CA2127440A1 - Method and composition for enhancing foam properties of fermented malt beverages - Google Patents

Abstract

There is disclosed a method and composition for enhancing the foam properties of a fermented malt beverage. Briefly stated, the method comprises adding to the beverage an isohumulone extract in the amount of between about 0.1 ppm and about 20 ppm by weight of the beverage and foaming proteins in the amount of between about 2 ppm and about 250 ppm by weight of the beverage. The fermented malt beverage foam-enhancing composition comprises a mixture of an isohumulone extract and foaming proteins, wherein the ratio of isohumulone extract to foaming proteins is between about 1 to about 1 and about 1 to about 2500. Further disclosed is a beverage composition with enhanced foam properties which comprises (a) a fermented malt beverage; (b) an isohumulone extract, the extract comprising between about 0.1 ppm and about 20 ppm by weight of the fermented malt beverage; and (c) foaming proteins, the proteins comprising between about 2 ppm and about 250 ppm by weight of the fermented malt beverage.

Description

~vo 93/15181 2 1 2 7 4 ~ ~ PCT'/US93/00932 S METHOD AND COMPOSlTlON FOR ENHANCING FOAM
PROPF,RTIES OF FERMENTED MALT BEVER~GES

Field of the In~!cn~i~n The present invention relates to a method and composition which enhances the foam properties of malt beverages. More particularly, the present invention relates to enhancing the amount and persistence of the foam head on a fermented malt beverage.
As used herein, the terrn "malt beverages" includes such foam-forming~ fermented malt beverages as beer, ale, dly beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic malt beverages and the like. Unless otherwise noted, the terrn "beer" shall be used throughout this specification as a generic terrn and refers to the entire group of fermented malt beverages.

B~ound ~the Invention The process of making fermented malt beverages is commonly referred to as brewing Generally, the principal raw materials used in making these beverages are water, hops and malt. In addition, adjuncts such as common corn grits, refined corn grits, and brewer's (milled) rice may be used as sources of starch. The starch will eventually be converted into dextrins and fermentable sugars.
For a number of reasons, the malt, which is produced princi-pally from selected varieties of barley typically has the greatest effect on the overall character and quality of the beer. First, the malt is the primary 9avoring agent in beer. Second~ the malt provides the major portion of the .

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WO 93/15181 PCI`/US93/00932.

fermentable sugars. Third, the malt provides the proteins which will contribute to the body and foam character of the beer. Fourth, the malt will provide the necessary en~natic activiy during mashing.
Hops also contribute significantly to beer quality, including flavoring. In particular, hops (or hop constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents, and aid in foam formation and stabilization.
MASHING. The brewing process begins by rnL~cing ground malt and warm water in large tubs until it forms a malt mash of porridge-like consistency. The temperature of the mash is then raised in steps, with time allowed at each step for the various enzymes to convert the starches into fermentable sugars.
If rice and corn adjuncts are to be used, they are separately cooked and a cooker mash is obtained. Production of the cooker mash involves the use of adjuncts along with a 10~b to 30% portion of the malt (or the addition of commercial enz~mes) in order to convert raw starch into fermentable sugars. The adjuncts (and the malt portion) are gradually brougbt to boilin~ and held there until the products are completely gelatinized. During the final stages of mashing (at the higher tempera-tures), the cooker mash and the malt mash are combined.
Mashing serves a three-fold purpose. First, it brings into soludon those substances of malt (and adjuncts) which are readily soluble in warm water. Second, it pem~its malt enzymes to act on insoluble substances and render them soluble. Third, it provides a far-reaching en~natic degradation of starches, proteins and gums into products of smaller size and lower molecular weight.
LAUTERING AND SPARGING: Lautering consists of the removal of the liquid, now termed wort, from the insoluble husks or "spent grains.N Lautering begins upon termination of the mashing process, where-by the ~mished mash is transferred to a lautering tub. There, it is allowed to rest for about ten to thirty minutes during which time the spent grains settle to the bottom~ The lautering tub is equipped with a false bottom SU~T~ SHEET

