I FIBROUS NORTH SUPPORT FOR THE FERMENTATION OF CLEAR BEER AND WINE
CROSS REFERENCE WITH RELATED REQUESTS The present invention claims the benefit of the filing date of the US Patent Application Series No. 60/271, 371 filed on February 23, 2001, in accordance with 35 U.S.C. § 1 1 9 (e), whose description is incorporated into the present invention as a reference. Background of the invention Although the fermentation of alcohol can be expressed through a series of biochemical reactions catalyzed by enzymes, the brewing of beer and wine are ancestral arts practiced centuries before the science of chemistry was born. The biochemistry of winemaking was demonstrated in 1 856 by Louis Pasteur, when he showed that the wine is produced when simple sugars are fermented in fruit juices, through the yeast Saccharomyces cerevisiae, which produces ethanol and carbon dioxide. The vinegar was produced after the fermentation of the alcohol, if the yeast cultures were contaminated by other organisms, such as acetic acid bacteria. Beer is processed in a similar way by fermenting the yeast from the carbohydrates present in cereal grains, such as barley. These carbohydrates, largely polysaccharides, are not degraded by yeast glycolytic enzymes, which act only on disaccharides and monosaccharides. This problem is overcome by making the "malt" of the grain. In malt processing, cereal seeds are allowed to germinate until they form the proper enzymes required to break the polysaccharides in the cell walls, as well as the starch and other food reserves of polysaccharides found within the seed cells. Germination is then stopped by controlled heating. The malt now contains enzymes such as alpha-amylase and maltase which have the ability to break down the starch into maltose, glucose and other simple sugars. In the next step, the brewer prepares the liquid by mixing the malt with water and crushing it. This allows the enzymes to break the cereal polysaccharides into simple sugars that are soluble in the liquid medium. Subsequently the cell matter is separated and the liquid is boiled with funnels to provide the taste of the beer. Subsequently, yeast cells are added. In the presence of oxygen, the yeast cells are activated, that is, they grow and reproduce in a very fast way. Ethanol is not formed until all the oxygen is used. Under anaerobic conditions, the yeast ferments the sugars in ethanol and carbon dioxide. This fermentation process is controlled in part by the concentration of the ethanol formed, the pH and the amount of sugar present. Once the fermentation has stopped, the unprocessed beer or wine (in the absence of the funnels) are ready for final processing, for example, the adjustment of the amount of "top layer", concentration of C02, addition of flavorings and removal of cells and particulate matter to form a clear final product. Soft drinks and liquors are prepared initially in the same way as beer, through the fermentation of a crushed cereal. The alcohol produced by the fermentation is then distilled to produce a product having an alcohol content of 30 to 50%. The only flavor present in the final product, for example, rum, vodka or gin, are those volatile compounds that accompany alcohol or which are added to concentrated alcohol. These methods require long periods of fermentation and a large processing and storage equipment. As can be seen, these requirements restrict production capacity and greatly increase the cost of both production and storage. Various processes have been tried to shorten the time required for alcohol fermentation and post-fermentation processing to create a clear product. NeverthelessIn almost all cases such efforts result in the deterioration of the aroma and taste of the final product, as well as time, cost and intense labor. Therefore, what is needed is a method for preparing a rapidly fermented and filtered (clear) alcohol product, which has all the desired flavor characteristics of a beverage processed through conventional means. Summary of the Invention The present invention provides a method and means for quickly producing alcohol and alcoholic beverages, including beer and wine, by fermentation, for example, from 1 8 to 48 hours, for example from 22 to 36 hours, which produces a product filtered, of course. One embodiment of the present invention is a method for producing an alcohol solution that includes contacting at least one microorganism, for example, Saccharomyces cerevisiae, which is immobilized on a fiber, for example, cellulose, including micro crystalline cellulose and wood chips. wood, or cellulose derivatives, for example, carboxymethylcellulose, ethylcellulose or methylceiulose, or a sponge such as sea sponge or vegetable sponge, the support with a fermentation mixture (feed material) to produce the alcohol (beverage) solution. Preferably, the fibrous support comprises cellulose, for example, sulfite-free paper. The alcohol beverage is prepared by preferably contacting a fermentation mixture with at least one alcohol-tolerant strain of S. cerevisiae and at least one flavoring organism, such as a yeast and / or a specific bacterium, e.g., a species of lactobacil