METHOD FOR FERMENTATION UNDER REDUCED PRESSURE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 62/797,674, filed January 28, 2019, which is hereby incorporated herein by reference in its entirety.

BACKGROUND

[0002] For decades, 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.). In brewing industries, 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).

[0003] Historically, industrial fermentation processes have been slow to change because of the high impact on S. cerevisiae and S. pastorianus viability, related closely to the different stresses during this process. However, in recent years, changes have started to occur such as high-gravity substrate brewing. As a consequence, processes have changed to improve beer quality, increase yield and optimize production rates. However these changes to the process require some adaptations by the yeast.

[0004] To withstand new stresses created during the modified fermentation the stress response mechanism in yeast must be understood. Due to environmental conditions S. cerevisiae and S. pastorianus respond to different kind of stress by synthesizing diverse chemical compounds and modifying their cell walls flexibility and permeability (Gibson, B. 2007). Some investigations have determined how a microorganism’s exposure to one type of stress can generate a resistance to another type of stress, such as induced barotolerance of the yeast caused by previous heat treatment (Iwahashi, H. 1991).

[0005] Despite advances in industrial fermentation, there is still a desire for improvements in efficiency and equipment utilization. These and other needs are satisfied by the present disclosure.

SUMMARY

[0006] In accordance with the purpose(s) of the disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to methods of fermentation wherein at least a portion of the fermentation step is conducted at a reduced pressure. In accordance with an aspect, 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. According to the disclosed method, 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.

[0007] According to one or more of the disclosed methods, the fermentation microorganism can be Saccharomyces cerevisiae or Sacchoromyces pastorianus, or an alcohol producing spp. According to some disclosed methods, the fermented product can be an alcoholic beverage, a distillation product, or a biofuel product. According to some disclosed methods, the liquid substrate can be a wort, or a biomass. According to some disclosed methods 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.

[0008] In accordance with another aspect, 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. In accordance with the disclosed methods, 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.

[0009] According to one or more of the disclosed methods, the reduced pressure can be 14.0 psia or below. According to some disclosed methods, the viability of the fermentation microorganisms in the fermentation composition is maintained at or above 90% during fermentation. According to some disclosed methods 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. According to some disclosed methods, 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. According to some disclosed methods, 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.

[0010] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0012] FIG. 1 shows a process flow diagram of an exemplary industrial brewing process, utilizing the disclosed fermentation methods.

[0013] FIG. 2 shows a pilot bioreactor apparatus as used in the Examples.

[0014] FIG. 3 shows a bioreactor apparatus used for yeast propagation in the Examples.

[0015] 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.

[0016] FIG. 5 shows data generated during an exemplary yeast propagation process, in accordance with Example 1 .

[0017] 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.

[0018] 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.

[0019] FIG. 8 shows data generated during an exemplary yeast propagation process, in accordance with Example 2.

[0020] 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.

[0021] 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.

[0022] FIG. 11 shows data generated during an exemplary yeast propagation process, in accordance with Example 3.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.

DETAILED DESCRIPTION

[0028] In view of the foregoing, 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. In the disclosed methods, 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.

[0029] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0030] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0032] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. [0033] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0034] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0035] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0036] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0037] As used herein,“comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,”“includes,”“included,”“involving,� ��“involves,”“involved,” and“such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of and“consisting of.” Similarly, the term“consisting essentially of is intended to include examples encompassed by the term“consisting of.

[0038] As used in the specification and the appended claims, the singular forms“a,”“an” and “the” include plural referents unless the context clearly dictates otherwise.

[0039] It should be noted that 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.

[0040] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, 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’. Likewise, the 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’. In addition, the phrase“about‘x’ to‘y’”, where‘x’ and‘y’ are numerical values, includes “about‘x’ to about‘y’”.

[0041] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, 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.

[0042] As used herein, 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. In such cases, it is generally understood, as used herein, that“about” and“at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, 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.

[0043] As used herein, 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.

[0044] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).

