Background of the Invention While alcoholic fermentation can be expressed by a series of enzyme- catalyzed biochemical reactions, brewing and wine-making are ancient arts practiced centuries before the science of chemistry was born. The biochemistry of wine-making was demonstrated in 1856 by Louis Pasteur when he showed that wine is produced when the simple sugars in fruit juices are fermented by the yeast Saccharomyces cerevisiae to yield ethanol and carbon dioxide. Vinegar was produced following alcohol fermentation if the yeast cultures were contaminated by other organisms, such as acetic acid bacteria.

Beer is made in a similar manner by yeast fermentation of the carbohydrates present in cereal grains such as barley. These carbohydrates, largely polysaccharides, are not degraded by the glycolytic enzymes in yeast, which can only act on disaccharides and monosaccharides. This problem is overcome by"malting"the grain. In malting, the cereal seeds are allowed to germinate until they form the appropriate enzymes required to break down the polysaccharides of the cell walls as well as the starch and other polysaccharide food reserves within the cells of the seeds. Germination is then stopped by controlled heating. The malt now contains enzymes such as alpha-amylase and maltase that are capable of breaking down the starch to maltose, glucose and other simple sugars. In the next step, the brewer prepares the liquid by mixing the malt with water and mashing. This allows the enzymes to break down the cereal polysaccharides into simple sugars which are soluble in the liquid medium. The remaining cell matter is then separated and the liquid boiled with hops to provide the flavor for the beer. The yeast cells are then added. In the

presence of oxygen, the yeast cells are"activated"; i. e., grow and reproduce very rapidly. No ethanol is formed until all of the oxygen is utilized. Under anaerobic conditions, the yeast ferments the sugars into 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.

After the fermentation has been stopped, the raw beer or wine (in the absence of hops) is ready for final processing, e. g., adjustment of the amount of "head", CO2 concentration, addition of flavorings, and removal of cells and particulate matter to form a clear end product.

Cordials and spirits are initially prepared in the same way as beer, by fermentation of a cereal mash. The alcohol produced by fermentation is then distilled to yield a product having an alcohol content of 30-50%. The only flavoring present in the final product, for example, rum, vodka, or gin, are those volatile compounds which accompany the alcohol or which are added to the concentrated alcohol.

These methods require long fermentation times and large processing and storage equipment. As can be readily seen, these requirements restrict output capacity and greatly increase the cost of both production and storage. Various process have been tried to shorten the time required for alcohol fermentation and post-fermentation processing to create a clear product. In almost all cases, however, such efforts result in deterioration of the aroma and flavor of the final product as well as being time, cost and labor intensive.

Thus, what is needed is a method for preparing a rapidly fermented and filtered (clear) alcoholic product which possesses all of the desirable flavor characteristics of a beverage processed by conventional methods.

Summary of the Invention The invention provides a method and means for rapidly producing alcohol and alcoholic beverages, including beer and wine, via fermentation, e. g., within 18 to 48 hours, for example within 22 to 36 hours, which yields a clear, filtered product. One embodiment of the invention is a method to produce an alcohol solution that includes contacting at least one microorganism, i. e., Saccharomyces cerevisiae, that is immobilized on a fibrous, e. g., cellulose,

including microcrystalline cellulose and wood chips, or derivatives of cellulose, e. g., carboxymethylcellulose, ethylcellulose, or methylcellulose, or sponge such as sea sponge or loofah sponge, support with a fermentation mixture (feedstock) to produce the alcohol solution (beverage). Preferably, the fibrous support comprises cellulose, e. g., sulfite-free paper. The alcoholic beverage is preferably prepared by contacting a fermentation mixture with at least one alcohol-tolerant strain of S. cerevisiae and at least one flavor-producing organism, such as a yeast and/or a specific bacteria, for example, a lactobacillus sp. 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 are contacted with the fermentation mixture. A high alcohol-tolerant high sugar- tolerant strain, such as S. cerevisiae 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 of up to only about 7 to 8% alcohol and 10 to 15% sugar. An alcohol-and sugar-tolerant strain can tolerate concentrations of alcohol of up to about 10% and concentrations of sugar of up to about 15%, e. g., strains 46 and 414. A"high"alcohol-and sugar-tolerant strain can tolerate concentrations of alcohol of greater than 10% up to about 20%, and concentrations of sugar of greater than 15% up to about 45%. As used herein"about"means that the sugar and/or alcohol contents can vary from the recited amounts within the deviations of the measurement techniques employed by the art. More preferably, flavor-producing microorganisms are also immobilized on the fibrous support. Preferred flavor-producing strains are lactobacillus species that include Lactobacillus plantarum, e. g., strains 133 or 88, and/or Lactobacillus delbruekii.

