How to Produce Agave Cultures for Tequila

Abstract

A method for promoting the growth of agave plants by propagating plant tissue from cell culture in the laboratory utilizes cultured cells taken from the leaves of agave plants and grown in a medium containing all the nutrients necessary for the production of new cells and for the maturation of those new cells. The cells are then exposed to photosynthetically active radiation and supplied with additional carbon dioxide, causing the cells to photosynthesize and form the sugars found in naturally growing agave plants. These cultured cells are provided with optimal amounts of light and carbon dioxide to promote growth rates that are unexpectedly and significantly higher than previously achieved.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 312,789, filed February 22, 2022, the entire contents of which are incorporated herein by reference.

[0002] background The present invention relates to the production of tequila, and more directly to a method of making tequila that provides an alternative to the 12 years normally required for the maturation of the agave plant. [Background technology]

[0003] Tequila is a distilled spirit commonly produced from the Weber's blue agave plant. The Weber's blue agave plant, or agave tequilana, is a large succulent plant with long, thorny leaves similar to the aloe vera plant. At the center of the blue agave plant is a corm called a piña. To distill tequila, the corm is roasted and juice is extracted, and the juice is fermented in barrels with yeast to produce tequila. Tequila is a popular distilled spirit and is used in a variety of cocktails, such as the margarita, paloma, and tequila sunrise.

[0004] Traditional tequila production is as follows:

[0005] 1. Harvesting the Agave: Modern tequila production begins with the traditional method of harvesting the blue agave plant, using a specialized blade called a coa to cut the leaves of the agave plant from its underground corm, the piña.

[0006] 2. Roasting the agave heart, or piña. To extract the fermentable sugars from the piña corm, the piña must be roasted. Traditionally, piñas were roasted in stone-lined pits, but today they are roasted either in clay and brick kilns called hornos or in large stainless steel ovens.

[0007] 3. Shredding the Piña and Extracting the Agave Juice: The roasted Piña is crushed and shredded to extract the sweet juice contained within, called the must. The must can be extracted in two ways: using an industrial mechanical shredder (the most common modern method), or one of the more traditional methods, which uses a large millstone called a tahona, which crushes the Piña and extracts the juice.

[0008] 4. Fermentation of the agave juice, or must. Next, the must must be fermented to turn into ethyl alcohol, which is then turned into a distilled spirit. The must is mixed with yeast and water in large fermenters. Either large stainless steel tanks or large wooden barrels are used for this process.

[0009] 5. Distillation of the fermented must. The agave juice is then distilled to purify the liquid and concentrate the alcohol in the mixture. Tequila is usually distilled twice: the first distillation produces a cloudy liquid called ordinario; the second distillation produces a clear silver tequila, which is ready for aging and bottling.

[0010] 6. Tequila Aging. All tequilas are aged for at least 14-21 days. Silver or blanco tequilas are aged the shortest. Aged tequilas come in three types: reposado ("nap", aged for 2 months to 1 year), añejo ("aged", aged for 1-3 years) and extra añejo (aged for more than 3 years). To produce more aged tequilas, the distilled blanco is poured into used oak barrels, which gives the tequila its golden color. There is also a fifth type of tequila called joven ("young") or oro ("gold"), which is a blend of silver and reposado tequilas.

[0011] The Mexican government has strict regulations regarding the production of agave plants, and by law, production is limited to certain Mexican states, including Jalisco, Nayarit, Guanajuato, Michoacán, and Tamaulipas. Furthermore, it takes 12 years for cultivated agave plants to grow large enough to produce tequila. These two restrictions, combined with a rapid increase in tequila sales and the entry of new tequila manufacturers, have created a severe shortage of agave plants. Currently, agave production is far below demand, and the situation is expected to worsen in the near future. Of all these issues, the biggest contributing factor to the shortage of agave plants is the plant's 12-year growing period. Until now, there was no way to obtain the required sugars and nutrients other than waiting 12 years for the plant to mature. Summary of the Invention [Means for solving the problem]

[0012] Summary of the Invention The present invention is a method for promoting the growth of agave plants by growing plant tissue from cell culture in the laboratory. A novel method has been developed that allows cells to be grown for short periods of time without being permanently part of the plant. Cultured cells are taken from the leaves of agave plants and grown in a medium that contains all the nutrients necessary for the production of new cells and the maturation of those new cells. The cells are then exposed to photosynthetically active radiation and supplied with additional carbon dioxide, causing the cells to photosynthesize and form the sugars found in naturally growing agave plants. These cultured cells are given optimal amounts of light and carbon dioxide to promote growth rates that are unexpectedly and significantly higher than previously achieved.

