WO2020159964A1 - Procédé de fermentation sous pression réduite - Google Patents

Procédé de fermentation sous pression réduite Download PDF

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Publication number
WO2020159964A1
WO2020159964A1 PCT/US2020/015379 US2020015379W WO2020159964A1 WO 2020159964 A1 WO2020159964 A1 WO 2020159964A1 US 2020015379 W US2020015379 W US 2020015379W WO 2020159964 A1 WO2020159964 A1 WO 2020159964A1
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Prior art keywords
fermentation
yeast
during
wort
psia
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Andrew John MACINTOSH
Mario Sergio GUADALUPE DAQUI
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/003—Fermentation of beerwort
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/006—Yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • yeast has been studied in different fields, such as: brewing, baking, wine making, medicine, etc. Recent investigations have started to look the adaptation and associated viability of yeast under atypical fermentation conditions (Landry, C. et al. 2005.).
  • Saccharomyces cerevisiae and Saccharomyces pastorianus are the dominant microorganisms responsible for the fermentation of the sugars dissolved in the substrate (Nicholas, M. 2016.; Harrison, M. 2009).
  • a method of fermentation comprises: combining in a vessel a fermentation microorganism and a liquid substrate comprising fermentable carbohydrates, to provide a fermentation composition; fermenting the fermentation composition within the vessel so that at least a portion of the fermentable carbohydrates have been converted to an alcohol, resulting in a fermented product comprising the alcohol.
  • the fermenting comprises: substantially depleting any oxygen in the fermentation composition; after substantially depleting the oxygen, reducing the pressure in the vessel to a reduced pressure of 7.4 psia or below; and continuing the fermentation under anaerobic conditions at the reduced pressure to produce the fermented product.
  • the fermentation microorganism can be Saccharomyces cerevisiae or Sacchoromyces pastorianus, or an alcohol producing spp.
  • the fermented product can be an alcoholic beverage, a distillation product, or a biofuel product.
  • the liquid substrate can be a wort, or a biomass.
  • the liquid substrate can have an original gravity of at least from about 1.048 to about 1.083 or sugar content from about 12°Plato to about 20°Plato. In other disclosed methods, the liquid substrate can have an original gravity of greater than about 1.083 or sugar content greater than about 20°Plato.
  • a method of fermentation comprises: combining in a vessel a fermentation microorganism and a substrate comprising fermentable carbon source, to provide a fermentation composition; fermenting the fermentation composition within the vessel so that at least a portion of the fermentable carbon source has been converted to a pharmaceutical substrate, cosmetic substrate, enzyme, or drug, resulting in a fermented product comprising the pharmaceutical substrate, cosmetic substrate, enzyme, or drug.
  • the fermenting further comprises: substantially depleting any oxygen in the fermentation composition; after substantially depleting the oxygen, reducing the pressure in the vessel to a reduced pressure of 14 psia or below; and continuing the fermentation under anaerobic conditions at the reduced pressure to produce the fermented product.
  • the reduced pressure can be 14.0 psia or below.
  • the viability of the fermentation microorganisms in the fermentation composition is maintained at or above 90% during fermentation.
  • the maximum number of microorganism cells in the fermentation composition is at least about 15% greater than the maximum number of microorganism cells in a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • the sugar concentration of the fermentation composition reaches its attenuation limit at least about 25% faster than that of a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • the fermented product has a concentration of ester volatiles or higher alcohol volatiles that is greater than a concentration of ester volatiles or higher alcohol volatiles of a fermented product produced during a comparable fermentation conducted under atmospheric pressure.
  • FIG. 1 shows a process flow diagram of an exemplary industrial brewing process, utilizing the disclosed fermentation methods.
  • FIG. 2 shows a pilot bioreactor apparatus as used in the Examples.
  • FIG. 3 shows a bioreactor apparatus used for yeast propagation in the Examples.
  • FIGS. 4A-4B show data generated during fermentation processes under partial vacuum conditions (FIG. 4B) as compared to control (atmospheric pressure) conditions (FIG. 4A), using wort having 14°P initial sugar content, in accordance with Example 1.
  • FIG. 5 shows data generated during an exemplary yeast propagation process, in accordance with Example 1 .
  • FIGS. 6-6B show data generated during fermentation processes under partial vacuum conditions as compared to control (atmospheric pressure) conditions, including (FIG. 6A) the number of yeast cells in suspension, and (FIG. 6B) ethanol and extract generated, using wort having 14°P initial sugar content, in accordance with Example 1.
  • FIGS. 7A-7B shows data generated during fermentation processes under partial vacuum conditions (FIG. 7B) as compared to control (atmospheric pressure) conditions (FIG. 7A), using wort having 14.5°P initial sugar content, in accordance with Example 2.
  • FIG. 8 shows data generated during an exemplary yeast propagation process, in accordance with Example 2.
  • FIGS. 9A-9B show data generated during fermentation processes under partial vacuum conditions as compared to control (atmospheric pressure) conditions, including, in FIG. 9A, the number of yeast cells in suspension, and, in FIG. 9B, the ethanol and extract generated, using wort having 14.5°P initial sugar content, in accordance with Example 2.
  • FIGS. 10A-10B show data generated during fermentation processes under partial vacuum conditions (FIG. 10B) as compared to control (atmospheric pressure) conditions (FIG. 10A), using wort having 15°P initial sugar content, in accordance with Example 3.
  • FIG. 11 shows data generated during an exemplary yeast propagation process, in accordance with Example 3.
  • FIGS. 12A-12B show data generated during fermentation processes under partial vacuum conditions as compared to control (atmospheric pressure) conditions, including, in FIG. 12A, the number of yeast cells in suspension, and, in FIG. 12B, the ethanol and extract generated, using wort having 15°P initial sugar content, in accordance with Example 3.
  • FIGS. 13A-13B show data generated during fermentation processes under partial vacuum conditions (FIG. 13B) as compared to control (atmospheric pressure) conditions (FIG. 13A), using wort having 30°P initial sugar content, in accordance with Example 4.
  • FIGS. 14A-14B show data generated during fermentation processes under partial vacuum conditions as compared to control (atmospheric pressure) conditions, including, in FIG. 14A, the number of yeast cells in suspension, and, in FIG. 14B, the ethanol and extract generated, using wort having 30°P initial sugar content, in accordance with Example 4.