In one embodiment of the invention, the rapid brewing of an alcoholic beverage is conducted by immobilizing the organisms on a fibrous support which resides in a single chamber or vessel to form a bioreactor and then allowing the fermentation mixture to flow in the reactor and onto and into the fibrous support (s). For example, the bioreactor may include one or more CO2 ports to remove gases and to provide a favorable environment for the immobilized cells thus maximizing the fermentation efficiency. The composition of organisms

present, e. g., bacteria and/or yeast, the column bed volume, the pH, the concentration of sugars and flavorings in the feedstock, and the flow rates are factors determining the time to process a beverage and its final characteristics.

Thus, the invention also provides a bioreactor comprising: (a) a fibrous support for immobilizing cells of a microorganism; and (b) a population of cells attached to or into a substantial portion of the surface of the support.

Brief Description of the Drawings Figure 1 is a diagram of an exemplary bioreactor for a dual purpose process system for rapid fermentation leading to clear beer and/or wine.

Detailed Description of the Invention The present invention provides a method and a bioreactor for use in the practice of the method, for production of alcohol and alcoholic beverages by fermentation with microorganisms which have traditionally been used for fermentation, that are immobilized on a fibrous support that is preferably cellulose, more preferably sulfite-free cellulose paper, e. g., which has FDA approval. Fermentation occurs upon mixing a fermentation mixture with the above-mentioned immobilized microorganisms in a chamber wherein alcohol is produced. The fibrous support advantageously removes undesired material, such as cells and particulate matter from the fermentation product produced according to the method of the invention which results in a filtered product, i. e., clear alcohol or a clear alcoholic beverage. Preferred organisms for use in the methods and bioreactor of the invention include at least one high alcohol-and sugar-tolerant strain of S. cerevisiae, as well as at least one flavor-producing strain of S. cerevisiae, and/or at least one flavor-producing strain of lactobacillus or other bacteria.

The term"alcoholic beverages"includes beverages made by fermentation of grains and fruit juices, such as beer, either"top"or"bottom"fermented varieties (0.5 to 8% alcohol); wine (12 to 21% alcohol); cordials (21 to 30% alcohol); and spirits (30-50% alcohol). For cordials and spirits, the end product is distilled after fermentation. The substrates employed for the manufacture of the beverage according to the present method vary depending on the type of beverage to be produced. The substrate for beer and spirits is a malt formed

from a cereal mash. The substrate for wine is usually crushed fruit or fruit juice.

Alcohol for use in increasing the alcohol content of a beverage or for uses other than consumption can also be produced using the method of the present invention on a variety of organic substrates. Alcoholic malt beverages having normal alcohol content but having reduced caloric content, which are made by adding extra enzymes during mashing to break down malt substances, can also be prepared using the method of the present invention.

The ratio of sugar to liquid for producing beer is about 2.2 lbs sugar and about 1.1 lbs malt in about 16.2 liters of liquid, which yields a preparation having about 3 to 4% alcohol. For wine, the ratio of sugar to liquid is greater than 0.2, the value depending on the percent alcohol required and the theoretical conversion of 100 g sugar to 50 g alcohol.

Wine is prepared according to the present invention using fruit crushed and filtered to form the substrate solution. Skins and seeds are removed prior to crushing according to the desired characteristics of the final product (i. e., white versus red wine). Variables in the final product result from differences in sugar content, source of the fruit, the fermenting organisms, the pH, the time of fermentation and the like. Once the wine reaches about 12 to about 20% alcohol, primary fermentation is complete and the wine can be readily consumed and/or aged or further processed according to taste and alcohol level.