us. In one embodiment, at least two, more preferably at least three, alcohol-tolerant, sugar-tolerant strains of Saccharomyces cerevisiae that are immobilized on the fibrous support contact the fermentation mixture. A highly sugar-tolerant, highly sugar-tolerant strain, such as S. cerevislae ATCC No. 20867, can survive in media having concentrations of up to about 20% alcohol and 45% sugar, in contrast to conventional strains that can tolerate concentrations only up to 7 to 8% alcohol and 1 0 to 1 5% sugar. An alcohol and sugar tolerant strain can tolerate alcohol concentrations of up to about 10% and sugar concentrations of up to about 15%, for example strains 46 and 414. A "highly" tolerant alcohol and sugar strain can tolerate concentrations of alcohol greater than 10% and up to approximately 20% and sugar concentrations greater than 15% and up to approximately 45%. As used in the present invention, the term "about" means that the sugar and / or alcohol contents may vary from the amounts mentioned within the deviations from the measurement techniques employed in the art. More preferably, flavor-producing microorganisms are also immobilized in the fibrous support. Preferred flavor-producing strains are Lactobacillus species including Lactobacillus plantarum, for example strains 133 or 88 and / or Lactobacilus delbruekii. In one embodiment of the present invention, rapid brewing of beer and an alcoholic beverage is carried out by immobilizing the organisms that are in a fibrous support, which reside in a single chamber or container to form a bioreactor and subsequently allow the Fermentation mixture flows in the reactor on and inside the fibrous support (s). For example, the bioreactor may include one or more C02 gates to remove gases and to provide a favorable environment for the immobilized cells, thereby maximizing fermentation efficiency. The composition of the organisms present, for example, bacteria and / or yeast, the volume of the bed of the column, the pH, the concentration of sugars and flavorings in the feeding material, and the flow indexes are determining factors the time to process a drink and its final characteristics. Thus, the present invention also provides a bioreactor comprising: a) a fibrous support for immobilizing the cells of a micro organism; and b) a population of cells adhered to or within a substantial part of the surface of the support. Brief Description of the Drawings Figure 1 is a diagram of an example bioreactor for a dual purpose process system for rapid fermentation, which produces a beer and / or light wine. Detailed Description of the Invention The present invention provides a method and a bioreactor to be used in the practice of the method for the production of alcohol and alcoholic beverages by fermentation with microorganisms that have traditionally been used for fermentation, which are immobilized on a support fibrous which is preferably cellulose, more preferably sulphite-free cellulose paper, for example, which is approved by the FDA. Fermentation occurs by mixing a fermentation mixture with the above-mentioned mobilized microorganisms in a chamber where alcohol is produced. The fibrous support conveniently removes unwanted material, such as cells and particulate matter from the fermentation product produced according to the method of the present invention, which results in a filtered product, for example, clear alcohol or a clear alcoholic drink. Preferred organisms for use in the methods and bioreactor of the present invention, include at least one strain tolerant to alcohol and higher sugar of S. cerevisiae, as well as at least one strain of S. cerevisiae that produces flavor, and / or at least a strain of lactobacillus or other bacteria that produce flavor. The term "alcoholic beverages" includes beverages made by the fermentation of grains and fruit juices, such as beer, either fermented varieties "in the upper part" or "in the bottom part" (from 0.5 to 8% alcohol); wine (from 12 to 21% alcohol); soft drinks (from 21 to 30% alcohol); and liquors (30 to 50% alcohol). For soft drinks and liquors, the final product is distilled after fermentation. The substrates employed for the manufacture of the beverage according to the method of the present invention, vary depending on the type of beverage that will be produced. The beer and liquor substrate is a malt formed from a crushed cereal. The substrate of the wine is usually crushed fruit or fruit juice. The alcohol that is used to increase the alcohol content of a beverage or for uses other than consumption, may be produced using a method of the present invention in a variety of organic substrates. Malt alcoholic beverages having a normal alcohol content, but having a reduced caloric content, which are elaborated by adding extra enzymes during crushing to break up the malt substances, can also be prepared using the method of the present invention. The ratio of sugar to liquid to produce beer is approximately 997.90 g (2.2 pounds) of sugar of approximately 498.95 (1.1 pounds) of malt in about 6.2 liters of liquid, which produces a preparation that has from about 3 to 4% alcohol For wines, the proportion