  • FIGS. 15A-15D show data generated during fermentation processes under partial vacuum conditions as compared to control (atmospheric pressure) conditions, more specifically volatiles analysis of samples taken during the respective fermentation processes, including, in FIG. 15A, the concentration of carbonyl compound detected, in FIG. 15B, the concentration of ester compound detected, in FIG. 15C, the concentration of higher alcohol detected, and in FIG. 15D, the final volatile concentration of the control and vacuum fermented products.
  • the present disclosure provides a method for fermentation, in which a fermentation microorganism converts a fermentable carbohydrate or other organic molecule to a desired substance such as an alcohol or a pharmaceutical ingredient.
  • a fermentation microorganism converts a fermentable carbohydrate or other organic molecule to a desired substance such as an alcohol or a pharmaceutical ingredient.
  • at least a portion of the fermentation is conducted under a vacuum or partial vacuum.
  • Benefits of using the disclosed methods include increased fermentation rates, and increased sugar consumption.
  • Another benefit of the disclosed vacuum fermentation methods includes improvements to volatile formation, such as increased concentration of desirable volatiles (such as higher alcohols and esters) in the final fermentation product.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. For example, if the value“10” is disclosed, then“about 10” is also disclosed. Ranges can be expressed herein as from“about” one particular value, and/or to“about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms a further aspect. For example, if the value“about 10” is disclosed, then“10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase“xto y” includes the range from‘x’ to‘y’ as well as the range greater than‘x’ and less than‘y’ ⁇
  • the range can also be expressed as an upper limit, e.g.‘about x, y, z, or less’ and should be interpreted to include the specific ranges of‘about x’,‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and‘less than z’.
  • phrase‘about x, y, z, or greater’ should be interpreted to include the specific ranges of‘about x’,‘about y’, and‘about z’ as well as the ranges of‘greater than x’, greater than y’, and‘greater than z’.
  • phrase“about‘x’ to‘y’”, where‘x’ and‘y’ are numerical values, includes “about‘x’ to about‘y’”.
  • a numerical range of“about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1 %, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1 %; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms“about,”“approximate,”“at or about,” and“substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined.
  • “about” and“at or about” mean the nominal value indicated ⁇ 10% variation unless otherwise indicated or inferred.
  • an amount, size, formulation, parameter or other quantity or characteristic is“about,”“approximate,” or“at or about” whether or not expressly stated to be such. It is understood that where“about,”“approximate,” or“at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • the terms“optional” or“optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
  • the disclosure relates to a method of fermentation, in which a fermentation microorganism converts a fermentable carbon source (e.g., carbohydrate or sugar) to a desired product such as an alcohol or a pharmaceutical ingredient.
  • a fermentation microorganism converts a fermentable carbon source (e.g., carbohydrate or sugar) to a desired product such as an alcohol or a pharmaceutical ingredient.
  • exemplary fermentation microorganisms include, for example, a bacteria, a mold, a fungus, a yeast, eukaryotic cells, or other organisms.
  • a“fermentable carbon source” or the like refers to a carbon source capable of being metabolized by the microorganisms disclosed herein for the production of a desired fermentation product.
  • Exemplary fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; C5 sugars such as xylose and arabinose; carbon substrates such as methane; and combinations and mixtures thereof.
  • “Fermentable sugar” as used herein refers to one or more sugars capable of being metabolized by the microorganisms disclosed herein for the production of alcohol, gas, acid, or other desirable product.
  • the fermentable sugars may be derived, for example, from a biomass source such as barley, rice, corn, cane, cellulosic, lignocellulsic material, or the like, and may be processed, for example, by liquefaction and/or saccharification to form a mash that is fermented by a microorganism.
  • Fermented products produced by the disclosed methods have such industrial applications as food, beverage, and pharmaceutical, as well as in general industrial applications.
  • the disclosed methods are described in more detail below in the context of an industrial brewing process. It will be understood, however, that the disclosed methods are not so limited and have practical application in many other processes.
  • the disclosure relates to a method of fermentation associated with an industrial brewing process used for the production of beer.
  • the production of beer involves steeping a starch source in water to produce a liquid (wort) containing fermentable sugar, and fermenting the wort with yeast.
  • yeast transforms fermentable sugars in the wort into ethanol and carbon dioxide, and the resulting fermented product is beer.
  • the typical brewing process contains a plurality of steps including, among others, wort production, yeast rehydration and propagation, and fermentation. The steps associated with the disclosed methods are described in more detail below.
  • wort production 110 a first step in an industrial brewing process 100 is wort production 110.
  • “wort” refers to the liquid extracted from the mashing process during the brewing of beer.
  • the wort is formed by the addition of water to malted and unmalted crushed grain such as, but not limited to, barley to form a slurry or mash in a mash tun. Through the action of naturally occurring enzymes this mash is then converted into the wort.
  • Wort contains fermentable sugars, such as maltose and maltotriose, which will be fermented by the brewing yeast to produce alcohol. Wort also contains amino acids to provide nitrogen to the yeast as well as more complex proteins that can contribute to beer head retention and flavour.
  • Wort production 1 10 can include one or more steps to make wort from starting grains, including, for example, milling, mashing, lautering, filtration, and/or boiling.
  • a typical wort production process 1 10 starts by making a malt from dried grain, such as sprouted barley. The malt is then run through a roller mill and cracked. This cracked grain is then mashed, that is, mixed with hot water and steeped, a slow heating process that enables enzymes in the malt to convert the starch into sugars.
  • the temperature of the mixture is increased to about 78 °C (170 °F).
  • Raising the temperature to this level stops the enzyme action, preserving the profile of sugars in the wort, and also reducing the viscosity of the mixture. Lautering is the next step in wort production 110, which means the sugar- extracted grist or solids remaining in the mash are separated from the liquid wort.
  • the liquid wort mixture is then boiled to sanitize the wort.
  • hops are added during this step, during which the bittering, flavor and aroma can be extracted from hops.
  • the wort is known as "sweet wort” until the hops have been added, after which it is called “hopped wort” or“bitter wort”.
  • the hops are added in three parts at set times.
  • the bittering hops, added first, are boiled in the wort for approximately one hour to one and a half hours. This long boil extracts resins, which provides the bittering.
  • the flavouring hops are added, e.g., about 15 minutes from the end of the boil.
  • the finishing hops can be added, e.g., toward the end of or after the boil. This extracts the oils, which provide flavour and aroma but evaporate quickly. Generally speaking, hops provide the most flavouring when boiled for approximately 15 minutes, and the most aroma when not boiled at all. At the end of boiling, the hot wort is quickly cooled to a temperature favorable to the yeast.
  • one or more adjunct grains may optionally be added to the mash.