Other alcoholic beverages are prepared in a fashion initially similar to beer, by the fermentation of a mash formed from cereal grains. These include whiskey, scotch whiskey, rum, gin, and vodka, as well as cordials. The final alcoholic content of these beverages is higher than for wine and beer, around 21 to 30% for cordials and greater than 30%, usually greater than 45%, for spirits.

The alcohol obtained by fermentation according to the present invention is distilled to produce concentrations of greater than about 20%. The alcoholic beverages produced by distillation, with the exception of whiskey and cordials, do not contain any flavorings other than volatiles associated with the alcohol.

Whiskey is traditionally flavored by storage in charred oak vats. Cordials can contain other flavorings such as fruit syrups.

The alcoholic content of all of these beverages can be increased by addition of distilled alcohol to the fermented product up to 20% or by increasing the amount of sugar present in the fermentation mixture.

When used here, the words"primary fermentation"mean a process of fermentation which is initiated by the addition of yeast to a liquid, e. g., fruit juice."Secondary fermentation"indicates the process of storage of unmatured malt beverage or wine following the primary fermentation until it acquires the characteristic aroma and flavor of the matured beverage. When the secondary fermentation is carried out at low temperature under pressure, the physico- chemical stability of the beverage is increased and the dissolution of carbon dioxide in the beverage is enhanced.

The present invention encompasses strains of S. cerevisiae including mutant strains of S. cerevisiae having high tolerance to sugar and alcohol. High sugar concentrations, required for high alcohol production, can cause cell death or plasmosis. Yeast normally cannot tolerate alcohol in concentrations in excess of about 7 to 8%. High alcohol tolerance is defined as the ability of a yeast strain to survive and proliferate in a media having an alcohol concentration in which normal yeast strains cannot survive and proliferate, i. e., greater than about 7 to about 8%. Moreover, normal yeast strains can tolerate about 10 to 15% sugar.

Sugar-tolerant yeast can tolerate concentrations in excess of about 20%, and preferably in excess of about 45%, sugar. An alcohol-tolerant, sugar-tolerant yeast strain was deposited with the American Type Culture Collection, Rockville, MD, on Sept. 17,1987 and assigned ATCC No. 20867. This yeast can tolerate concentrations of up to about 20% alcohol.

Known S. cerevisiae yeast strains may be utilized to develop mutants which are capable of growing in a high sugar, high alcohol-containing medium, employing basic techniques relating to the mutation of microorganisms known to those skilled in the art, see, for example, U. S. Patent No. 4,029,549 and the references contained therein. Typically, the organisms are exposed to a mutating agent and then screened for the features which are desired, for example, growth in a high alcohol, high sugar-containing mixture.

Other strains of organisms which constitute part of the present invention include two flavor-producing S. cerevisiae strains, for example, strain 46 and strain 414 which are also alcohol-tolerant and sugar-tolerant strains, including the beer-flavoring strain deposited with ATCC on Sept. 17,1987, and assigned ATCC No. 20866; and flavor-producing and aging strains of microorganisms, such as lactobacillus spp., for example, L. plantarum 133, L. plantarum 88 and L. delbruekii 17. Microorganisms useful in the practice of the methods of the invention are maintained in culture using the methods and materials available to those skilled in the art.

By fermenting a malt liquor mixture or fruit juice mixture in the presence of a flavor-producing strain of Saccharomyces cerevisiae or other bacteria such as lactobacillus and a high alcohol-tolerant mutant strain of Saccharomyces cerevisiae, the rate of fermentation can be accelerated to complete fermentation of beer or wine within about 18 to 48 hours, e. g., 22 to 36 hours.

The microorganisms are immobilized in and on fibrous supports having high surface area. These supports may be used in combination with rigid inert supports such as glass or organic porous beads, although other materials such as ceramics and alumina (only for use in production of fuel alcohol), and other types of supports, may be utilized. Caution must be exercised to avoid supports that might determinably affect flavor. Preferred average bead diameter for rigid inert supports is 0.4 to 0.45 cm, although beads having a diameter between 0.25 and 0.6 cm are acceptable. The supports have sufficient surface area for attachment of at least 109 to 1012 cells/g support.