of sugar to liquid is greater than 0.2, depending on the value of the percentage of alcohol required and the theoretical conversion of 1 00 g of sugar to 50 g of alcohol. The wine is prepared according to the present invention, using crushed and filtered fruit to form the substrate solution. The husks and seeds are removed before crushing, according to the desired characteristics of the final product (for example, white wine versus red wine). The variables in the final product result from the differences in the sugar content, the source of the pulp, the fermentation organisms, the pH, the fermentation time and the like. Once the wine reaches from about 12 to about 20% alcohol, the primary fermentation is completed, and the wine can be consumed and / or aged easily or processed in an additional way according to the desired taste and alcohol level. Other alcoholic beverages are prepared in a form initially similar to that of beer, by fermenting a crush formed from cereal grains. These include whiskey, scotch, rum, gin and vodka, as well as soft drinks. The final alcohol content of these beverages is higher than that of wine and beer, around 21 to 30% for soft drinks and above 30%, usually above 45% for liquors. The alcohol obtained by fermentation according to the present invention is distilled to produce concentrations of more than about 20%. Alcoholic beverages produced by distillation, with the exception of whiskey and liquors, do not contain any flavorings other than the volatiles associated with alcohol. Whiskey is traditionally flavored by storage and in charred oak barrels. Soft drinks may contain other flavorings, such as fruit syrups. The alcohol content of all these beverages can be increased by the addition of distilled alcohol to the fermented product of up to 20% or by increasing the amount of sugar present in the fermentation mixture.
When used in the present invention the words "primary fermentation" mean a fermentation process, which starts by the addition of yeast to a liquid, for example, fruit juices. "Secondary fermentation" indicates the process of storing a malt beverage or unripe wine after primary fermentation until it acquires the characteristic aroma and flavor of a mature beverage. When the secondary fermentation is carried out at low temperatures under pressure, the physico-chemical stability of the beverage is increased and the dissolution of the carbon dioxide in the beverage is improved. The present invention comprises strains of S. cerevisiae including mutant strains of S. cerevisiae which are highly tolerant to sugar and alcohol. The higher sugar concentrations required for higher alcohol production can cause cell death or plasmosis. Yeast usually can not tolerate alcohol in concentrations that exceed approximately 7 to 8%. High tolerance to alcohol is defined as the ability of a yeast strain to survive and proliferate in a medium that has a concentration of alcohol in which normal yeast strains can not survive and proliferate, for example, which is higher to approximately 7 to 8%. In addition, normal yeast strains can tolerate about 10 to 15% sugar. The sugar-tolerant yeast can tolerate concentrations exceeding about 20%, and preferably exceeding about 45% sugar. An alcohol-tolerant, sugar-tolerant yeast strain was deposited with the American Type Culture Collection, Rockville, MD, on September 7, 1987, and assigned the ATCC number 20867. This yeast can tolerate concentrations up to approximately 20% alcohol. Known yeast strains of S. cerevlsiae can be used to develop mutants that have the ability to grow in medium containing higher alcohol, higher sugar, employing basic techniques that are related to the mutation of micro organisms known to those skilled in the art, see, for example, US Patent No. 4,029,549 and references herein. Normally, the organisms are exposed to a mutation agent and subsequently classified according to the desired characteristics, for example, growth in a mixture containing higher alcohol, higher sugar. Other strains of organisms that are part of the present invention include two strains of S. cerevisiae that produce flavor, for example, strain 46 and strain 414 which are also alcohol tolerant and sugar tolerant strains, which include the strain that gives flavor to the beer deposited with the ATCC on September 7, 1987 and with the assigned number ATCC No. 20866; and a strain of microorganisms to produce flavor and aging, such as the lactobacillus species, for example, L. plantarum 133, L. plantarum 88 and L. delbruekii 17. Microorganisms useful in the practice of the methods of the present invention are They maintain in crops using the methods and materials available to those skilled in the art. By fermenting a mixture of malt liquor or a mixture of fruit juice in the presence of a strain of Saccharomyces cerevisiae that produces flavor or other bacteria, such as lactobacillus and a strain of alcohol-tolerant Saccharomyces cerevisiae, the index can be accelerated of fermentation to complement the fermentation of the beer or wine in approximately 18 to 48 hours, for example from 22 to 36 hours. The microorganisms are immobilized within and