had pipe access and tube out to a current line and current siphon, with an in-line check valve, through one meter and eighty-three centimeters (six feet) of flexible stainless steel housing, for a solenoid valve connected to a timer and foot switch, followed by a flow measurement valve for a sanitary collector adjustment. While it was vaporized, the sanitary collector setting was connected to a ball valve that connected to the current exhaust manifold. The ball valve was closed after vaporizing the line and the ball valve was decoupled from the harvest line once it entered a lamination flow hood in order to keep the entire line sterile. The cultures Cordyceps sinensis, Tuber melanosporum and Morchella angusticeps were proliferated in 4 l bottles, in 1.5 l of the same medium used in the pre-dilution of the bioreactor. These cultures were proliferated for six days and were used to inoculate sterile green coffee bean bags. The broad beans were mycelia for 7 days, in which their odor gave the desired taste profile of the drink produced from the roasted mycelia broad beans, to which they were dried on the 8th day to a moisture content of 13%. EXAMPLE 7
[000177] A fungus suitable for use in the methods of the present invention has been prepared by the following methods. The following strains of G. lucidum were purchased commercially from the Pennsylvania State University mushroom culture collection: 496 Ling ZHI; Singapore commercial line; 7/85; 502 IFO # 8436; IFO-Japan; 7/30/85; 510 Red Oak, State College, PA; D.J. Royse; 9/85; 549 Y.H. Park, ASI-Korea; 5/12/85; 550 Y.H. Park, ASI-Korea; 5/12/85; 551 Y.H. Park, ASI-Korea; 5/12/85; 580 Y.H. Park, ASI-Korea; 10/2/85; 607 Y.H. Park, ASI-Korea; 2/19/85; 617 Y.H. Park, ASI-Korea; 2/25/85; 618 Y.H. Park, ASI-Korea; 2/25/85; 619 Y.H. Park, ASI- Korea; 2/25/85; 620 Y.H. Park, ASI-Korea; 2/25/85; 621 Y.H. Park, ASI-Korea; 2/25/85; 622 Y.H. Park, ASI-Korea; 2/25/85; 623 Y.H. Park, ASI-Korea; 2/25/85; 624 Y.H. Park, ASI-Korea; 2/25/85; 625 Y.H. Park, ASI-Korea; 2/25/85; 626 Y.H. Park, ASI-Korea; 2/25/85; 627 Y.H. Park, ASI-Korea; 2/25/85; 665 Chemo; Philippines; 3/6/86; 669 Y.H. Park, ASI-Korea; 3/25/86; 686 B. W. Yoo; 4/28/86; 724 T. Mitchel, Lawn PSU Forestry Bldg. 9/16/90; 806 Alice Chen; Buffalo, NY; 4/94; 807 Alice Chen; North Caroline; 4/94; 841 White Oak; Campus PSU; J. Peplinski; 8/99. The above strains were grown using the medium described in this document which comprises green coffee bean extract (see Example 9). Many strains were unable to grow and / or died in between. Surprisingly, the inventors found that the strain of G. lucidum 806 Alice Chen; Buffalo, NY, was able to proliferate in the medium comprising green coffee bean extract and was selected for further use in accordance with the present invention. EXAMPLE 8
[000178] Fungi (including the strain of G. lucidum 806, C. sinensis and T. melanosporum as described in this document, also H. erinaceus, T. versicolor, L. edodes, T. matsutake, F. velutipes, A blazei, G. frondosa, P. nameko, L. officinalis, M. hortensis, M. angusticeps, A. auricula, T. fuciformis, I. obliquus, F. fomentarius and L. sulfureus) were maintained in a culture comprising an undefined medium including green coffee bean extract. The experiments showed that the use of the medium including green coffee bean extract for culture maintained the fungus' ability to tolerate, proliferate, metabolize, remove or reduce caffeine or undesirable flavor components. It was also found that successive propagations of fungi as defined above caused an increase and / or improvement in the ability of the fungus to tolerate, proliferate, metabolize, remove or reduce caffeine or decrease components of undesirable taste, which results from training or adaptation of the fungus to the environment indefinite including extract of green coffee beans. Such fungi with altered, improved and adapted properties, as described in this document, in relation to the initial strains, both selected or unselected, were developed. These adapted strains were deposited with ATCC, as described elsewhere in this document.
[000179] The undefined medium including extract of green coffee beans was prepared as follows: 0.9 kg (2 pounds) of pulverized green coffee beans was mixed with 946 ml (% gallon) of water at room temperature. The mixture was left for extraction for 20 minutes with stirring and then filtered three times through fine mesh. Separately, about 5 organic potatoes were placed in 10 l of water and autoclaved for 20 minutes to soften the potatoes. The potatoes were then sprayed with a potato masher and then filtered through fine mesh three times. 1 l of unsweetened commercial fruit juice was added. These solutions were combined and autoclaved. This revenue has also been expanded or reduced as needed.
[000180] The washed green coffee beans were soaked in water and the moisture content was increased to about 30%. At other times, the moisture content has been increased to around 60%. At that time, the bean was well dissipated from chlorogenic acid, as evidenced by the lack of green seen in the bean. Some chlorogenic acid was left in the bean, although most of it was obviously and evidently removed. The removal of chlorogenic acid from the green coffee beans allowed good myceliation at moisture contents of 30% and higher, while the green coffee beans which did not have a chlorogenic acid removal step required a moisture content of 60% for good mycelia.
[000181] Liquid culture: The culture comprising the fungus for use in inoculating the prepared green coffee beans was stirred with sparse air and a motorized shovel to create a turbulent environment and to shear hyphae with pure mechanical force. The double stirring method was superior to each method individually, since the sparse air created the greatest turbulence in the upper half of the culture, while less affecting the bottom, which was agitated by a motorized paddle. In return, the paddle can be rotated at a lower RPM speed and still obtain the hypha ball size obtained in a faster RPM agitation in the absence of sparse. The hypha size was about 2 to 5 microns in diameter. Undamaged mycelium and appropriate morphology in the prepared fungi were prepared by this method and used for culture and / or mycelia. EXAMPLE 9
[000182] Perigord Black Italian Winter Truffles [Truffle 1 (T1) and Truffle 2 (T2)], were aseptically manipulated in the mycology laboratory environment to obtain several new Truffle cultivars for commercial work, including isolates of opposite conjugate types, by the following 4 methods.
[000183] Method 1: whole pieces intact and fresh (less than a week old) from T1 (pieces of truffle meat approximately 10 mm by 20 mm in size) were placed on agar oak (an indefinite agar medium) general based on oak leaves and oak sticks) in 50 Petri dishes, which were sealed with parafilm to allow the cultures to incubate at room temperature (23 ° C) for 5 weeks. After 5 weeks, T1 pieces were seen to regenerate in mycelia described by the following cultivars: (a) vigorous white mycelium that has purple hues in 6 Petri dishes, (b) vigorous golden mycelium with purple hues in 3 plates, (c ) vigorous white and golden mycelium in 6 plates, (d) fragile and non-vigorous purple and golden mycelium in 2 plates and (e) fragile white and golden mycelium non-vigorous in 2 plates. The rest of the Petri dishes (37 dishes) were discarded after becoming covered by contamination (bacterial and fungal). Isolates (a), (b) and (c) were determined to have commercial potential. The (a) vigorous white isolate is the maternal conjugated mycelium of the original Truffle mushroom, while the isolate (b) is the paternal conjugated mycelium of the original Truffle mushroom and (c) is a cultivar derived from asexual recombination or anastomosis of both conjugated types (a) and (b). After 5 to 6 weeks of growth on oak-agar, cultures of (c) vigorous white and golden mycelium began to produce tiny (0.1mm to 2.0mm) truffle fruits. After the structures were grown in fresh Petri dishes and similar agricultural media, these crops produced ever-growing black truffle mushrooms.