w~ o 93/15181 2 1 2 7 ~ ~ O PCr/USs3/00932 containing numerous perforations and an outlet leading to the true bottom of the tub. The wort is drawn off and circulated through the grains until a clear product is obtained. The clear wort is then pumped into a brewing kettle. Hot water is run through the spent grains to rinse out, or sparge, any remaining wort.
BOILING AND HOPPlNG OF WORT: The wort is boiled vigorously for one to two and one-half hours in the brew kettle. ln addition, hops may be added a~ various stages of the boiling process, depending on the nature of the final product that is sou~ht.
Wort boiling serves a number of objectives, including (1) concentration of the sparged wort, (2) complete inactivation of en~nes that may have survived the final mashing process, (3) coagulation and precipitation of highmolecular weight proteins and solids (termed 'rkettie break" or "hot breakn), (4) extraction of desirable hop constituents, and (5) sterilization of the wort.
COOLING, FERME~TATION AND STORAGE: After boiling, the wort is strained to remove the solids, or "trub." Next, the wort is cooled generally to a temperature of about 47F.
Fermentation is started when the wort is pitched with the proper amount of a pure yeast culture. After 24 hours, fermentation is established and proceeds at an accelerated rate. Fermentation continues for usually about 7 to 10 days. During this period, the wort temperature must be controlled, since the fermenta~ion process causes the temperature of the wort to rise. Once the yeast has metabolized all the fermentable ingredients in the wort, it settles to the bottom and is subsequently recovered for use in pitching other brews. As the fermentation process comes to a conclusion, the temperature of the wort begins to drop. The fermented wort (termed "green beer") is drawn off for storage in a tank, or "ruh." l~ere, its temperature is lowered to about 36F.
MATURATION, PROCESSING AND PACKAGlNG: The "ruh" beer may be allowed to remain in the "ruh" tank for compledon of the maturation process, or it may be transferred into a separate maturation aJBE~ITUrE SHEET

wo 93/15181 Pcr/uss3/00932 2 ~27tL~ ~pon further settling of any remaining yeast and other solids.
Depending on the particular brewery, the beer is allowed to age from about 14 days to about 3 months. During this period, the beer clarifies and its flavor develops. Upon maturation, the becr generally undergoes ~lltration and pasteurization.
The beer can undergo a single or a double pass filtration process. The double pass filtration consists of two steps, a primary (coarse) filtration and a polish (fine) filtration. In the case of the cold-filtered "draft" beers, a microfiltration system is used to remove any possible contuninants, thcreby avoiding the need for pasteurization. Filtered beer is subsequently stored in a finishing tank.
To prepare the beer for consumption, it is carbonated to a specified level. Then, depending on the form of packaging, the beer is pasteurized. ~ Beer packaged in cans ~nd bottles is usually pasteurized, ~` 15 ~ wbile ~beer packaged in kegs (and sometimes bottles) remains unQ~ur~ed. After final processing of the packaged product (e.g.
labelin& etc.), tbe beer is ready for shipping to the consumer.
THE ATI~IBUTES OF FERMENlED MALT BEVER~GES:
Malt beverages, especially beer, possess attributes readily discernable by 20~ ~ ~ the consumer. These attributes include foam, flavor and darity. Of these, foam is probably paramount, as consumers are continually subjected to the sigbt of a foamy glass or stein of beer through the advertising media. Also, foam is typically the first attribute they become aware of as beer is poured or drawn. A number of beers will produce a relatively good foam immedi-ately after pouring, but the foams so produced may not be as persistent as ' is usually desired by consumers. For example, a loose, large-bubbled foam may tend to settle quickly without showing any of the desirable characteris-tics generally associated with a good beer foa~n.
The formadon of a good so-called "head" of foam whcn beer is poured into a glass is considered by many to be a visual gauge of bccr quality. A Sne, creamy stable foam bas a definite psychological appeal to many consumers. Consequently, a number of consumers desire a product SUE~TUTE SHEET