  • Adjunct grains include, for example, oat, wheat, corn, rye, sorghum, and rice. Adjunct grains can be used for example to create varietal beers such as wheat beer and oatmeal stout, to create grain whiskey, or to lighten the body. Adjunct grains may first need gelatinization and cooling before adding to the mash.
  • one or more additional fermentable carbohydrates or sugars may be added. Fermentable carbohydrates and sugars include, for example, sucrose, dextrose (glucose), fructose, maltose, lactose, maltodextrin, and the like.
  • the result of the wort production 110 is a liquid wort 1 16 comprising fermentable sugars.
  • the liquid wort 1 16 can be used in the yeast propagation step 130, and/or the fermentation step 140, as described further below.
  • yeast rehydration 120 another step in the disclosed industrial brewing process 100 is the yeast rehydration 120.
  • yeasts are aerobic facultative anaerobe type microorganisms (Bekatorou et. al. 2006) which means they can utilize different pathways to consume the nutrients from the media they are in, depending on the environmental conditions.
  • a catabolic reaction should occur to obtain energy from the degradation of the organic molecules. The energy then produced by the catabolic pathways lets the anabolic process occur, in which not only internal structures are made, but cellular growth and multiplication can also be obtained.
  • yeasts Because of the facultative characteristic of the yeasts, the final products of the reactions are different whether in presence of oxygen or not. With the presence of oxygen, yeasts are going to generate new cells or biomass, using as a principal pathway the glycolysis. In this pathway, yeasts transform a six-carbon molecule such as glucose into two molecules of pyruvate to produce energy.
  • the tricarboxylic acid (TCA) cycle known as Krebs cycle has an important role in this pathway, because the major redox reactions occur within it obtaining as a final product water, carbon dioxide and energy (Piskur, J. et al. 2006).
  • TCA tricarboxylic acid
  • Krebs cycle has an important role in this pathway, because the major redox reactions occur within it obtaining as a final product water, carbon dioxide and energy (Piskur, J. et al. 2006).
  • yeasts Without the presence of oxygen, yeasts perform a different and important process for the beer industry, called fermentation.
  • the main aim in fermentation is to transform sugars into ethanol and carbon dioxide.
  • the products of this process can contribute to the characteristic flavors of the beer (Briggs, D. et al. 2004).
  • the exception to these generalizations is the“Crabtree effect” where yeast will ferment in the presence of oxygen under specific conditions. This is accounted for in the brewing industry by the inhibition of synthesis of respiratory enzymes due to the high concentration of glucose (Deken, R. 1966).
  • ADY Active Dry Yeast
  • Fluidized bed drying is the most common process applied in the industry because is generally less stressful to the yeast and it gives a higher viability and a higher capacity for yeast to generate biomass (Jenkins, D. 2011). Freeze drying removes water via sublimation of a frozen culture under no pressure (Kawamura, S. 1995). Spray drying produces a powder from rapidly drying the slurry solution droplets using a hot air stream. (Morgan, C. 2006).
  • the survival rate during this process can be determined, at least in part, by factors such as: osmotic pressure, temperature and the medium (Laroche, C. and Cervais, P. 2003.). For example, it has been found that for Lager strains, a rehydration process at 25°C resulted in 73% viability, as compared to 67% viability at 30°C; while for Ale strains opposite results were obtained, in which a rehydration process at 25°C resulted in 72% viability, as compared to 80% viability at 30°C; the results indicating that Lager strains are lest thermos tolerant than Ale strains. (Jenkins, D. 201 1.).
  • the result of the yeast rehydration step 120 is a hydrated yeast 126, which can then be used in the yeast propagation step 130.
  • the disclosed industrial brewing process 100 includes the step of yeast propagation 130.
  • yeast propagation is the process by which yeast growth is cultivated to produce a sufficient quantity of yeast for fermentation. Propagation conditions should be such that a maximal amount of yeast is produced which provides optimal fermentation performance once pitched.
  • yeast propagation is influenced by several factors, including oxygen, pH, temperature, and wort composition.
  • oxygen is important for good yeast growth and is the driving force behind many aspects of yeast metabolism including fermentation.
  • Oxygen is quickly absorbed by yeast and is used to synthesize unsaturated fatty acids and sterols which form the cell membrane. These molecules are important for both growth and fermentation and serve as a means of storing oxygen within the cell. They are also necessary for increasing cell mass (growth), improving the overall uptake of nutrients, and determining alcohol tolerance. Oxygen also stimulates synthesis of molecules necessary for yeast to metabolize and take up maltose, the primary sugar in wort.
  • the media used for a yeast propagation process can range from cheap agricultural and industrial wastes such as molasses (Bekatorou, A. et al. 2006) to the actual wort substrate used for the fermenting process.
  • the media used for propagation is the liquid wort 1 16 that results from the wort production 1 10.
  • the wort composition can affect yeast growth and is important in maintaining and storing viable, stable yeast.
  • wort should contain all of the ingredients necessary for yeast propagation.
  • one or more additional nutrients or salts may be added to the wort mixture to improve yeast growth. Nutrients serve to increase the nitrogen content of the wort and yeast.
  • Typical yeast nutrients comprise ammonium phosphate-based nutrients, amino acid/peptide and vitamin-based nutrients, or a combination thereof.
  • metal ions can be added to the propagation mixture.
  • zinc can be added.
  • pH Another factor that affects yeast growth is pH. Generally speaking, yeast grow well at acidic pHs, such as between pH 4 to pH 6. Normal wort is acidic with a pH near 5.2. During propagation the pH can drop to about 3.5 to about 4.5, or from about 3.8 to about 4.1. In some disclosed methods, the pH may be adjusted prior to, during, or after propagation. For example, in some disclosed methods, further acidification of the wort can help to prevent bacterial infection, because most bacteria cannot tolerate acidic pH, while yeast can survive at very low pH, as low as 2.0.
  • the temperature of the propagation step is determined, at least in part, by the type (strain and species) of yeast used. For example, for an S. cerevisiae yeast, propagation of a lager yeast may be conducted at a temperature of from about 12 °C to about 20°C; while for an S. pastorianus yeast, propagation of a lager yeast may be conducted at a temperature of from about 20 °C to about 30°C.
  • S. cerevisiae yeast propagation of a lager yeast may be conducted at a temperature of from about 12 °C to about 20°C
  • S. pastorianus yeast propagation of a lager yeast may be conducted at a temperature of from about 20 °C to about 30°C.
  • One having ordinary skill in the art would understand how to determine a suitable propagation temperature range, based on the selected yeast strain and species.