[000184] Method 2: fresh intact spores (less than a week old) of T1 (0.01 g to 0.1 g of spore nodules of a mass of approximately 0.1 mm by 0.1 mm in size) were placed in oak agar media tempered in Petri dishes (40 dishes), which were sealed with parafilm for 5 weeks to allow the spores to germinate at room temperature (23 degrees C). After 5 weeks, the spores were seen to germinate on mycelia in 10 plates; however, all mycelium that germinated from the spores, although viable, were extremely fragile and grew slowly.
[000185] Method 3: Fresh intact whole pieces (less than a week old) of T1 (40 g pieces of meat approximately 10 mm by 20 mm in size) were placed in 2 bottles each containing 400 ml of sterile water and 50 ug / ml of each of the following antibiotics: Ampicillin, Penicillin, Chloramphenicol, Erythromycin, Neomycin, Streptomycin, capped, but not tight, in which they were allowed to be incubated undisturbed for 5 weeks at room temperature (23 ° C) with occasional mild agitation. After 5 weeks, the intact pieces were removed from the antibiotic buffer and melted on general undefined agar medium (which contains the same levels of antibiotics), based on oak leaves and branches, in 50 Petri dishes which were then sealed with parafilm to allow cultures to incubate for 2 weeks at room temperature (23 ° C). After 2 weeks of agar regeneration, the T1 pieces regenerated in the following mycelial cultivars: (a) white mycelium in 12 Petri dishes, (b) golden mycelium in 4 plates, (c) white and golden mycelium in 2 plates . The rest of the Petri dishes (32 dishes) were discharged after showing no proliferation or after becoming covered by fungal contamination. The isolates obtained using method 3, although quite similar to the isolates of method 1, were very vigorous.
[000186] Isolates (a), (b) and (c) from Method 1 were prepared as described in this document and used for substrate myceliation. The substrates included prepared green coffee beans. Myceliation of the prepared green coffee was allowed to continue until the desired coffee flavor for the resulting roasted coffee was reached, about 14 days. The resulting mycelia green coffee beans were roasted, infused, served, and tasted by trained tasters. It was found that when the isolate described for method 1 was used isolate (a), a pleasant flavor / aroma, reminiscent of flowers, was achieved in mycelia coffee. When the isolate described for method 1, isolate (c) was used, a pleasant flavor / aroma, reminiscent of truffles, was achieved. EXAMPLE 10
[000187] Mycelia coffee products including mycelia green coffee beans and bases produced by the methods of the present invention contained added polysaccharides and beta glucans. An analysis showed that Robusta coffee bases produced by the methods of the invention had 30.54 mg dextran per gram of coffee bases. This result provided the amount of total polysaccharide in the substrate through a spectrophotometric method based on a modified phenol-sulfuric acid approach. The analysis also showed that Robusta coffee bases produced by the methods of the invention had beta glucans in 0.432%, when measured by the MYBG method using strong hydrolysis conditions to hydrolyze beta glucan with quantification by spectrophotometric method. This represents an advantage over consuming beta-glucans from Reishi mushrooms, since these mushrooms are non-culinary mushrooms for reasons of bitterness, woodiness, and hardness, or in tablet form. EXAMPLE 11 COMPARISON OF TASTE 1, SUMATRAN, PERUVIAN, AND HONDURAN ARABIC BEADS
[000188] A coffee professional and owner of a coffee roasting business (taster) and a trained employee of tested taste, in a double-blind trial, performed a comparison of superior standard coffee beans with the use of Sumatran, Peruvian, and Honduran Arabica (control beans) with coffee beans produced by the methods of the present invention (mycelia beans). Both mycelia and control beans were roasted on the day of the test. These were served side by side with the control, using standard coffee tasting techniques.
[000189] The effects of enhancing the taste of mycelia have been confirmed. The tasters experimented with mycelial and normal infusions of each variety in this blind taste test. Notes were taken. At the conclusion of the tasting, the coffee beans used for each cup were identified.
[000190] Commenting primarily on mycelia, Sumatra has been described as having a fuller, more complex and less bitter taste than the control Sumatra. The tasters stated that this was the only process, of which they were aware, that actually removed a taste deficiency and really accentuated the taste.
[000191] Peruvian mycelia showed an accentuation of remarkable flavor too, being less bitter, softer, and a cup markedly "better" when mycelia. Despite being a high quality bean, the Peruvian control proved "simple" by comparison.
[000192] Of the two tasters, one taster was able to feel a difference in the Honduran infusion. The methods of the invention resulted in the removal of the bitter compounds found in coffee resulting in a better tasting cup of coffee. TASTE COMPARISON 2 ARABICA FAVAS
[000193] A taste test for mycelia coffee was carried out in a cafeteria. The barista / roaster (taster) distributed the formal tasting of his internal Arabica Sulawesi coffee (of Indonesian origin). The beans were selected from the inventory and both mycelia and control beans (Arabica) were roasted on the day of tasting. The results showed that mycelia coffee (produced by the methods of the invention) had an improved taste profile.
[000194] The taster described mycelia coffee as less acid, softer, fuller, more complex and with a better taste in general than the original bean. Several other participants in the taste test also noted the enhanced flavor found in mycelia coffee. TASTE COMPARISON 3 ROBUST FAVAS
[000195] A coffee professional and owner of a coffee roasting business (taster) and a trained employee of tested taste, in a double-blind trial, performed a comparison of coffee beans from control Robusta coffee beans with beans Robust coffee beans produced by the methods of the present invention. Coffee made from 100% Robusta beans is generally considered to be imbibable due to Robusta's high acidity and bitterness. Thus, Robusta coffee beans are not typically used alone, instead Robusta beans are typically pre-processed (for example, steamed), then mixed with more expensive beans to make them palatable.
[000196] This lower quality bean is considered to be imbibable by some due to its bitterness, but Robusta is used commercially in coffee blends to reduce the price. The use of Robusta is growing and the Robusta segment of the coffee market has grown from 39% of the market in 2008 to 41% of the market in 2013.
[000197] The tasters agreed that mycelia Robusta is "effortlessly" a cup of coffee better than non-mycelia. One tester commented that "you have proved that your technology undoubtedly works"; the other taster commented that he is a self-proclaimed coffee snob and "I would drink this mycelia Robusta daily", simultaneously pointing out that the non-mycelia coffee was impossible to digest. More specifically, he noticed a notable lack of bitterness and acidity in the cup, with a more full-bodied taste. The tasters pointed out that a non-professional coffee taster would be able to taste and appreciate the difference, and that mycelia Robusta has great market value. Other employees of the roasting company also noticed the difference.
[000198] The taste test results clearly demonstrated that instant processes accentuate the taste of coffee. The results showed that the processes of the invention removed taste deficiencies such as the bitterness from both Arabica and Robusta coffee beans and enhanced their flavor and value. The processes result in a smoother, fuller and more complex tasting coffee with any of Robusta or Arabica beans.