[0013] Once the culture matures and produces sugars, for example at around 8% sugar in the liquid medium, yeast is added and the entire cell culture suspension is fermented until the sugars are completely converted to ethanol. In a preferred embodiment, citric acid is added to lower the pH of the liquid medium to a range of 3.5-4.5. This lowering of the pH promotes the photosynthetic process as follows: the energy required for the light reaction of photosynthesis is used to abstract hydrogen from water molecules to convert NADP to NADPH, and by lowering the pH to a level where the cells can still survive, more hydrogen ions are available to the chloroplasts, thereby reducing the amount of light energy required to split the water molecules.

[0014] The ethanol is then removed, along with other aromatic compounds produced, through distillation to produce tequila. This process reduces the 12-year waiting period to about 24 weeks, dramatically increasing production and process efficiency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The process of obtaining tequila according to the present invention is divided into four stages, as described below: The following description should be considered as the inventor's best mode for carrying out the invention, but it will be understood that there are variations in the ingredients and conditions under which the final product is still obtained, and does not exclude the above-mentioned variations that a person skilled in the art would recognize.

[0016] Initiation of Agave-derived callus cultures: Preparation of callus induction medium Prepare a solution by adding sucrose (3%), a 0.004% stock solution of naphthalene acetic acid (NAA, 1%), and powdered Murashige and Skoog basal medium (0.44%) to distilled water to make a total of 100%.

[0017] Callus induction medium is prepared using Murashige and Skoog (MS) medium obtained from Sigma, combining 3% sucrose and 1% naphthalene acetate (from a concentrated stock solution of 0.004% w / v). The prepared medium is preferably adjusted to pH approximately 5.75 and solidified using 0.2% phytagel. The medium is then autoclaved at approximately 121 °C for 20 min and poured into sterile plastic plant tissue culture dishes.

[0018] Initiating Agave-derived Callus Cultures: Sterilization of Plant Tissue Agave leaf tissue is sterilized by immersion in a 70% ethanol solution for 2 minutes, followed by immersion in a 10% bleach solution for 10 minutes. The agave is washed multiple times with sterile (autoclaved) distilled water and aseptically cut into disks in a sterile clean bench. The agave slices are placed on the prepared plates containing callus induction medium, and the plates are then sealed with a film such as Nesco film. The sealed plates are then placed in a dark box with an air temperature of approximately 27°C. Callus formations will begin to be visible after approximately 30 days under these conditions.

[0019] Media preparation for established cultures In the third step of the present invention, the callus formations are introduced into a growth-promoting medium. The previously indicated medium from the first step (distilled water, sucrose, Murashige-Skoog basal powder and NAA) is repeated with the addition of 0.01% of a vitamin stock solution containing 0.05% pyridoxal hydrochloride, 0.10% thiamine dichloride and 0.05% g of nicotinic acid. The basal medium is obtained by mixing 3% sucrose, 0.44% powdered MS, 1% NAA stock and 0.01% vitamin stock with distilled water to make a total of 100%. The components are mixed using a magnetic stirrer until all the dry components are dissolved, and then the pH of the medium is adjusted to 5.75, first using a 1M solution of NaOH and then a 0.1M solution of NaOH. Using a 250 ml Erlenmeyer flask, 50 ml of medium is introduced and then the mouth of the flask is sealed with metal foil. Each sealed flask is sterilized in an autoclave at 121 °C and 103 kPa for about 25 minutes. The flask is then placed in a clean bench and allowed to cool to ambient temperature.

[0020] Inoculation and subculture of established cultures The final step in this process is to proceed with subculture from the inoculation and established culture. First, the clean bench and equipment are sterilized with 70% ethanol solution, and in the case of equipment (tweezers, spatulas, etc.), they are heated to red heat and then allowed to cool in the sterile clean bench. In the clean bench, the metal foil is removed from each flask of prepared medium, and a small piece of friable callus about the size of a thumbnail is removed from the plant tissue using sterile tweezers. The friable callus is broken apart and added to the flask as finely dispersed cells. In a preferred embodiment, about 5 grams of tissue is added to 50 ml of medium (10% w / v). After the callus is added to the flask, the mouth of the flask is flamed and then covered with a sheet of sterilized metal foil. The sealed flask is placed on a shaker at 120 rpm in a dark room at an ambient temperature of 27° C. The flask is left until a thick dispersed cell suspension culture is observed (about 2 weeks).