  • the result of the yeast propagation 130 is a cultivated yeast 136, which can be “pitched” to the fermentation process 140.
  • the propagation step produces a composition having any necessary or desired amount of yeast, where the yeast has viability of greater than about 90%, or greater than about 95%.
  • the disclosed industrial brewing process 100 includes the step of fermentation 140.
  • fermentation is an exothermic process in which the main goal is to use the yeast to metabolize and convert sugars mainly into alcohol and carbon dioxide (Briggs, E. et al. 2004).
  • the nature of the fermentation process was well-established in the late 19th century. More recently, improvements in the equipment to be able to crop the yeast once the fermentation process has finished, resulted in the modern standard cylindroconical vessel.
  • yeast and wort are combined to provide a fermentation mixture, in which the yeast metabolizes the fermentable sugars to produce ethanol and carbon dioxide.
  • “fermentation mixture” means a mixture of one or more of water, sugars, dissolved, solids, yeast producing alcohol, produced alcohol, and other constituents.
  • Other terms used to reference the fermentation mixture include “fermentation medium,” “fermentation composition,”“fermentation broth,” and“fermented mixture.”
  • fermentation is affected, at least in part, by temperature, pH and wort composition.
  • Another factor that can affect fermentation is the presence of volatiles such as exogenous ethanol.
  • pH One factor that can affect fermentation is pH.
  • yeast can survive at very low pH, e.g., as low as 2.0.
  • fermentation can be relatively slow at low pH.
  • Typical wort is acidic, e.g., having a pH near 5.2.
  • the pH can drop below 4.0.
  • the pH may be adjusted prior to, during, or after fermentation. For example, if the pH of the fermentation composition is too low, the pH may be buffered, e.g., with a small amount of calcium carbonate, which can in some cases accelerate fermentation.
  • the wort can be further acidified, e.g., to help to prevent bacterial infection.
  • acidification during fermentation can slow down fermentation, so acidification is typically done after fermentation is complete.
  • Wort composition also can affect fermentation performance.
  • standard brewing wort contains most of the ingredients necessary for fermentation.
  • a starting wort can be one of several different worts, and may be selected depending upon a number of factors including, for example, the type of yeast being utilized, and the desired fermentation product.
  • the starting wort can have a certain initial original gravity related to the amount of solutes dissolved, mostly fermentable sugars that are metabolized into ethanol (and other metabolites) throughout the fermentation.
  • The“original gravity” of the wort is a measure of the specific gravity of the wort measured before fermentation, and is the ratio of the density of a sample to the density of water.
  • the sugar content of the composition is sometimes expressed using the Plato scale, in which a Plato degree is generally calculated using the following equation:
  • a normal gravity wort typically has an original gravity of about 1.048 (12°P).
  • a High-Gravity (HG) wort that is commonly used in the industry may have an original gravity of between about 1.065 (16°P) to about 1.074 (18°P).
  • a Very High Gravity (VHG) wort refers to one having an even higher concentration of fermentable sugar dissolved into the wort, for example, one having an original gravity greater than about 1.083 (20°P).
  • VHG worts are typically used in distillation or biofuel industries where the undesirable off flavours and volatiles produced as a result of the very high sugar concentration will not be detrimental to the final product.
  • the selection of the wort may be at least partially dependent upon the selection of the yeast.
  • Some ale yeast strains have been bred to be more ethanol-tolerant than industrial Lager yeasts.
  • the selected yeast strain is a Lager yeast strain intended for a normal original gravity fermentation
  • the original gravity of the wort may range between about 1.048 (12°P) to about 1.083 (20°P), or about 1.057 (14°P) to about 1.079 (19°P), or about 1.065 (16°P) to about 1.074 (18°P).
  • the original gravity of the wort may be greater than about 1.083 (20°P), or it may range between about 1.083 (20°P) to about 1.129 (30°P). This strain can also produce undesirable flavors and volatiles during fermentation that limits its use in the brewing industry, making it more useful in the distillation/biofuel industries.
  • the reduction of sugar is a measurable parameter that indicates the progress of the fermentation process.
  • a change in the specific gravity of the wort is an indication of how much sugar has been consumed by the yeast, i.e., converted to alcohol. This parameter is related to the attenuation limit gravity, which refers to the lowest gravity value after the fermentation started. Knowing the amount of sugar in the wort before and after fermentation, the amount of alcohol formed during the fermentation can be determined.
  • the fermentation step is conducted under a low pressure, or vacuum. Fermentation has been performed under continuous or semi-continuous vacuum pressure.
  • the use of vacuum during fermentation resulted in an improvement in the yeast’s behavior, which was believed to be related to the elimination of sources of stress such as the ethanol concentration through the boiling point change under low pressures (Cysewski, G. and Wilke, C. 1977).
  • the yeast faced another type of stress caused by the non-volatile compounds such as higher (less volatile) alcohol formation. It was found that under a continuous vacuum fermentation process, the ethanol productivity increased almost 6 times compared to the ethanol productivity obtained during a conventional fermentation (at atmospheric pressure) (Cysewski, G. and Wilke, C. 1977).
  • the fermentation step involves reducing the pressure of the fermentation mixture to below about 14.0 psia, or below about 13.0 psia, or below about 12.0 psia, or below about 11.0 psia, or below about 10 psia, or below about 9 psia, or below about 8 psia, or below about 7.4 psia, or below about 7.0 psia, or below about 6.5 psia, or below about 6.0 psia, or below about 5.5 psia or below about 5.0 psia, or below about 4.5 psia, or below about 4.0 psia, or below about 3.5 psia, or below about 3.0 psia, or below about 2.5 psia, or below 2.0 psia, or below about 1.5 psia, or below 1.0 psia
  • the fermentation is conducted at a pressure greater than about 0.65 psia.
  • the temperature of the fermentation step is determined, at least in part, based on the pressure of the fermentation step, and the desired fermentation results.
  • a higher temperature causes a higher rate of fermentation but also causes the generation of more undesirable byproduct.
  • One having ordinary skill in the art would understand how to determine a suitable fermentation temperature based on the fermentation pressure, and the desired fermentation results.
  • the fermentation step is concluded when the desired attenuation has been achieved.
  • the fermentation step is concluded when the sugars in the fermentation mixture have been depleted to within less than about 15% or about 10% or about 5% of the attenuation limit for the fermentation mixture.
  • a fermentation process can produce a fermentation product with a higher concentration of ester and higher alcohol volatiles as compared to a fermentation product made under atmospheric pressure.