[0045] 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. Exemplary fermentation microorganisms include, for example, a bacteria, a mold, a fungus, a yeast, eukaryotic cells, or other organisms.

[0046] As used herein 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.

[0047] “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.

[0048] 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. A. METHOD OF INDUSTRIAL BREWING

[0049] In one aspect, the disclosure relates to a method of fermentation associated with an industrial brewing process used for the production of beer. Generally speaking, 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. During the fermentation process, 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.

1. WORT PRODUCTION

[0050] Referring to FIG. 1 , a first step in an industrial brewing process 100 is wort production 110. As used herein,“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.

[0051] 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. During a typical wort production 1 10 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.

[0052] The liquid wort mixture is then boiled to sanitize the wort. In some processes, hops are added during this step, during which the bittering, flavor and aroma can be extracted from hops. In beer production, the wort is known as "sweet wort" until the hops have been added, after which it is called "hopped wort” or“bitter wort". In a typical process, 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. Next, the flavouring hops are added, e.g., about 15 minutes from the end of the boil. Then, 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.

[0053] During the wort production process 1 10, 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. During the wort production process 110, 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.

[0054] 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.

2. YEAST REHYDRATION

[0055] Referring still to FIG. 1 , another step in the disclosed industrial brewing process 100 is the yeast rehydration 120.

[0056] As an initial matter, selection and propagation of yeast can affect many aspects of the industrial brewing process 100. 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. To synthetize new structures such as proteins, enzymes, etc. 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.

[0057] 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). [0058] 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).

[0059] The use of Active Dry Yeast (ADY) has gained more popularity and acceptance within the brewing industries (Normand, C. 2007.); it has become increasingly attractive because of the advantages presented in the beer processing (Jenkins, D. 201 1.). The yeast used throughout this study was rehydrated dried yeast.

[0060] For many years, the only dry strain used was the ale strain S. cerevisiae known as ADY, however it was until this last decade that manufacturers had introduced the Active Dry Lager Yeast, S. pastorianus, known as ADLY (Normand, C. 2007.). Some literature has mentioned that the principal advantage of the dry yeast is that they could be stored for a long period of time (Fels, S. 1999. Felsher, A. 1955.), without any considerable problem with the yeast viability. Furthermore, the process applied to obtain dry yeast has increased the benefits in the yeast physiology, creating substances such as trehalose, which make the yeasts more resistant and protect them from some stresses (Normand, C. 2007.).

[0061] However, some of the disadvantages mentioned by the literature were the variation in some fermentation profiles (Normand, C. 2007; Jenkins, D. 201 1.), also negative consequences in the quality of the final product such as: abnormal flocculation, haze formation and less stable foam structure (Fin, D. 2002.).

[0062] Common ways to obtain dry yeast include fluidized bed drying, freeze drying and spray drying. 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).

[0063] The rehydration of the dry yeast prior to fermentation is an important step to improve yeast’s viability. According to some authors, 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.).

[0064] The result of the yeast rehydration step 120 is a hydrated yeast 126, which can then be used in the yeast propagation step 130.

3. YEAST PROPAGATION

[0065] Referring to FIG. 1 , the disclosed industrial brewing process 100 includes the step of yeast propagation 130. Generally speaking, 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.

[0066] The propagation of yeast is influenced by several factors, including oxygen, pH, temperature, and wort composition.

[0067] One factor that affects yeast growth is oxygen. The oxygen content, or aeration, 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.

[0068] 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. In typical brewing processes, 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. In a typical brewing process, wort should contain all of the ingredients necessary for yeast propagation. In some brewing methods, 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. In some methods, metal ions can be added to the propagation mixture. For example, in some methods, zinc can be added. [0069] 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.

[0070] Another factor which affects yeast growth and metabolism is temperature. Typical brewing yeasts will grow and ferment at temperatures up to 98 °F (37 °C). These high temperatures are not optimal for yeast propagation or fermentation, since they produce numerous undesirable volatile and flavor compounds, as well as affect the overall viability and stability of the yeast. 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. 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.