The immobilized organisms are suspended in a bioreactor of a length and diameter proportional to the amount of feedstock to be fermented. The system can be designed to ferment the feedstock to yield a desired concentration of alcohol on a single or on multiple passes through the reactor using a peristaltic pump. The reactor preferably consists of one or more hollow"columns"or "chambers", e. g., a cylindrical column. In one embodiment, the flavor- producing organisms are placed in a first chamber (bottom) and the high alcohol- tolerant yeast plus the juice (for wine) in the second (top) chamber, so that the feedstock can be fed into the bottom chamber then to the top chamber via the

pump to obtain the desired flavoring while not killing the flavor-producing organisms through exposure to high concentrations of alcohol. In a preferred embodiment, all the immobilized microorganisms are placed in a single column or chamber. The system can be designed to ferment the substrate to yield a desired concentration of alcohol on a single or on multiple passes through the reactor.

Batch Processing of an Alcoholic Beverage Using High Alcohol-Tolerant Yeast Strains In one embodiment, a flavor-producing organism (or mixture of organisms) and a high alcohol-tolerant strain of S. cerevisiae such as ATCC 20867, is immobilized on a fibrous support and mixed with a liquid substrate in a fermentation chamber. The mixture is heated for up to about 2 hours, at a temperature of between 25 °C and 40°C, preferably about 30°C, while an oxygen- containing gas is bubbled through the mixture to activate the culture. The fermentation temperature in the fermentation vessel is then lowered to between about 24° and 35°C, preferably about 28°C, and maintained at the lower temperature level for between about 14 to 48 additional hours to complete the fermentation. The fermented beverage is then withdrawn from the fermentation vessel and the spent yeast separated from the beverage.

General batch processing is further described by the following non- limiting"recipe". One and one-tenth pounds of heated liquid malt extract is poured into a 5 gallon vat fermenter. Two and one-half pounds sugar and about 1/2 tbsp. salt are added to the malt extract in the fermenter. The fermenter is then filled with about 16.2 liters freshly distilled hot water.

About equal amounts, approximately 0.5 liter, of a fibrous immobilized flavor-producing yeast strain and high alcohol-tolerant yeast strain are added to a concentration effective to permit rapid fermentation, about 10'° cells/ml of slurry. The fermentation is allowed to proceed at about 30°C (the useful temperature range is between about 25° and 40°C), for a time between about 1 and 2 hours, bubbling oxygen gas or a stream of air in a constant stream through the liquor to activate the culture. The temperature in the fermenter is then

lowered to about 28°C (the acceptable range is between 24° and 35°C), and maintained at this level for a period of between about 14 and 18 hours.

After a period of about 18 hours (the range is between about 12 and 48 hours or when the fermentation is complete), the malt beverage, a completely fermented beer, is withdrawn from the top.

For wine and beer, one would use approximately 1/3 alcohol-tolerant yeast, 1/3 lactobacilli, and 1/3 flavoring yeast. If higher alcohol content is desired, more sugar can be added at intervals during the fermentation process.

A procedure for preparing beer according to the method of the present invention has the following steps: Placing the malt liquor fermentation mixture in a fermentation vessel; heating the mixture for between about 1 and 2 hours at a temperature of between 38° and 40°C ; adding one half of a total amount of hops, e. g., one third of a can of malt with hops, to the fermentation vessel; heating the hop-fermentation mixture at a temperature of between about 50° and 55 °C for an additional 0.5 to 1 hours; elevating the fermentation temperature of the liquor to between about 60° and 65°C and holding the temperature at that level for between about 30 and 35 more minutes, then adding the other half of the hops; increasing the vessel temperature to between about 70° and 75°C, holding this temperature for between about 25 and 35 more minutes; and finally increasing the vessel temperature 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.