  • the most important volatile compounds are higher alcohols and esters. Esters are often desirable to the final product. However, these compounds can be highly style-dependent. Higher alcohols are precursors of flavor-active esters present in the beer. In comparison, there are some volatiles, such as Vicinal Diketones (VDK), that are considered undesirable and usually represent a quality defect in the final product.
  • VDK Vicinal Diketones
  • the disclosed vacuum fermentation process can result in an increase in the volatile concentration of the higher alcohols and esters in the resulting beer product.
  • the fermentation product made in the disclosed vacuum fermentation process may have a concentration of higher alcohols that is at least 10% greater, or at least 12% greater, or at least 14% greater, or at least 15% greater, or at least 16% greater than the concentration of higher alcohols in a comparable fermentation product made under atmospheric pressure.
  • the fermentation product made in the disclosed vacuum fermentation process may have a concentration of esters that is at least 20% greater, or at least 25% greater, or at least 30% greater, or at least 35% greater than the concentration of esters in a comparable fermentation product made under atmospheric pressure.
  • the disclosed methods can result in an increase in the fermentation rate as compared to a comparable process performed under atmospheric pressure.
  • the disclosed methods result in a significant increase in the maximum number of suspended yeast cells in the fermentation mixture and increase in the rate of fermentation.
  • the maximum number of microorganism cells in the fermentation mixture is at least about 15% greater, or at least about 20% greater, or at least about 25% greater than or at least about 30% greater than, or at least about 35% greater than, or at least about 40% greater than, or at least about 45% greater than, or at least about 50% greater than, or at least about 55% greater than, or at least about 60% greater than, or at least about 65% greater than, or at least about 70% greater than, or at least about 75% greater than, or at least about 80% greater than, or at least about 85% greater than, or at least about 90% greater than, or at least about 95% greater than, or at least about 100% greater than that of a fermentation mixture produced during a comparable fermentation conducted under atmospheric pressure.
  • the rate of sugar consumption using the disclosed vacuum fermentation is greater than the rate of sugar consumption of a similar fermentation process performed under atmospheric conditions.
  • the sugar concentration of the fermentation mixture reaches its attenuation limit at least about 20% faster, or at least about 25% faster, or at least about 30% faster, or at least about 35% faster than a comparable fermentation conducted under atmospheric pressure.
  • the disclosed methods do not significantly change other factors affecting fermentation, including pH and viability of the yeast.
  • the viability of the yeast in the fermentation mixture is maintained at greater than 90%, or greater than 91 %, or greater than 92%, or greater than 93%, or greater than 94%, or greater than 95%, or greater than 96% throughout the disclosed fermentation process.
  • the viability of the yeast in the disclosed fermentation method is at least about the same as the viability of the yeast in a similar fermentation method conducted at atmospheric pressure.
  • Using the disclosed methods can have a significant economic impact on the industrial brewing processes and industry.
  • the methods can result in increasing the efficiency of existing fermenting facilities, utilizing more effectively the existing fermenter space.
  • Increasing the rate of fermentation enables an increase in the production output of a facility.
  • increasing the sugar concentration up to 50% more beer could be made with the existent equipment which has the potential to save more than 600 million dollars worldwide (this process is called high gravity substrate fermentation).
  • the result of the fermentation step 140 is the desired fermentation product 146.
  • the fermentation product contains the desired products of the metabolite.
  • the desired product would be alcohol (ethanol), and the fermentation product would comprise the desired amount of the alcohol, which is the metabolic product of the sugar in the wort.
  • the fermentation product 146 resulting from fermentation 140 may be subject to one or more additional processes, such as, for example, conditioning, filtration, distillation, packaging, etc.
  • the disclosed methods can be employed in any process in which a microorganism capable of fermentation such as yeast, fungi, mold, or bacteria converts a fermentable carbohydrate (sugar) to alcohol.
  • a microorganism capable of fermentation such as yeast, fungi, mold, or bacteria converts a fermentable carbohydrate (sugar) to alcohol.
  • alcoholic beverages are made from a process that includes fermentation.
  • Exemplary alcoholic beverages include wine, cider, perry, brandy, mead, whiskey, vodka, rice wine, rum and the like.
  • Another exemplary process is a biofuel production process, in which a fermentation microorganism such as yeast, fungi, mold, or bacteria converts a fermentable carbohydrate into an alcohol.
  • the fermentable carbohydrate can be derived for example from an agricultural product or byproduct, such as sugarcane, corn, sugarbeets, sorghum, pearl millet, grapes, rice, cassava and the like.
  • the resulting alcohol can be used as a fuel source.
  • Distilled alcoholic products including distilled spirit, are alcoholic beverages that are obtained by distillation from different possible sources such as wine or other fermented fruit or starches.
  • Distillation industry is another field where vacuum fermentation can be applied, improving rates and efficiency of the process. When fermenting under vacuum partial pressure, the boiling point of liquids decreases. Thus, removing alcohol from the original liquid is significantly easier during the fermentation process.
  • Yet another exemplary process is a pharmaceutical manufacturing process in which a fermentation microorganism can be used to convert organic materials into any of a number of pharmaceutical products or intermediates.
  • Microorganisms that can be used in these exemplary processes include, for example, prokaryotes such as bacteria (e.g. Escherichia coli, Staphylococcus aureus) and Streptomycetes (e.g. Streptomyces spp, Actinomyces spp), eukaryotes such as filamentous fungi (e.g., Nigrospora spp, Aspergillus spp,) and yeast (e.g. Saccharomyces cereviciae, Pichia pastoris).
  • prokaryotes such as bacteria (e.g. Escherichia coli, Staphylococcus aureus) and Streptomycetes (e.g. Streptomyces spp, Actinomyces spp), eukaryotes such as
  • Pharmaceutical molecules that can be produced by fermentation include, for example, smaller molecules such as short peptides and low molecular weight organic molecules, larger molecules including proteins and nucleic acids (DNA, RNA) and macromolecules such as lipids and carbohydrate polymers, plus various combinations of product types, for example lipopolysaccharides, lipopeptides, peptidoglycans.
  • Yet another exemplary process is a cosmetic manufacturing process.
  • the cosmetic industry has currently adopted fermentation processes and techniques to produce ingredients or products. Natural ingredients such as black tea, ginseng, seaweed, bamboo sap extract, red clove flower, hibiscus and various herbs and flowers are a common substrate in this industry. Cosmetics made from fermentation can use one or more natural ingredients that have undergone a fermentation process. Since natural ingredients are used, beneficial effects to even sensitive skin have been reported.