wo 93~15181 212 7 4 4 0 Pcr/uss3/oos32 that, when poured into a glass, will form a stable head that will persist until the beer is consumed. In addition, it is desirable that the beer foam cling to the vessel as it slides down the glass surface, leav~ng behind an adherent curtain of foam, sometimes referred to as 'lace" or 'lacing of the glass."
In addition, an increasing number of consumers desire an all-natural beer product which demonstrates the above qualities yet is entirely free of artificial additives or supplements.
Suffice it to say that a great many brewmasters seek to improve the foa n qualities of their beers. As such, it has long been the practice of brewers to attempt to improve the beer foam through the use of additives.
For example, U.S. Patent No. 2,478,988 discloses the addition of propylene glycol alginate (PGA) to the beverage to increase the duration and persistence of the foam. Concerning this particular additive, lackson, et al., ~Mechanism of Beer Foam Stabilization by Propylene Glycol Algi-nate,n J. Inst. Brew. 86:34 (1980) suggest that the foam stabilizing action of PGA results from the electrostatic interaction between carboxyl groups on the glycol alginate molecules and the peptide amino groups found at the foam bubble walls.
U.S. Patent No. 3,223,529 discloses the addition of a mixture of PGA and a water-soluble zinc salt to improve the foam characteristics of beer.
U.S. Patent No. 3,266,902 discloses the addition of zinc and ma~ganese salts to PGA to provide a distinct improvement in foam qualities, including an increase in the tendency of the foam to '1ace" on the glass wall.
U.S. Patent No. 3,526,510 discloses the use in certain chemi-cally pasteurized beers of a compound as a "foam stabilizer" and "curtain former." This compound is selected from the group consisting of sodium dioctyl sulfosuccinate, sodium dihexyl sulfosuccinate, sodium diamyl sulfosuccinate~ disodium N-octadecyl-sulfosuccinamate and tetrasodium N-(1,2-dicarboxyethyl)-N-octadecyl-sulfo-succinamate~
: .
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WO 93/15181 PCI'/US~3/00932 212~440 6-- U.S. Patent No. 3,966,976 discloses a process for preparing malt beverages havmg good foam stability and improved lace and cling through the addition of a polysaccharide colloid S-10.
Notably, each of the above methods or compositions for the improvement of foam quality, with the exception of the polysaccharide colloid S-10, involved the use of additives not naturally found in beer.
It has also been known that certain compounds found naturally in beer can contribute to good foam quality. For example, these foam positive materials generally include iso-alpha acids such as isohumulone and reduced isohumulone; partially hydrolyzed proteins;
carbohydrates; ethanol; air; nitrogen; carbon dioxide; and some metal ions.
See Bishop, et aL, "A Scientific Basis for Beer Foam Formation and Cling,"
JIB 80: 68-80 (1974); Aubert et al., "Aqueous Foams," Scientific American 254: 74~2 (1986); Roberts, R.T., "Colloidal Aspects of Beer Foam,"
Brewers Digest 52: 50-58 (1977); Slack, P.T. and Bamforth, C.W., ''rhe Fractionation of Polypeptides from Barley and Beer by Hydrophobic lnteraction Cbromatography; The Influence of Their Hydrophobicity on ~oam Stability," II~ 89: 397 401 (1983). Of these, Roberts, ~, has sug-gested that the proteinaceous material in beer is the major foaming agent.
Asano and Hashimoto, 'lsolation and Characterization of Foaming Proteins in Beer," A~B(~Jn~al 38: 129-136 (1980) reported the presence in beer of "foaming proteins" responsible for foaming in beer.
These protein were ~ound to consist of three fractions having molecular weights in the range of 15,000 to 1,000,000 Daltons.
Bamforth and Cope, "Original Approaches to lmproving the Foam of Beer,'t Proc. E~3C Con~ress, 515-522 (1985), su8gested the addition of polypeptides to beer to ensure the presence of an adequate level of pro-teins in beer, either by extracting foam promoters from raw materials and restoring them to the finished beer, or by using existing commercial sources of protein, such as egg white, gluten, and soya. However, the authors had little success in extracting the foam promoters from some of the raw SUESTITI~ SHEET