Adding to the fermentation vessel water and a mixture of malt flavor- producing strain of S. cerevisiae and at least one, preferably two, and more preferably three, high alcohol-tolerant, sugar-tolerant strains of S. cerevisiae capable of accelerating the fermentation of the malt liquor; heating the mixture for between 30 and 35 minutes at a temperature of between 24° and 30°C while bubbling an oxygen-containing gas therethrough; lowering the temperature in the fermentation vessel to between about 26° and 28°C, and maintaining the lower temperature level for between about 24 to 48 hours to complete the fermentation.

The beverage is then withdrawn from the fermentation vessel. The alcoholic

malt beverage produced according to this invention can be clarified, salts and/or CO2 added, and bottled for distribution according to known procedures in the art.

Processing of an Alcoholic Beverage in a Bioreactor Containing Microorganisms Immobilized on a Fibrous Support Immobilizing large numbers of microorganisms in the bioreactor accelerates fermentation by processing the beverage at a continuous rate without the addition of new yeast. In one embodiment for processing beer, yeasts that have traditionally 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, e. g., ATCC 20867, strain 46 and strain 414, which are alcohol-and sugar-tolerant, at least one which is a flavor-producing strain, and at least one flavor-producing strain of bacteria, are immobilized a fibrous support, e. g., sulfite-free paper in a single chamber or section of the bioreactor. The desired volume of immobilized cells is at least about 109 to about 10'2, more preferably at least about 10'°, cells/ml slurry. The total number of immobilized cells can be determined using standard procedures. For example, the yeast cells in an appropriate dilution of supernatant are counted following immobilization using a hemocytometer (Levy and Levy-Hausser corpuscle counting chamber, Hausser Sci., Philadelphia, Pennsylvania). See also Hamdy et al. (Biomass, 21, 189 (1990)). The feedstock is fed into the bioreactor and passed through the immobilized organisms. After reaching a desired percentage of alcohol, which may take up to about 24 to about 48 hours, the fermented liquor is withdrawn, bottled, or further processed, as necessary.

The viability of strains on substrates may be determined by the modified methylene blue of Hamdy (1990) (also see Manual of Microbiological Methods, Society of Am. Microbiol., 1957). Resazurin may also be used (Latham and Sharpe, In : Isolation of Anaerobes, 5th ed., Sharpten and Board (eds.), pp. 133- 147, Academic Press, New York (1971)). A change in color in the cell means that there are viable cells. Factors affecting yeast immobilization include incubation time; pH; media; substrate form, composition and size; the amount of sugar, type of support, and the like. Any cellulose-based support form may be

used, although maintaining adequate surface area for attachment of the cells and minimizing shear force caused by the combination of the flow of the fermentation mixture and the evolution of CO2 in the bioreactor are considerations in the design of materials.

The construction of bioreactors and processing of an alcoholic beverage in bioreactors is further described by the following non-limiting example.

EXAMPLE 1 Dual Purpose Bioreactor for Rapid Fermentation of Beer or Wine Leading to a Clear Filtered End-Product The yeast cells are allowed to grow for 48 hours at 37°C (stationary phase) in a flask of malt-glucose broth placed inside a metabolic shaker. This broth contains g/1 deionized water as follows: yeast extract, 5; malt extract, 20.0; glucose, 10.0; sucrose, 5.0; tryptone, 1.5; peptone, 1.5; KH2PO4, 1.0; NH4Cl, 2.0, and NaCl, 2.0; and trace minerals (e. g., Mg, Ca, Zn, Cu, S, Mn and/or Fe).

A feedstock for making dark beer containing one pound of malt extract includes 0.6 pounds of sugar; 1 gram of salt; and 1.2 gallons of hot water. The feedstock was fed into a bioreactor chamber as shown schematically in Figure 1.

The temperature of the circulating feedstock can be externally controlled between about 25°C and about 37°C by means of a pump circulating water via a water bath to the column jacket. The pH of the feedstock was maintained at between about 4.5 and 5.5.

Varying the flow rate allows one to improve the efficiency of the system.