on fibrous supports having a superior surface area. These supports can be used in combination with rigid inert supports, such as porous glass or organic granules, although other materials such as ceramics and alumina can be used (only to be used in the production of a combustion alcohol) and other types of supports . Care must be taken to avoid that the supports can affect in a decisive way the flavor. The preferred average granule diameter for rigid inert supports is 0.4 to 0.45 cm, although granules with a diameter between 0.25 and 0.6 cm are acceptable. The supports have a sufficient surface area to damage 1 09 to 1012 cells / g support. The immobilized organisms are suspended in a bioreactor with a length and diameter proportional to the amount of feed material that will be fermented. The system can be designed to ferment the feedstock to produce a desired concentration of alcohol in a single step or in multiple passes through the reactor using a peristaltic pump. The reactor preferably consists of one or more "columns" or "hollow chambers", for example, a cylindrical column. In one embodiment, the organisms that produce flavor are placed in a first chamber (bottom part) and the upper alcohol tolerant yeast in addition to the juice (for wine) in the second chamber (top), so that the feed material can be fed in the chamber of the bottom part and later to the chamber of the upper part through the pump to obtain the desired flavor, while the organisms that produce the taste through high exposure are not exterminated. concentrations of alcohol. In a preferred embodiment all immobilized microorganisms are placed in a single column or chamber. The system can be designed to ferment the substrate to produce a desired alcohol concentration in a single step or in multiple passes through the reactor. Processing batches of an alcoholic beverage using superior alcohol tolerant yeast strains In one embodiment, the organism that produces the taste (or mixtures of organisms) and a strain of S. cerevisiae highly tolerant to alcohol, such as ATCC 20867, is immobilized in a fibrous support and mixed with a liquid substrate in a fermentation chamber. The mixture is heated for up to about two hours, at a temperature of 25 ° C and 40 ° C, preferably about 30 ° C, while an oxygen-containing gas is bubbled through the mixture to activate the mixture. culture. The fermentation temperature in the fermentation vessel is subsequently lowered between 24 and 35 ° C, preferably about 28 ° C, and maintained at a lower temperature level for approximately 14 to 48 additional hours to complete the fermentation. The fermented beverage is then removed from the fermentation vessel and the spent yeast is separated from the beverage. The general batch processing is described further through the following "recipe" without limitation. One and one tenth pounds of heated liquid malt extract are poured into a barrel fermenter of 1 8,927 It (5 gallons). Two and a half pounds of sugar and about ½ teaspoon of salt are added to the malt extract in the fermenter. Subsequently the fermenter is filled with approximately 1 6.2 liters of hot water distilled recently. Approximately equal amounts, approximately 0.5 liters, are added of a strain of yeast that produces fibrous immobilized flavor and an alcohol-tolerant yeast strain at an effective concentration to allow rapid fermentation, about 1010 cells / ml of paste. The fermentation is allowed to proceed at a temperature of about 30 ° C (the range of useful temperature is between about 25 ° and 40 ° C), for a time between about 1 and 2 hours, by bubbling oxygen gas or a stream of air in a constant stream through the liquor to activate the crop. Subsequently the temperature of the fermenter is lowered to approximately 28 ° C (the acceptable range is between 24 ° and 35 ° C), and it is maintained at this level for a period of approximately 14 and 18 hours. After a period of approximately 18 hours (the range is between approximately 12 and 48 hours or when the fermentation is completed), the malt beverage is extracted from the top, a beer completely fermented. For wines and beer, approximately 1/3 of alcohol-tolerant yeast, 1/3 of lactobacillus, and 1/3 of flavoring yeast may be used. If a higher alcohol content is desired, more sugar can be added at intervals during the fermentation process. A process for preparing beer according to the method of the present invention has the following steps: Place the malt liquor fermentation mixture in a fermentation vessel; heat the mixture for between 1 and 2 hours at a temperature between 38 ° and 40 ° C; add one half of a total funnel amount to the fermentation vessel with a funnel, eg, 1/3 of a malt can;