wo g3/~sl8l 2 1 2 7 ~ 4 0 Pcr/US93/oo932 materials investigated, including barley, and thus focused upon ex~raction of hydrolyzed albumen from egg whites as a suitable source of foam protein.
With respect to the is~alpha acids generally, Asano and Hashimoto, "Contribution of Hop Bitter Substances to Head Formation of S Beer," Rep. Res. Lab. Kirin Brewerv Co.. Ltd. 19: 9-16 (1976) reported that isobumulone can intcract with positively charged amino groups of polypep-tides in beer during foaming to provide a more stable foam. A similar interaction betwcen melanoidin and beer polypcptides has becn suggested by lackson and Wainwright, "Melanoidins and Beer Foam," Proceedin~s.
BC 36:192 (t978).
~` Reccntly, Baker, ~mpact of Post-Filtration Addition of Selected Hop Extracts on Becr Foam and Clarity," MBAA Tech. Quart. 27:
~` 33-38 (1990) found that beers treated with either an iso-alpha acid (LAA) or a reduced iso-alpha acid (RLAA) showed no significant increase in foam collapse times beyond addition of 10 mg of Bitterness UnitstL of finished ~; beer of IAA or addidon of 15 mg/L of RIAA, respectively. Foam coUapse bmes of beers treated with other reduoed isohumulone extracts such as tetrahydro iso-alpha acid (THLAA), hexahydro iso-alpha acid (HHIAA) or a combination of IAA/HHIAA increased significantly with each incremen-tal addition of the extracts. However, foam cling showed no signi~lcant improvement beyond 5 mg/L of IAA, ~IIAA, HHLAA, or a 65/35 volu-metric blend of LAA/HHIAA, or 10 mg/L of RLAA. ln addition, the turbidity of each treated beer increased as the level of extract was increased, and the clarities were generally acceptable at additional rates up to lff mg/L for each extract.
.
SUMMARY OF THE lNVENTlON
Briefly stated, ~he present invention is a method and compo-sition for enhancing the foam properties of a fermented malt beverage.
Tbe method almprises adding to the beverage an isobumulone extract in the amount of between about 0.1 ppm and about 20 ppm by weight of the beverage. The method further comprises adding to the beverage foaming SU~llTI~ SHEET

wo 93/15181 Pcr/uss3/00932 proteins in the amount of between about 2 ppm and about 250 ppm by weight of the beverage. The isohumulone extract and the foaming proteins may be added in-line after final filtration, either simultaneously, succes-sively, or by a single injection of two pre-blended components.
. The fersnented malt beverage foam-enhancing composition comprises an isohumulone extract and foaming proteins. In particular, the composition comprises an isohumulone extract and foaming proteins;
wherein the ratio of isohumulone extract to foaming proteins is between about 1 to about 1 and about 1 to about 2S00.
Further disclosed is a beverage composition with enhanced foam properties which comprises (a) a fermented malt beverage; (b) an isohumulone extract, the extract comprising ben~een about 0.1 ppm to about 20 ppm by weight of the fermented malt beverage; and (c) foaming proteins, the proteins comprising between about 2 ppm to about 250 ppm ~ by weight of the fermented malt beverage.
It should be noted that the phrase "isohumulone extract" as used in this specification and appended claims is intended to have a reladvely broad meaning and to refer to all isohumulone extracts, regular or reduced, which contribute to the enhancement of beer foam, whether th~y be derived from the hop itself or from non-hop or commercial source,s.
Further included within the meaning are all forms of synthetic isohumulone extracts, regular or reduced, which may be produced.
ln addition, it is further noted that the phrase "foaming proteins'~ as used in this speàfication and the appended claims is intended to have a relatively broad meaning and to refer to proteins which contri-bute to the enhancement of beer foam, whether they be derived from the beer itself or from non-beer or commercial sources, as more fully described below.
The method and composition of the present invention is advantageous in that a way is provided of enhancing the foam proper~ies of a fermented malt beverage. In particular, the present invention provides for the use of ingredients found naturally in beer to achieve a remarkable S~JBST~UrE SHEET