  • a method of fermentation comprising: combining in a vessel a fermentation microorganism and a liquid substrate comprising fermentable carbohydrates, to provide a fermentation composition; fermenting the fermentation composition within the vessel so that at least a portion of the fermentable carbohydrates have been converted to an alcohol, resulting in a fermented product comprising the alcohol; wherein the fermenting comprises: substantially depleting any oxygen in the fermentation composition; after substantially depleting the oxygen, reducing the pressure in the vessel to a reduced pressure of 14.0 psia or below; and continuing the fermentation under anaerobic conditions at the reduced pressure to produce the fermented product.
  • Aspect 2 The method of Aspect 1 , wherein the fermentation microorganism is Saccharomyces cerevisiae, Saccharomyces pastorianus or an ethanol-producing bacterial spp.
  • Aspect 3 The method of Aspect 1 or 2, wherein the fermented product is an alcoholic beverage, a distillation product, or a biofuel product.
  • Aspect 4 The method of any one of Aspects 1 to 3, wherein the liquid substrate has an original gravity from about 1.048 to about 1.083 or sugar content from about 12°Plato to about 20°Plato, or an original gravity of about 1.057 (14 °P) to about 1.079 (19°P), or about 1.065 (16°P) to about 1.074 (18°P), or an original gravity of greater than about 1.083 (20°P) or an original gravity of about 1.083 (20°P) to about 1.129 (30°P).
  • Aspect 5 The method of any one of Aspects 1 to 4, wherein the liquid substrate comprises a wort or a biomass.
  • Aspect 6 The method of any one of the foregoing Aspects, wherein the reduced pressure is 12 psia or below, or 10 psia or below, or 8 psia or below, or 6 psia or below, or 4 psia or below, or 3.5 psia or below.
  • Aspect 7 The method according to any one of the foregoing Aspects, wherein the viability of the fermentation microorganisms in the fermentation composition is maintained at or above 90% during fermentation.
  • Aspect 8 The method according to any one of the foregoing Aspects, wherein during the fermentation, the maximum number of microorganism cells in the fermentation composition is at least about 15% greater than the maximum number of microorganism cells in a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • Aspect 9 The method according to any of the foregoing Aspects, wherein during fermentation the sugar concentration of the fermentation composition reaches its attenuation limit at least about 25% faster than that of a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • Aspect 10 The method according to any one of the foregoing Aspects, wherein the fermented product has a concentration of ester volatiles or higher alcohol volatiles that is greater than a concentration of ester volatiles or higher alcohol volatiles of a fermented product produced during a comparable fermentation conducted under atmospheric pressure.
  • a method of fermentation comprising: combining in a vessel a fermentation microorganism and a substrate comprising fermentable carbon source, to provide a fermentation composition; fermenting the fermentation composition within the vessel so that at least a portion of the fermentable carbon source has been converted to a pharmaceutical substrate, cosmetic substrate, enzyme, volatile, or drug, resulting in a fermented product comprising the pharmaceutical substrate, cosmetic substrate, enzyme, volatile, or drug; wherein the fermenting further comprises: substantially depleting any oxygen in the fermentation composition; after substantially depleting the oxygen, reducing the pressure in the vessel to a reduced pressure of 14.0 psia or below; and continuing the fermentation under anaerobic conditions at the reduced pressure to produce the fermented product.
  • Aspect 12 The method according to Aspect 11 , wherein the reduced pressure is 12 psia or below, or 10 psia or below, or 8 psia or below, or 6 psia or below, or 4 psia or below, or 3.5 psia or below.
  • Aspect 13 The method according to Aspect 1 1 or 12, wherein during the fermentation, the maximum number of microorganism cells in the fermentation composition is at least about 15% greater than the maximum number of microorganism cells in a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • Aspect 14 The method according to any one of Aspects 11 to 13, wherein during fermentation the sugar concentration of the fermentation composition proceeds at a rate that is at least about 25% faster than that of a fermentation composition produced during a comparable fermentation conducted under atmospheric pressure.
  • Aspect 15 The method according to any one of Aspects 1 1 to 14, wherein the viability of the fermentation microorganisms in the fermentation composition is maintained at or above 90% during fermentation.
  • Aspect 16 The method according to any one of Aspects 1 1 to 15, wherein the fermented product has a concentration of ester volatiles or higher alcohol volatiles that is greater than a concentration of ester volatiles or higher alcohol volatiles of a fermented product produced during a comparable fermentation conducted under atmospheric pressure.
  • a BioFlo ® 310 Benchtop Bioreactor (commercially available from Eppendorf North America, Hauppauge, NY), such as the one shown in FIG. 3, was employed in fermentation or propagation processes as indicated herein.
  • This equipment had microprocessor control of pH, dissolved oxygen (DO), agitation, temperature, pump feed, antifoam, foam level.
  • the equipment vessel had a 5L capacity consisting of a stainless steel headplate with a variety of ports to attach different probes to control a number of parameters, a detachable glass tube vessel body, and a stainless- steel bottom jacket.
  • a Bio Pilot 130 Liter bioreactor (commercially available from Applikon Biotechnology, The Netherlands), such as the one shown in FIG. 2, was employed for fermentation processes as described below.
  • the Bio Pilot 130 Liter system is a stainless-steel stirred jacket tank reactor with a 130 Liters capacity. Inside the vessel there was a sparging and stirring device to control the aeration and agitation respectively.
  • the reactor consisted of a stainless steel headplate with different sizes ports to monitor parameters such as pressure.
  • a condenser was attached to the headplate, which had a cooling and a hot phase.
  • the reactor had a sight glass and 7 ports in the bottom-front side of the vessel, which could be used to measure parameters such as: temperature, pH, dissolved oxygen can be monitored.
  • a sampler device was attached to one of the front ports, to perform the external analyses during the fermentation.
  • the procedure for counting yeast cells was based on the method described by the American Society of Brewing Chemists (ASBC) Yeast-4. This method was applied to calculate the number of cells per mL after the rehydration process, during the propagation and fermentation. A hemocytometer with a cover slip was used and it was observed through a microscope with a 400x magnification combining a 40x objective lens and 10x eyepiece.
  • ASBC American Society of Brewing Chemists
  • Chambers filling A 45° angle between the pipette tip and the chamber was used to fill the chambers. Hemocytometer’s chamber was filled with 10.0 pL of sample each and left it rest to let the yeast settle.