heat with a funnel the fermentation mixture at a temperature between about 50 ° and 55 ° C for an additional 0.5 to 1 hour; raise the fermentation temperature of liquor to between approximately 60 ° and 65 ° C and keep the temperature at a level between approximately 30 and 35 minutes more, then add the other half of the intervals; increase the temperature of the container to between approximately 70 ° and 75 ° C, maintaining this temperature for approximately 25 and 35 more minutes; and finally increasing the temperature of the vessel to between about 80 and 90 ° C, boiling for between about 30 and 35 minutes and then rapidly cooling the mixture to between about 25 ° and 35 ° C. Add to the fermentation vessel water and a mixture of the strain of S. cerevisiae that produces malt flavor and at least one, preferably two and more preferably three strains of S. cerevisiae highly tolerant to alcohol and tolerant to sugar with the ability to accelerate the fermentation of malt liquor; heat the mixture to between 30 and 35 minutes at a temperature between 24 ° and 30 ° C while bubbling an oxygen-containing gas; the temperature in the fermentation vessel at between about 26 ° and 28 ° C, and keep the temperature level low for between about 24 and 48 hours until the fermentation is complete. Subsequently, the drink is extracted from the fermentation vessel. The alcohol malt beverage produced in accordance with the present invention can be rinsed, salts and / or CO 2 can be added, and bottled for distribution according to procedures known in the art. Processing of an Alcohol Drink in a Bioreactor Containing Immobilized Microorganisms in a Fibrous Support By immobilizing large numbers of microorganisms in the bioreactor, fermentation is accelerated by processing the beverage in a continuous range without the addition of a new yeast. In a modality for processing beer, the yeasts that have traditionally been used to produce alcoholic beverages are immobilized on a cellulose support. In a preferred embodiment for processing beer, a mixture of three strains of yeast, for example ATCC 20867, strain 46 and strain 414, which are tolerant to alcohol and sugar, at least one of which is a strain that produces flavor, and at least one strain of bacteria to produce flavor, are immobilized on a fibrous support, for example, sulfite-free paper in a single chamber or section of the bioreactor. The desired volume of immobilized cells is from at least 109 to about 1012, more preferably at least about 1010 cells / ml paste. The total number of immobilized cells can be determined using standard procedures, for example, the yeast cells are counted in an adequate dilution of supernatant after immobilization using a hemocytometer (Levy and Levy-Hausser corpuscle count chamber, Hausser Sci. , Philadelpía, Pennsylvania). Also see the publication by Hamdy et al. (Biomass, 21, 189 (1990)). The feedstock is fed into the bioreactor and passed through the immobilized organs. After reaching a desired percentage of alcohol, which can take from about 24 hours to about 48 hours, the fermented liquor is extracted, bottled or further processed, as necessary. The viability of strains on substrates can be determined through the modified methylene blue of Hamdy (1990) (see also the Manual of Microbiological Methods, Society of Microbiol., 1957). Resazurin can also be used (Latham and Sharpe, In: Isolation of Anaerobes, 5th ed., Sharpten and Borrad (eds.), Pages 133-147, Academic Press, New York (1 971)). A change in color in cells means they are viable cells. Factors that affect the immobilization of yeast include incubation time, pH, medium; form, composition and size of the substrate; amount of sugar, type of supports and the like. Any form of cellulose-based support can be used, although maintaining a suitable surface area for adhesion of the cells and minimizing the shear force caused by the combination of the flow of the fermentation mixture and the evolution of C02 in the reactor, are considerations that are taken into account in the design of the materials. The construction of bioreactors and processing of an alcohol beverage in bioreactors is further described through the following non-limiting example. EXAMPLE 1 Dual Purpose Bioreactor for Rapid Fermentation of Beer or Wine Which Leads to a Final Product Clear Filtration Cells Yeast are allowed to grow for 48 hours at a temperature of 37 ° C (stationary phase) in a flask of a malt-glucose broth placed inside a metabolic stirrer. This broth contains g / l of deionized water as indicated below: yeast extract, 5; Malt extract, 20.0; glucose, 10.0; sucrose, 5.0; Tryptone, 1.5; peptone, 1.5; KH2P04, 1 .0; NH4CI, 2.0, and NaCI, 2.0; and oligominerals (for example, Mg, Ca, Zn, Cu, S, n and / or Fe). A dark brewing feedstock containing 453.59 g (1 pound) of malt extract includes, 272.15 (0.6 pounds) of sugar; 1 gram of salt; and 4,542 (1.2 gallons) of hot water. The feed material was introduced into a bioreactor chamber as shown schematically in Figure 1. The temperature of the circulating feed material can be controlled externally between about 25 ° C and about 37 ° C by means of a pump with circulating water through a water bath for the sheath of the column. The pH of the feed material was maintained between about 4.5 and 5.5. By varying the flow rate it is possible to improve the efficiency of the system. In general, decreasing the flow rate increases efficiency. Decreasing the flow rate also provides a means for minimizing the shear forces within the bioreactor and thereby decreasing the number of discharged organisms (washed cells) in