W O 93/15181 PC~r/US93/00932 ....
2~274~0 improvement in the foam characteristics of the beer, especially with regard to the beer's Cling, Collapse Rate, and Sigma Foam Value. Moreover, these enhanced qualities are preferably obtained with or~y an insignificant increase in undesirable chill-haze formation upon pasteurization.
These and other advantages of the present invention will be better understood upon reading the following detailed description of the preferred embodiments, together with the examples below.

DETAILED DESCRIPIlON OF
THE PREFERRED EMBODIMENTS
ln the method aspect of the present invention, an isohumulone extract ~nd foaming proteins are added to a malt beverage.
Preferably, the isohumulone extract and the foaming proteins are added to the beer in the later stages of the brewing process in-line after primary lS filtration and before final filtration or while the beer is in the finishing tank. The components are typically not added earlier in the brewing pro-cess primarily because of the possibili~ of being precipitated out during the kettle boil, fermentation or maturation. When added during finishing, the deleterious effects of the brewing process are avoided. Pasteurization has not been shown to have any negative effects. *hese components may be added either simultaneously, successively, or by a single injection of the two pre-blended components.
l'he isohumulone extract used in the present invention may be a reduced isohumulone extract. In that case, the reduced isohumulone extract is preferably a hexahydro isohumulone extract. In general, the choice of whether to use a regular or reduced isohumulone extract depends upon which foaming protein solution is combined, and there is presently no general rule on which is preferred with regard to the entire class of foaming proteins. Presently, the best method to decide which isohumulone extract is preferred in combination with a given foaming protein solution is by experimentation, which experimentation is not undue. Suitable proce-dures for carIying out such experimentation is set forth in the Exarnples.

SuB~lTurE SHEET

wo 93/15181 Pcr/us93Joo932 2~2~l 4 40 Generally, isohumulone extract is obtained by isolating humulone from hop using either hexane or liquid CO2 as a solvent. The humulone is then isomerized by heat distillation to convert humulone to isohumulone, an iso-alpha acid. Reduced isohumulones, which are a reduced form of iso-alpha acids or isomerized beta acids include dihydro iso-alpha acid (RHOLAA), tetra hydro iso-alpha acid (TIAA) and hexa-hydro iso-alpha acid (HHLAA).
The isohumulone extract may be obtained from commercial sources. Suitable isohumulone extracts are ones such as those sold by Kalsec, lnc., of K~alamazoo, Michigan under the designations "HEXAl~NE" and ~SOLONE~" A similar product is available from the English Hop Processing Co., Ltd., Tonbridge, Kent, England, under the designation 'lSOHOP CO2N". The isohumulone extracts were available in 20% and 30~o by weigbt liquid solutions.
The method of the present invention further comprises adding to the beverage foam~ing proteins. Preferably, the foaming proteins are of the naturally-occurring variety which can be isolated using known protein sxtraction procedures from a number of sources, and then added to the beverage, either simultaneously, successively, or by a sing~e injection with 0 ` an isohumulone extract. These naturally-occurring foaming proteins can be isolated using known protein extraction procedures from wheat gluten, malted barley, malted wheat~ ale yeast, and lager yeast according to the methods and techniques set forth in Examples 4-7 below. Of these, the malted sources are most preferred.
ln order to prepare the foam-enhancing composition, an isohumulone extract is combined with foaming proteins~ In accordance with a preferred embodiment, the isohumulone extract and the foaming proteins are in a solution comprising water, sugars~ alcohols and other constituents found naturally in beer and which inherently result from the extraction methods and techniques set forth in the Exa~nples. More preferably, the isohumulone extract and foaming proteins are concentrated and packaged for use by brewers. Most preferably, the foam-enhancing .