In general, decreasing the flow rate increases the efficiency. Decreasing the flow rate also provides a means for minimizing shear forces within the bioreactor and thereby decreases the number of organisms dislodged (washed out cells) from the supports thereby allowing for long-term operation of the system.

The process was conducted in a bioreactor chamber containing a mixture of yeast and other cultures immobilized onto/into a modified sterile cellulose paper having no sulfite residue (Rayonier, Inc.) was employed. Cellulose and wood pulp are approved by the FDA for use in food and food contact applications. Cultures such as those described below and in U. S. Patent No.

4,929,452 and Serial No. 09/086,020 may be employed, however, the invention is not limited to those strains as any other strain (s) which can be immobilized on a fibrous support can be used.

The fermentation in the bioreactor occurred in a vertical (Figure 1) hard plastic chamber containing a slurry of pulp-paper onto which three different yeast strains and lactobacillus cells were immobilized at a cell density of about 109 to 10'2, e. g., 10'°, cells per ml of slurry. The substrate (feedstock) was fed to the chamber by a peristaltic pump equipped with an overflow device and the effluent was filtered during recycling in the chamber to remove the yeast cells.

The substrate, such as malt extract with hops for beer or fruit juice for wine, was continuously fed from a six liter glass vessel to the chamber at a desired rate, e. g., 25 to 96 ml per minute, and recycled. This rate can be set automatically using a variable speed peristaltic pump. This process was continued for 24 hours for beer and 22-48 hours for wine. The clear final end product was intrinsically filtered in the chamber by the continuous recycling of the effluent through the paper-slurry and collected (recycled) in the same original vessel, where samples were tested for neutral alcohol (distilled alcohol) and a reading of the refractive index was taken to determine concentration.

A preferred embodiment of the invention is a process which employs a combination of the following: S. cerevisiae (ATCC 20867), an alcohol-and sugar-tolerant strain; S. cerevisiae 46 (an alcohol-and sugar-tolerant, flavor- producing strain); S. cerevisiae 414 (an alcohol-and sugar-tolerant, flavor- producing strain); and Lactobacillus plantarum 133 (flavor and aging); L. plantarum 88 (flavor and aging); and/or L. delbruekii 17 (flavor and aging).

These six strains, or a combination thereof, may be used to make red and white wines and beer, depending on substrate used. For example, such a mixture includes: S. cerevisiae 20867 + S. cerevisiae 46 L. plantarum 133. L. plantarum 88.

+ S. cerevisiae 414 and L. delbruekii 17 60-65% 40-35% equal components equal components

For red wine, a preferred mixture 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 with the paper-slurry and immobilized yeast- cultures may be employed for home use or can be scaled up for industry.

Furthermore, this dual process for the bioreactor system can be utilized for more than 10 fermentations, e. g., for a period of more than 3 months. The pulp-slurry paper in the chamber filters the wort, while the immobilized cultures ferment the substrate present in the wort into ethanol, as the substrate is continuously recycled through the bioreactor. The amount of alcohol produced depends on the sugar level and only the CO2 gas is allowed to escape via an exit port on top of the chamber to the vessel. This CO2 can be collected for future use if so desired.

In particular, the bioreactor of the present invention rapidly ferments sugar to ethanol, e. g., one mole of glucose can produce at least 2.0 moles of ethanol and 2.0 moles of CO2 gas, and produces a clear end product, e. g., wine or beer depending on the substrate used.

A second fermentation for CO2 synthesis of the clear beer can be performed in capped bottles or in a container to which an aliquot of yeast cultures and sugar are added leading to desired carbonation. A non-toxic combination of specific chemicals with known concentration, such as sodium carbonate, citric acid and fructose, can be used instead of sugar and yeast to achieve the desired carbonation of the beer or sparkling wine. The yeast cells can be stored for several months at room temperature (about 24°C) in a sterile citrate buffer (pH 3.5) after which they can be activated with non-toxic phosphates and employed in fermentation.

All publications and patents are incorporated by reference herein, as though individually incorporated by reference, as long as they are not inconsistent with the present disclosure. The invention is not limited to the exact details shown and described, for it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention defined by the claims.