[0071] The result of the yeast propagation 130 is a cultivated yeast 136, which can be “pitched” to the fermentation process 140. According to various disclosed methods, 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%.

4. FERMENTATION

[0072] Referring to FIG. 1 , the disclosed industrial brewing process 100 includes the step of fermentation 140. Generally speaking, 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.

[0073] In fermentation, yeast and wort are combined to provide a fermentation mixture, in which the yeast metabolizes the fermentable sugars to produce ethanol and carbon dioxide. As used herein,“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.”

[0074] Like propagation, 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.

[0075] One factor that can affect fermentation is pH. As mentioned above, yeast can survive at very low pH, e.g., as low as 2.0. However, fermentation can be relatively slow at low pH. Typical wort is acidic, e.g., having a pH near 5.2. During propagation and fermentation the pH can drop below 4.0. In some disclosed methods, 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. In some embodiments, the wort can be further acidified, e.g., to help to prevent bacterial infection. However, acidification during fermentation can slow down fermentation, so acidification is typically done after fermentation is complete.

[0076] Wort composition also can affect fermentation performance. In terms of fermentation, 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. In the brewing industry, 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:

Plato degree (°P) = 1000 Specific Gravity— l)/4

[0077] In the brewing industry, a normal gravity wort typically has an original gravity of about 1.048 (12°P). In comparison, 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.

[0078] 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. Where 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). When a selected yeast strain is intended for a very high gravity fermentation, 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.

[0079] During fermentation, the reduction of sugar (extract) 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.

[0080] According to the disclosed industrial brewing process and methods, at least a portion of the fermentation step is conducted under a low pressure, or vacuum. Fermentation has been performed under continuous or semi-continuous vacuum pressure. In previous studies 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). In absence of the stress caused by the ethanol, 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).

[0081] During the first hours of a conventional fermentation process, the oxygen dissolved in the substrate decreases due to the growth of the yeast. Some literature emphasized the importance of maintaining oxygen levels during a vacuum fermentation process, for the purpose of promoting formation of sterols and fatty acids and for a successful beer production (Gibson et. al. 2007). As previously proposed, one way to promote these oleic compounds, was to enrich the substrate during the fermentation with different supplement such as: ergosterol (Finn, R. and Ramalingam, A. 1977), or a mixed solution of ergosterol, oleic acid and Tween 80 (Ramalingham and Finn 1977). An alternative way to promote these oleic compounds, was to sparge oxygen directly to the substrate during the fermentation process at vacuum pressures (Cysewski, G. and Wilke, C. 1977). [0082] Unlike the previously reported methods, the disclosed methods do not involve introducing additional oxygen or enriching the substrate. Rather the method involves allowing the dissolved oxygen that is present in the fermentation mixture to deplete, and then applying a vacuum pressure to the composition for the remainder of the fermentation step, without supplying additional oxygen.

[0083] According to the disclosed methods, after the oxygen is depleted in the fermentation mixture, 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 for the remainder of the fermentation. Generally speaking, a lower pressure provides a greater benefit to the fermentation result. However, reducing the pressure causes a reduction in the boiling point of the fermentation mixture, making it difficult to achieve the desired fermentation results. According to some embodiments, the fermentation is conducted at a pressure greater than about 0.65 psia.

[0084] According to the disclosed methods, 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. Generally speaking, 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.

[0085] According the disclosed methods, the fermentation step is concluded when the desired attenuation has been achieved. For example, according to some of the disclosed methods, 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.

[0086] Surprisingly, in applying the disclosed methods, it has been found that fermentation under the disclosed vacuum conditions differs significantly from a similar fermentation process performed under atmospheric conditions. In particular, by using the disclosed methods, a fermentation process can produce a fermentation product with a higher sugar content compared to a fermentation product made under atmospheric pressure.