权利要求:
Claims (12)
[0001]
1. Method for the preparation of a mycelium coffee product characterized by the fact that it comprises: a) providing prepared green coffee beans that comprise the steps of: i. supplying green coffee beans; ii. sterilize green coffee beans to provide prepared green coffee beans; b) supply a fungal component selected from the group comprising G. lucidum, H. erinaceus, T. versicolor, L. edodes, T. matsutake, F. velutipes, A. blazei, G. frondosa, P. nameko, L officinalis, M. hortensis, M. angusticeps, A. auricula, T. fuciformis, I. obliquus, F. fomentarius, L. sulfureus, C. sinensis, and T. melanosporum, prepared as a submerged fungal tissue culture; c) inoculate the green coffee beans prepared with the prepared fungal component; and d) cultivating the prepared green coffee beans and the fungal component prepared to allow mycelia to prepare the mycelia coffee product, in which the mycelia coffee beans are capable of being used to prepare a palatable coffee drink for human consumption.
[0002]
2. Method according to claim 1, characterized in that the step of supplying prepared green coffee beans further comprises at least an aqueous washing of the green coffee beans to reduce the amount of chlorogenic acid.
[0003]
3. Method according to claim 1, characterized by the fact that the stage of supplying prepared green coffee beans further comprises hydrating the green coffee beans.
[0004]
4. Method, according to claim 1, characterized by the fact that the green coffee beans are hydrated to a humidity level of 60% or to a humidity level of 30%.
[0005]
5. Method, according to claim 1, characterized by the fact that the fungal component prepared is G. lucidum.
[0006]
6. Method, according to claim 5, characterized by the fact that the fungal component prepared is strain 806 of G. lucidum.
[0007]
7. Method, according to claim 1, characterized by the fact that the fungal component prepared is C. sinensis.
[0008]
8. Method according to claim 1, characterized by the fact that the prepared fungal component is prepared by a method which comprises maintaining a strain or fungi in an undefined medium comprising an aqueous green coffee bean extract and a source power.
[0009]
9. Method, according to claim 1, characterized by the fact that the cultivar stage is performed for about 7 days.
[0010]
10. Method according to claim 1, characterized by the fact that the green coffee beans are from Coffea arabica.
[0011]
11. Method, according to claim 1, characterized by the fact that the green coffee beans are from Coffea robusta.
[0012]
12. Mycelial coffee product characterized by the fact that it is prepared by the method, as defined in claim 1.