  • Cell counting range To obtain an accurate value of cell counting, five squares of each chamber were selected. The number of yeast cells counted on the entire chamber area (1 mm 2 ) were greater than or equal to 75 cells.
  • C is the initial number of yeast cells expressed in V is the volume of slurry needed expressed in mL
  • C 2 is the final number of yeast cells after pitching expressed in - c ⁇ -
  • V 2 is the substrate’s volume to pitch expressed in mL.
  • the yeast viability assessment determines the percentage of living yeast cells during fermentation and is a good indicator to understand if any change or changes affects yeast cells health and therefore their ability to perform an adequate fermentation process.
  • the method used for assessing the viability was based on the ability of methylene blue to stain dead yeast cells into a dark blue color while living cells do not stain since the viable cells contain an enzyme that decolorizes the dye.
  • the yeast viability was assessed with the methylene blue technique (Painting, K. 1990). The hemocytometerwas used, and the counting yeast cell procedure was applied. The results are expressed in percentage of living yeast cell according to the Equation II, below.
  • the objective of this Example was to demonstrate the behavior of yeast within a fermentation, using a wort with initial sugar content of 14.0°P, and under partial headspace vacuum pressure (approximately 3.5 psia), as compared to a similar fermentation at atmospheric pressure (14.7 psia), to demonstrate the effect of vacuum pressure on the ethanol production rates and yeast viability.
  • the Lager wort made was used for both the propagation and fermentation steps. Prior propagation, the wort was sterilized 121 °C for 15 minutes.
  • the Active Dry Yeast (ADY) selected for this Example was“Diamond” Lager yeast (S. pastorianus), commercially available from Lallemand Inc., Canada.
  • the yeast rehydration process was followed according to the method described by Jenkins, D. 201 1.
  • a tube was filled with 10 mL of tap water and sterilized for 15 minutes at 121 °C. After the sterilization process, the tube was attemperate at 25°C in a water bath and 1 g of ADY was added, the slurry was then mixed and left to rest for 15 minutes, after this time the tube was gently mixed again and let it rest for 45 additional minutes in the same water bath at 25 °C.
  • the propagation step was conducted in the BioFlo ® 310 Benchtop Bioreactor, described above.
  • the yeast growth, yeast viability, and substrate sugar concentration were monitored and controlled to the parameters shown in Table 3, below. Cells in suspension, viability, sugar concentration and pH were observed and/or calculated using the methods described herein, and recorded in FIG. 5.
  • FIG. 5 shows the mean of each parameter monitored from each of the propagation steps performed prior the fermentation processes. The standard deviation of each sample is shown.
  • the yeast in each propagation began with a low viability after the rehydration process but increased above 90% throughout the propagation.
  • the sugar concentration decreased during the process mainly due to the yeast metabolism of sugars to promote yeast growth.
  • the final count of the yeast cells in suspension was between 140-180 million cells per milliliter with an initial yeast cells in suspension of approximately 15 million of cells per milliliter, this represents a 10-fold increase during the propagation period.
  • the fermentation step was conducted under partial headspace vacuum pressure (approximately 3.5 psia), labeled as“Vacuum” or“V” processes, as compared to a similar fermentation at atmospheric pressure (14.7 psia) labeled as“Control” or“C” processes.
  • the substrate preparation, yeast rehydration, yeast preparation, and initial pitching were performed consistently across all samples, as described above.
  • the fermentation process was performed in the BioPilot 130L fermenter at 15°C.
  • the temperature and pressure of the reactor were controlled, while monitoring parameters such as: sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability.
  • yeast cells were allowed to grow after pitching, as per the control fermentation at atmospheric pressure. This facilitated the normal formation of the protective compounds such as: glycerol, trehalose, and the like due to the presence of oxygen.
  • the dissolved oxygen parameter was monitored carefully during the first 12 hours (approximately) of fermentation until the oxygen was completely depleted. Once depleted, a vacuum pressure was applied to the vacuum processes to maintain a 3.5 psia pressure, while the control processes were maintained at atmospheric pressure (approximately 14.7 psia).
  • FIG. 4A shows the data generated during the control fermentation process.
  • FIG. 4B shows the data generated during the vacuum fermentation process.
  • FIG. 6A shows a comparison in number of cells in suspension for both control and vacuum processes, and
  • FIG. 6B shows a comparison in ethanol and extract content for both the control and vacuum processes.
  • the substrate preparation, yeast rehydration and yeast propagation were conducted as described in Example 1 , above. Cells in suspension, viability, sugar concentration and pH were observed and/or calculated during propagation, using the methods described herein and are shown in FIG. 8.
  • the substrate was prepared using the same procedures and industrial Lager wort recipe as for Example 1 , however the sugar content was increased to 14.5°P.
  • the fermentation process was performed in the BioPilot 130L fermenter at 15°C.
  • the temperature and pressure of the reactor were controlled, while monitoring parameters such as: sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability.
  • yeast cells were allowed to grow after pitching, as per the control fermentation. This facilitated the normal formation of the protective compounds such as: glycerol, trehalose, and the like due to the presence of oxygen.
  • the dissolved oxygen parameter was monitored carefully during the first 12 hours (approximately) of fermentation until the oxygen was completely depleted. Once depleted, a vacuum pressure was applied to the vacuum processes to maintain a 3.5 psia pressure, while the control processes were maintained at atmospheric pressure (approximately 14.7 psia).
  • FIG. 7 A shows the data generated during the control fermentation process.
  • FIG. 7B shows the data generated during the vacuum fermentation process.
  • FIG. 9A shows a comparison in number of cells in suspension for both control and vacuum processes, and
  • FIG. 9B shows a comparison in ethanol and extract content for both the control and vacuum processes.
  • the substrate preparation, yeast rehydration and yeast propagation were conducted as described in Example 1 , above. Cells in suspension, viability, sugar concentration and pH were observed and/or calculated during propagation, using the methods described herein and are shown in FIG. 11.
  • the substrate was prepared using the same procedures and industrial Lager wort recipe as for Example 1 , however the sugar content was increased to 15°P
  • the fermentation process was performed in the BioPilot 130L fermenter at 15°C.
  • the temperature and pressure of the reactor were controlled, while monitoring parameters such as: sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability.
  • yeast cells were allowed to grow after pitching, as per the control fermentation. This facilitated the normal formation of the protective compounds such as: glycerol, trehalose, and the like due to the presence of oxygen.
  • the dissolved oxygen parameter was monitored carefully during the first 12 hours (approximately) of fermentation until the oxygen was completely depleted. Once depleted, a vacuum pressure was applied to the vacuum processes to maintain a 3.5 psia pressure, while the control processes were maintained at atmospheric pressure (approximately 14.7 psia).