[0087] By using the disclosed methods, 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. For example, during beer fermentation, the most important volatile compounds, with respect to sensory characteristics, 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. 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. This benefit also can be valuable to industries in which the volatile metabolites produced during fermentation are the final product, such as in flavor and pharmaceutical manufacturers. Therefore, in some aspects, 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. In some aspects, 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.

[0088] In addition, 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. According to the disclosed methods, 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.

[0089] According to the disclosed methods, 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. According to the disclosed methods 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.

[0090] Without intending to be bound by a particular theory, it is hypothesized that the removal of the carbon dioxide by vacuum is one potential mechanism for the improved results. As a product of fermentation, carbon dioxide is produced inside the yeast cell. (Swart C.W., et al., 2012) Accumulation of carbon dioxide within the yeast cell can cause stress, reducing its effectiveness. Applying vacuum during fermentation can increase the pressure gradient across the yeast cell wall, enabling release of the carbon dioxide from the yeast cells. Removal of the carbon dioxide can reduce a significant stressor on the yeast, enabling faster fermentation and increased sugar consumption.

[0091] In addition, the disclosed methods do not significantly change other factors affecting fermentation, including pH and viability of the yeast. In embodiments of the disclosure, 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. According to the embodiments, 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.

[0092] 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. And by 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).

[0093] The result of the fermentation step 140 is the desired fermentation product 146. In the disclosed methods the fermentation product contains the desired products of the metabolite. For example, for an exemplary alcoholic beverage 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.

[0094] 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.

B. OTHER APPLICATIONS OF DISCLOSED METHODS

[0095] While the disclosed fermentation methods are described above with reference to industrial brewing, they are not limited to this application. Having read the present disclosure, a skilled artisan would recognize that the disclosed methods also have potential utility in other fermentation processes in which a fermentation microorganism converts a fermentable carbon source into another desired substance. The disclosed methods may similarly result in a reduction in fermentation time, and increase in capacity in such processes.

[0096] For example, 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. Essentially all 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.

[0097] 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.

[0098] 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.

[0099] 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). 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.

[00100] 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.

[00101] The present disclosure will be better understood upon reading the following numbered aspects, which should not be confused with the claims. Any of the numbered aspects below can, in some instances, be combined with aspects described elsewhere in this disclosure and such combinations are intended to form part of the disclosure.

[00102] Aspect 1. 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.

[00103] Aspect 2. The method of Aspect 1 , wherein the fermentation microorganism is Saccharomyces cerevisiae, Saccharomyces pastorianus or an ethanol-producing bacterial spp.

[00104] Aspect 3. The method of Aspect 1 or 2, wherein the fermented product is an alcoholic beverage, a distillation product, or a biofuel product.

[00105] 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).

[00106] Aspect 5. The method of any one of Aspects 1 to 4, wherein the liquid substrate comprises a wort or a biomass.

[00107] 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.

[00108] 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.

[00109] 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.

[00110] 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.

[00111] 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.

[00112] Aspect 1 1. 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.

[00113] 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.

[00114] 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.

[00115] 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.

[00116] 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.

[00117] 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.

[00118] Before proceeding to the Examples, it is to be understood that this disclosure is not limited to particular aspects described, and as such may, of course, vary. Other systems, methods, features, and advantages of foam compositions and components thereof will be or become apparent to one with skill in the art upon examination of the drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. It is also to be understood that the terminology used herein is forthe purpose of describing particular aspects only, and is not intended to be limiting. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

C. EXAMPLES

[00119] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. EQUIPMENT USED

[00120] 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.

[00121] 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.

2. METHODS

[00122] Counting yeast cells

[00123] 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.

[00124] The following aspects of the method were followed:

[00125] 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.

[00126] Borders: Once the hemocytometer was loaded, placed in the microscope platform, and focused with the magnification previously mentioned, the borders considered to be counted were the top and the left borders, while the right and the bottom borders were not considered

[00127] 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.

[00128] Pitching calculation.

[00129] The volume of slurry needed to pitch the substrate prior the propagation process or the fermentation process, was calculated by Equation I below.