[0021] Subculture After this culture period, the metal foil is removed from the flask of prepared medium in a sterile clean bench, as well as from the flask containing the dispersed cell suspension culture (produced by the initial inoculation). A sterile wide-hole spatula is used to scoop up the cells and add them to fresh medium. In a preferred embodiment, about 5 grams of tissue is added to 50 ml of medium. Citric acid may be added to the medium to lower the pH to a range of 3.5 to 4.5, more preferably to pH 4.0. The mouth of the flask is covered with a sheet of sterile metal foil. The flask is then placed on a shaker at 120 rpm in a lighted room at an ambient temperature of 27°C. After 14 days, the cell suspension culture can be used for further subcultures or the cells can be harvested and lyophilized.

[0022] Establishment of intermediate cultures The final step in this process is the preparation of 20 liter culture bags. This begins with making the medium using 3% sucrose, 0.44% powdered MS, 1% NAA stock and 0.01% vitamin stock in 100% total distilled water. Using a magnetic stirrer, mix until all dry ingredients are dissolved, then adjust the pH using 1M and 0.1M NaOH solutions to a pH of 5.75. Using three sterile 20L (nominal) containers, aseptically add the medium to each bag using a peristaltic pump, tubing and a 0.2 micron sterile filter. This transfer should be done in a sterile clean bench. Using a sterile 50ml syringe, transfer 200ml of cells from a 250ml flask into the medium in the bag using the addition port on the container. After the mixture has been cultured in the lab for 4 weeks, place the culture container under PAR light with 1W light output per liter of culture. Air is added to the liquid through an aeration system at an air flow rate of 200 ml / min. Carbon dioxide is added to the liquid through an aeration system at a CO2 flow rate of 50 ml / min. The mixture is incubated for approximately six weeks until the total sugar content, measured by dry weight, is equal to or greater than six percent (6%). At that point, all light and gas flows are stopped and brewer's yeast is added to the liquid. It is then allowed to ferment for a period of approximately five to ten days. After fermentation, the ethanol produced can be removed by distillation. Water can be added to the distillate to reduce the ethanol content to that expected for tequila.

[0023] While the above process represents the inventor's best mode for carrying out the invention, those skilled in the art will recognize modifications and permutations of this process that still yield tequila in a much shorter period than the normal growth and development of the agave plant. The present invention is intended to encompass all such modifications and permutations that would be apparent to those skilled in the art, and nothing herein should be construed as excluding any variation of the method unless expressly so stated. Rather, the present invention is not limited to the above description, but should be considered as including any modifications and permutations within the scope of the claims that use their ordinary and accustomed meanings that are not inconsistent therewith.

Claims

1. 1. A method for culturing agave cells for the production of tequila, comprising: (a) preparing a callus induction medium comprising sucrose, naphthalene acetic acid and water; (b) placing agave cells on said induction medium and culturing callus formation; (c) establishing the mixture by placing the callus formations on an enhancing medium containing sucrose, naphthalene acetate and water to which has been added a vitamin stock solution, and adjusting the mixture to a pH of 5.5 to 6; (d) heating the mixture of (c); (e) cultivating a subculture from a promotion medium using the culture obtained from the mixture of (d) and adding citric acid to a pH of 3.5 to 4.5 and introducing light to the subculture; (f) adding carbon dioxide and yeast and fermenting; wherein the ethanol concentration is adjusted to produce tequila.

2. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the callus induction medium comprises Murashige and Skoog medium.

3. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the callus induction medium has a ratio of sucrose to naphthaleneacetic acid of 3:

1.

4. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the callus induction medium is solidified with phytagel.

5. 2. The method of culturing agave cells for the production of tequila according to claim 1, further comprising sterilizing the agave prior to introducing the cells into the callus induction medium.

6. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the agave cells and the callus induction medium are sealed in a container and placed in the dark.

7. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the vitamin stock solution comprises pyridoxal hydrochloride, thiamine dichloride and nitric acid.

8. 2. The method of culturing agave cells for the production of tequila according to claim 1, wherein the ratio of culture to promotion medium in step (e) is 5 grams to 50 mL.

9. 10. The method of culturing agave cells for the production of tequila as described in claim 1, wherein 1 watt of light is used per liter of culture.

10. 2. The method of culturing agave cells for the production of tequila as described in claim 1, wherein the total sugar content as measured by dry weight is six percent (6%) or greater.