  • FIG. 10A shows the data generated during the control fermentation process.
  • FIG. 10B shows the data generated during the vacuum fermentation process.
  • FIG. 12A shows a comparison in number of cells in suspension for both control and vacuum processes, and
  • FIG. 12B shows a comparison in ethanol and extract content for both the control and vacuum processes.
  • an exemplary fermentation process for a high gravity substrate was carried out under partial vacuum (3.5 psia, labelled“Vacuum” or“V”) and atmospheric (14.7 psia, labelled“Control” or“C”) conditions.
  • the objective of this Example was to demonstrate the effects of vacuum on a high gravity (20°P initial sugar content) and very high gravity (30°P initial sugar content) fermentation process.
  • the Active Dry Yeast (ADY) selected for this Example was“Belle Wu Belgian Huawei-Style” yeast ( Saccharomyces cerevisiae var. diastaticus), from Lallemand, Inc.
  • the yeast rehydration process was followed according to the method described by Jenkins, D. 201 1.
  • a tube was filled with 10 mL of tap water and sterilized for 15 minutes at 121 °C. After the sterilization process, the tube was attemperate at 30°C in a water bath and 1 g of ADY was added, the slurry was then mixed and left to rest for 15 minutes, after this time the tube was gently mixed again and let it rest for 45 additional minutes in the same water bath at 30 °C.
  • Phase 1 After rehydration, 3.0 pl_ of yeast slurry were transferred aseptically into three 125-mL flask containing 50-mL of YEPD broth using a 1.0 pL disposable sterile loop. A cotton ball was added to top of each flask to protect from contamination while allowing aerobic incubation.
  • Table 6 shows the parameters controlled during the phase 1 of the yeast propagation.
  • Phase 2 After the first 24 hours, the resulting slurry was centrifuged in sterile 10-mL tubes and the supernatant was discarded. The centrifuge parameters were 3,000xg for 3 min. Yeast pellets were resuspended in sterile distilled water. The centrifugation and resuspension of the resulting yeast/water slurry was repeated two more times, as described above, for a total of three washes.
  • Washed yeast was pitched at 15 x 10 s viable cells/mL into six flasks of 250-mL volume capacity, each containing 100-mL of YEPD broth.
  • Table 7 shows the parameters controlled during the phase 2 of the yeast propagation.
  • yeast viability was 100% after phase 2 of propagation.
  • the final count of the yeast cells in suspension increased 10-fold during the propagation period.
  • the fermentation process was performed in the BioFlo ® 310 Benchtop Bioreactor at 30°C. During fermentation, the temperature and pressure of the reactor were controlled, while monitoring parameters such as: sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability.
  • yeast cells were allowed to grow after pitching, as per the control fermentation. This facilitated the normal formation of the protective compounds such as: glycerol, trehalose, and the like due to the presence of oxygen.
  • the dissolved oxygen parameter was monitored carefully during the first 4 hours (approximately) of fermentation until the oxygen was completely depleted. Once depleted, a vacuum pressure was applied to the vacuum processes to maintain a 3.5 psia pressure, while the control processes were maintained at atmospheric pressure (approximately 14.7 psia).
  • an industrial lager wort recipe was fermented with Saccharomyces pastorianus at 15°C and maintained pressure of 3.5 psia (modified process).
  • a control fermentation process was conducted simultaneously, in which the same industrial lager wort recipe was fermented with Saccharomyces pastorianus at 15°C but at a maintained pressure of 14.7 psia.
  • Samples were taken from each process every 24 hours during the fermentations. The samples were analyzed for rate, yeast health, and volatile production. Volatiles analysis was performed using a Stratum purge and trap unit and individual compounds were identified using a GC-MS.
  • the final vacuum-fermented beer product had a significantly higher concentration of the same volatile compounds as compared to the final control- fermented beer product.
  • the concentration of higher alcohols and esters were 16% and 37% higher under vacuum conditions than atmospheric conditions, respectively, likely due to the high rate of sugar consumption under vacuum.
  • Swart CW Dithebe K, Pohl CH, Swart HC, Coetsee E, van Wyk PW, Swarts JC, Lodolo EJ, Kock JL.

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Abstract

Dans un aspect, l'invention concerne un procédé de fermentation, dans lequel un micro-organisme convertit une source de carbone fermentable en un produit de fermentation, tel qu'un alcool. Dans le procédé décrit, au moins une partie de la fermentation est conduite à des pressions réduites. Dans les conditions décrites, le taux de fermentation peut augmenter, et le taux de consommation de sucre peut augmenter. Le présent abrégé est destiné à être utilisé comme outil d'exploration à des fins de recherche dans ce domaine technique particulier et ne se limite pas à la présente invention.
PCT/US2020/015379 2019-01-28 2020-01-28 Procédé de fermentation sous pression réduite Ceased WO2020159964A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100316761A1 (en) * 2006-03-17 2010-12-16 Jean-Luc Baret Nutritional Supplement for Simultaneous Saccharification and Fermentation Medium for the Manufacture of Ethanol
WO2011045365A1 (fr) * 2009-10-14 2011-04-21 Purac Biochem Bv Procédé de fermentation sous pression réduite
US8173390B2 (en) * 2008-10-10 2012-05-08 The Board Of Trustees Of The University Of Illinois Method for facilitating fermentation of high solids compositions
US20130143277A1 (en) * 2009-12-23 2013-06-06 Danisco Us Inc. Methods for Improving the Efficiency of Simultaneous Saccharification and Fermentation Reactions
US20180036706A1 (en) * 2012-10-10 2018-02-08 Xyleco, Inc. Processing materials

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100316761A1 (en) * 2006-03-17 2010-12-16 Jean-Luc Baret Nutritional Supplement for Simultaneous Saccharification and Fermentation Medium for the Manufacture of Ethanol
US8173390B2 (en) * 2008-10-10 2012-05-08 The Board Of Trustees Of The University Of Illinois Method for facilitating fermentation of high solids compositions
WO2011045365A1 (fr) * 2009-10-14 2011-04-21 Purac Biochem Bv Procédé de fermentation sous pression réduite
US20130143277A1 (en) * 2009-12-23 2013-06-06 Danisco Us Inc. Methods for Improving the Efficiency of Simultaneous Saccharification and Fermentation Reactions
US20180036706A1 (en) * 2012-10-10 2018-02-08 Xyleco, Inc. Processing materials

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