CiV = C ₂ V ₂ Equation I

[00130] Where 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 ^- , and V ₂ is the substrate’s volume to pitch expressed in mL.

[00131] Yeast viability

[00132] 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.

Number of viable yeast cells

% Viability = Equation II

Total yeast cells counted

3. EXAMPLE 1. EXEMPLARY INDUSTRIAL BREWING PROCESS: FERMENTATION OF AN INDUSTRIAL LAGER WORT (14.0°P)

[00133] 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.

[00134] Substrate Preparation:

[00135] The wort used for this Example was made based upon an industrial Lager wort recipe in Table 1 , below.

Table 1. Industrial Lager wort recipe

Ingredients Quantity

Malt

American 2-row 14.37 kg

American 6-row 14.37 kg

Adjunct

American flaked rice 9.61 kg Hops

Hallertau Hersbrucker 147.91 g

Hallertau Hersbrucker 295.83 g

Water

Mashing water 160 L

Lautering water 40 L

[00136] The hops were divided in two parts, and introduced as described in Table 2, below.

Table 2. Process to make substrate

Process Temperature Time

Mashing 65°C 90 min

Lautering 79°C 10 min

Boiling

Phase 1 Boiling point 20 min

Phase 2 Boiling point 70 min

[00137] The Lager wort made was used for both the propagation and fermentation steps. Prior propagation, the wort was sterilized 121 °C for 15 minutes.

[00138] Yeast Rehydration:

[00139] 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.

[00140] Yeast Propagation

[00141] After the rehydration time, the slurry in each tube was mixed one last time before it was pitched in the substrate. The counting cell procedure and pitching were used.

[00142] 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.

Table 3. Propagation controlling Parameters

Concentration Temperature Agitation

cel I/m L _ Rpm 15 x 10 ⁶ 20 100

[00143] 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.

[00144] Fermentation

[00145] 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.

[00146] To reduce the variability due to any process factor other than fermentation pressure, the substrate preparation, yeast rehydration, yeast preparation, and initial pitching were performed consistently across all samples, as described above.

[00147] For this example, the fermentation process was performed in the BioPilot 130L fermenter at 15°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.

[00148] After pitching the yeast cells to both the control and vacuum fermentation processes, and before applying partial vacuum pressure to the vacuum process, 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). Fermentation proceeded for up to 120 hours, during which sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability were monitored and recorded. 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.

[00149] Referring to FIGS. 4A-4B and 6A-6B, the results showed that the fermentation under vacuum differed significantly from the control in the number of suspended yeast cells, and the rate of fermentation. When fermented under vacuum, the fermentation occurred more rapidly, with an increase of 15% in the number of cells in suspension. The rate of sugar consumption under the vacuum fermentation was greater than the control (at atmospheric pressure), thus increasing the rate of ethanol production when vacuum was applied. The removal of C0 ₂ under vacuum, is the hypothesized mechanism for these results. The ending pH (3.9), and viability of the yeast (above 90%) during vacuum fermentations did not differ significantly from the respective controls. Applying vacuum pressure during the process, makes it reach the highest ethanol concentration in a shorter time compared at atmospheric conditions. The total reduction in fermentation time was approximately 15 hours from the control process.

4. EXAMPLE 2. EXEMPLARY INDUSTRIAL BREWING PROCESS: FERMENTATION OF AN

INDUSTRIAL LAGER WORT (14.5°P)

[00150] In this Example, an exemplary fermentation process was carried out under partial vacuum (3.5 psia, labelled“Vacuum” or“V”) and atmospheric (14.7 psia, labelled“Control” or “C”) conditions, to demonstrate the effects of vacuum on the fermentation process. Both control and vacuum conditions were repeated at initial sugar content of 14.5°P.

[00151] 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.

[00152] For this example, the fermentation process was performed in the BioPilot 130L fermenter at 15°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.

[00153] After pitching the yeast cells to both the control and vacuum fermentation processes, and before applying partial vacuum pressure to the vacuum process, 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). Fermentation proceeded for up to 120 hours, during which sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability were monitored and recorded. 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.