the supports, thus allowing long-term system operation. The process was carried out in a bioreactor chamber containing a yeast mixture and other immobilized cultures were employed on / within a modified sterile cellulose paper having no sulfite residues (Rayonier, Inc.). Cellulose and wood pulp were approved by the FDA for use in food and food contact applications. Crop can be used, such as will be described below and in US Patent No. 4,929,452 and Series No. 09 / 086,020, however the present invention is not limited to these strains since any other strains that can be used can be used. be immobilized in a fibrous support. Fermentation in the bioreactor occurred in a vertical durol plastic chamber (Figure 1) containing a pulp-paper paste on which three different strains of yeast and lactobacillus cells were immobilized at a cell density of approximately 109 to 1012, by Example 1 010, cells per milliliter of pulp. The substrate (feed material) was fed into the chamber through a peristaltic pump equipped with an overflow apparatus and the effluent was filtered during recycling in the chamber to remove the yeast cells. The substrate, such as malt extract with beer funnels or fruit juice for wine, was fed continuously in a desired range from a six liter glass container to the chamber, eg, 25 to 96 ml per minute, and It was recycled. This range can be adjusted automatically using a variable speed peristaltic pump. This process continues for 24 hours for beer and 22 to 48 hours for wine. The clear end product was intrinsically filtered in the chamber by continuous recycling of the effluent through the pulp-paper and collected (recycled) in the same original container, where the samples were tested for neutral alcohol (distilled alcohol) and a refractive index reading was taken to determine the concentration. One embodiment of the present invention is a process that employs a combination of the following: S. cerevisiae (ATCC 20867), a strain tolerant to alcohol and sugar; S. cerevisiae 46 (a strain that produces flavor, tolerant to alcohol and sugar); S. cerevisiae 414 (a strain that produces flavor, tolerant to alcohol and sugar); and Lactobacillus plantarum 133 (taste and aging); L. plantarum 88 (taste and aging); and / or L. delbruekii 1 7 (flavor and aging). These six strains, or a combination thereof, can be used to make red and white wine and beer, depending on the substrate used. For example, the mixture includes:
S.cerevisiae 20867 ÷ S. cerevisiae 46 L plantarum 133. L plantarum 88. + S. cerevisiae 414 v L. delbruekii 17 60-65% 40-35% equal components equal components
For red wine, a preferred blend includes: S. cerevisiae 20867 + S. cerevisiae 46 + S. cerevisiae 414 + L. plantarum 133 + L. plantarum 88. For white wine, a preferred mixture includes: S. cerevisiae 20867 + S. cerevisiae 46 + S. cerevisiae 414 + L. delbruekii 17. For beer, a preferred mixture includes: S. cerevisiae 20867 + S. cerevisiae 46 + S. cerevisiae 414 + L. plantarum 133. This bioreactor system can be used with the pulp-paper and immobilized yeast cultures for domestic use or can be scaled for industry. In addition, this double process of the bioreactor system can be used for more than 10 fermentations, for example during a period of more than 3 months. The pulp-paste paper in the chamber filters the unfermented malt infusion, while the immobilized cultures ferment the substrate present in the unfermented malt infusion in ethanoi, since the substrate is continuously recycled through the bioreactor. The amount of alcohol produced depends on the level of sugar and only the C02 gas is allowed to escape through an outlet door located at the top of the chamber towards the container. This C02 can be collected for future use, if desired. In particular, the bioreactor of the present invention rapidly ferments the sugar to ethanol, for example, one mole of glucose can produce at least 2.0 moles of ethanol and 2.0 moles of CO 2 gas, and produces a clear end product, for example, wine or beer depending on the substrate used. A second fermentation can be carried out for the synthesis of C02 of clear beer out in capped bottles or in a container in which an aliquot of yeast and sugar cultures is added, which leads to a desired carbonation. A non-toxic combination of specific chemicals with a known concentration, such as sodium carbonate, citric acid and fructose, can be used in place of sugar and yeast to achieve the desired carbonation of the beer or bubbling wine. The yeast cells can be stored for several months at room temperature (approximately 24 ° C) in a sterile citrate buffer (pH 3.5) after which they can be activated with non-toxic phosphates and used in fermentation. All publications and patents are incorporated herein by reference, since they are considered incorporated individually as reference, provided they are not inconsistent with the present disclosure. The present invention is not limited to the exact details shown and described herein, for which it should be understood that many modifications and variations may be made while remaining in the spirit and scope of the present invention defined by the appended claims.