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法律状态:
2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A23F 5/02 (2006.01) |

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2019-09-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-08-04| B09A| Decision: intention to grant|

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2020-12-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361802256P| true| 2013-03-15|2013-03-15|

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US13/844,685|US9068171B2|2012-09-06|2013-03-15|Method for myceliating coffee|

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US13/859,719|2013-04-09|

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US13/859,719|US9427008B2|2012-09-06|2013-04-09|Method of myceliation of agricultural substates for producing functional foods and nutraceuticals|

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US13/874,832|2013-05-01|

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US13/874,832|US20140065131A1|2012-09-06|2013-05-01|Extract of a myceliated agricultural substrate and its use as a nutraceutical composition|

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US201361844498P| true| 2013-07-10|2013-07-10|

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US61/844,498|2013-07-10|

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US201361857671P| true| 2013-07-23|2013-07-23|

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US201361866371P| true| 2013-08-15|2013-08-15|

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US201361867501P| true| 2013-08-19|2013-08-19|

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US201314020781A| true| 2013-09-06|2013-09-06|

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US14/020,512|US20140170264A1|2012-09-06|2013-09-06|Improved method for myceliating raw coffee beans including removal of chlorogenic acids|

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US14/020,512|2013-09-06|

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US14/020,781|2013-09-06|

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US201361878037P| true| 2013-09-15|2013-09-15|

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US201361896097P| true| 2013-10-27|2013-10-27|

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PCT/US2014/029989|WO2014145256A1|2013-03-15|2014-03-15|Myceliated coffee products and methods for making|

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