[00154] The results showed that the fermentation under vacuum differed significantly from the control. Referring to FIGS. 9A-9B, fermentation under vacuum pressure occurred more rapidly with an increase in the number of suspended yeast cells of approximately 25%, as compared to fermentation under atmospheric pressure. Fermentation under vacuum pressure increased the rate of fermentation, reaching the attenuation level in a shorter time than under atmospheric conditions. The rate of sugar consumption and ethanol production under the vacuum fermentation were faster than under atmospheric pressure, reaching the attenuation level about 20 hours. The pH (approximately 3.9) and viability of the yeast (approximately 95%) did not differ significantly between the control and vacuum samples.

5. EXAMPLE 3. EXEMPLARY INDUSTRIAL BREWING PROCESS: FERMENTATION OF AN INDUSTRIAL LAGER WORT (15.0°P)

[00155] In this Example, an exemplary fermentation process was carried out under partial vacuum (3.5 psia, labelled“Vacuum” or“V”) and atmospheric (14.7 psia, labelled“Control” or “C”) conditions, to demonstrate the effects of vacuum on the fermentation process. Both control and vacuum conditions were repeated at initial sugar content of 15.0°P.

[00156] 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

[00157] For this example, the fermentation process was performed in the BioPilot 130L fermenter at 15°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.

[00158] After pitching the yeast cells to both the control and vacuum fermentation processes, and before applying partial vacuum pressure to the vacuum process, 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). Fermentation proceeded for up to 120 hours, during which sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability were monitored and recorded. 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.

[00159] The results showed that the fermentation under vacuum differed significantly from the control. Referring to FIGS. 12A-12B, fermentation under vacuum pressure occurred more rapidly with an increase in the number of suspended yeast cells of approximately 100%, as compared to fermentation under atmospheric pressure. Fermentation under vacuum pressure increased the rate of fermentation, reaching the attenuation level in a shorter time than under atmospheric conditions. The rate of sugar consumption and ethanol production under the vacuum fermentation were faster than under atmospheric pressure, reaching the attenuation level about 30 hours. The pH (approximately 3.9) and viability of the yeast (approximately 95%) did not differ significantly between the control and vacuum samples.

6. EXAMPLE 4. VERY HIGH GRAVITY FERMENTATION

[00160] In this Example, 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.

[00161] Substrate Preparation

[00162] The wort used for this Example was made based upon an industrial very high gravity wort recipe in Table 4, below.

Table 4. Very high gravity wort recipe

Ingredients Quantity

Malt

Pale barley 14.59 kg

Adjunct

American flaked rice 4.43 kg

Hops _ Hallertau Hersbrucker 148.00 g

Hallertau Hersbrucker 74.00 g

Water 50L

[00163] The hops were divided in two parts, and introduced as described in Table 5, below.

Table 5. Process to make substrate

Process Temperature Time

Mashing 65°C 90 min

Lautering 79°C 10 min

Boiling

Phase 1 Boiling point 20 min

Phase 2 Boiling point 70 min

[00164] The wort made was used only for both the fermentation step.

[00165] Yeast Rehydration

[00166] The Active Dry Yeast (ADY) selected for this Example was“Belle Saison Belgian Saison-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.

[00167] Yeast Propagation

[00168] Yeast propagation was followed according to the method described by the American Society of Brewing Chemists (ASBC) Yeast- 14. This process was divided in two phases, described below.

[00169] 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.

[00170] Table 6 shows the parameters controlled during the phase 1 of the yeast propagation.

Table 6. Phase 1 propagation controlling parameters

Volumen

Temperature Agitation Time

Pitched

mI- °C_ Rpm_ Hour 3.0 30 100 24

[00171] 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.

[00172] 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.

Table 7. Phase 2 propagation controlling parameters

Volumen

Temperature Agitation Time

Pitched

FL °C _ Rpm Hour

15.0 x 10 ^s 30 100 24

[00173] Yeast was washed as mentioned above.

[00174] The 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.

[00175] Fermentation

[00176] For this example, 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.

[00177] After pitching the yeast cells to both the control and vacuum fermentation processes, and before applying partial vacuum pressure to the vacuum process, 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). Fermentation proceeded until no more fermentable sugars remained, during which sugar concentration, pH, ethanol concentration, number of yeast cells in suspension and yeast viability were monitored and recorded. The results for the very high gravity (30°P initial sugar content) fermentation are reported in FIGS. 13A-13B and 14A-14B.

[00178] It was observed that the fermentation under vacuum did not differ significantly from the control, with a high gravity wort (20°P). It is theorized that the yeast used in this example was selected for its tolerance to higher gravity worts, and was not under high stress in these conditions with the 20°P wort, which made the benefits of the vacuum less measurable. However, fermentation under vacuum differed significantly from the control within a very high gravity wort (30°P). Referring to FIG. 14A, fermentation under vacuum pressure occurred more rapidly with an increase in the number of suspended yeast cells of approximately 40%, as compared to fermentation under atmospheric pressure. Referring to FIG. 14B, fermentation under vacuum pressure increased the rate of fermentation, with the sugar concentration and ethanol production reaching attenuation in 100 hours (approximately 4 days) before the control fermentation under atmospheric conditions. Meanwhile, the resulting pH (approximately 4.1) and viability of the yeast did not differ significantly between the control and vacuum samples.

7. EXAMPLE 5. RAPID BEER FERMENTATION: VOLATILE FORMATION UNDER VACUUM

FERMENTATION

[00179] In this Example, the effect of vacuum (1/3 atmospheric) fermentation on the volatile content of an industrial lager product was evaluated. The objective of this example was to determine the volatile compound formation throughout an exemplary typical lager beer fermentation process under partial vacuum as compared to a comparable standard fermentation process at atmospheric pressure. This was accomplished by controlling the temperature and pressure of a 30L pilot fermentation.

[00180] For this experiment, 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.

[00181] The volatiles identified in the control process samples and vacuum process samples were classified in three different groups as shown in Table 8. Higher alcohols (Linalool, Isopentyl alcohol, Butyl alcohol, Isoamyl alcohol, Isobutanol, 2-Phenylethanol); esters (Ethyl Acetate, Isoamyl acetate, Ethyl caproate, Ethyl caprylate, Phenethyl ester); and carbonyl compounds (Acetaldehyde, Isoamyl aldehyde, Isobutanal). For each sample, the concentration of volatiles was quantified, as summarized in FIGS. 15A-15D. Table 8. Volatile Compounds

Compound Aroma Type

Acetaldehyde Green leaves, fruity Carbonyl compound Isoamyl aldehyde Chocolate, peach, fatty Carbonyl compound Isobutanal Grainy like germinating malt Carbonyl compound Isoamyl acetate Banana, estery Ester Phenethyl acetate Flowey, fruity, roses, honey Ester Ethyl acetate Sour apple, solvent, fruity, sweetish Ester Ethyl caproate Sour apple, fruity Ester Ethyl caprylate Apple, anissed Ester Linalool Flowery Higher alcohol

Isopentyl alcohol Bitter Higher alcohol Butyl alcohol Alcohol, fuel oil, varnish Higher alcohol Isoamyl alcohol Alcoholic, banana Higher alcohol Isobutanol Solvent Higher alcohol 2-Phenylethanol Roses, sweetish, perfumed Higher alcohol

[00182] Referring to FIG. 15D, 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.

[00183] This result can be exploited by industries where the volatile metabolites produced are the product including flavor and pharmaceutical companies. Additionally, as vacuum fermentation shows a higher generation of volatile compounds, this will have a significant impact upon the flavor profile of the beer - a potentially positive and important aspect within fermentation industries where product consistency is of the utmost importance.

D. REFERENCES

[00184] The following references, which are cited herein, are incorporated by reference in their entirety, to the extent they are consistent with the present disclosure.

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[00185] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.