al~E SHEET

WO 93/15181 2 1 2 7 ~ 4 0 P~r/US93/oo932 composition is spr~y dried and packaged in powdered form for use by brewers.
The optimal amounts of isohumulone extract and foaming proteins to be combined were studied using a shaker (HRV) test as S outlined in Example.8 below It should be noted that this test must be . separately run for a given foarning protein solution in order to determinethe optimum isohumulone extract concentration to be used with each parti-cular protein. For example, a particular foarning protein solution may contain more foaming proteins per rnilliliter of solution as would a polymeric amino acid solution produced according to the methods and tochniques set forth in Example 2. In that instance, because there are presumably many more active sites for reaction with isohumulone extract in the first solution than in the polymeric amino acid solution, more iso-hurnulone extract would be required to achieve an optimum level.
~ In performing the shaker (HRV) test (Exarnple 8) on the various foaming proteins obtained according to the methods and techniques set forth in Examples 2-7 below, thè following optimal ranges for isohumu-lone and reduced isohumulone a~tract concentrations were determined. It should be noted that while the following ranges are described in terms of combination with a particular foaming protein, the method of the present, imention contemplates that the isohumulone extract and the foaming pr~
teins may be added simultaneously, sequentially, or by a single injection of the two pre-blended components.
For the foaming protein solution derived from wheat gluten, preferably between about 0.1 ppm and about 20 ppm by weight of the beverage of isohumulone extract is combined with the protein solution prepared according to the methods and techniques set forth in Example 4.
More prefera~ly, the amount of isohumulone extract combined is between about 2.8 ppm and about 8.5 ppm of the beverage.
For the foaming protein solution derived from ale or lager yeast, preferably between about 0.1 ppm and about 20 ppm by weight of ~` ~ the beverage of isohumulone extract is combined with the protein solution SHEET

wo 93/15181 Pcr/uss3/00932 ~2rl 440 ,~
prepared according to the methods and techniques set forth in Example S.
More preferably, the amount of isohumulone extract combined is between about 3.3 ppm and about 16.9 ppm of the beverage.
For the foaming protein solution derived from malted barley, S preferably between about O.I ppm and about 20 ppm by weight of the beverage of isohumulone extract is combined with the protein solution prcpared according to the methods and techniques set forth in Example 6.
More preferably, the amount of isohumulone extract combined is between about 0.85 ppm and about lO.S ppm of the beverage.
For the foaming protein solution derived from malted wheat, preferably between about 0.1 ppm and about 20 ppm by weight of the beverage of isohumulone extract is combined with the protein solution pNpared according to the methods and techniques set forth in Example 7.
More preferably, the amount of isohumulone extract combined is between lS about 0.25 ppm and about 5.10 ppm of the beverage.
It should be noted that for each of the above foaming prouins, the amount of isohumulone extract to be added is the same regardless of whether the extract is regular or reduced.
In addition, certain prepared polymeric amino acids can be used in place of the naturally-occurring foaming proteins for addition to the beverage. As used throughout this specification and the appended claims, the phrase "polymeric amino acid" refers to a polypeptide or protein con-sisting entirely of an unspeci~led number of single, repeating amino acids units. For example, polymeric Iysine consists entirely of repeating units of the amino acid lysine.
For the polvmeric amino acids, preferably between about 0.1 ppm and about 20 ppm by weight of the beverage of isohumulone extract is combined with the protein solution prepared according to the methods and techniques set forth in Example 2. More preferably, the amount of iso-humulone extract combined is between about 2.8 ppm and about 8.5 ppm of the beverage.

: SU~lTrUTE SHEET

wo 93/15181 2 1 2 7 4 ~ O Pcr/US93/oo932 At present, because all the mechanisms by which the poly-meric als~ino acids and the isohumulone extract interact tO enhance the foam of fermented beverages are not completely understood, the best means for selecting the particular polymeric amino acid is by experi-S mentation. In this regard, the most effective test for selecting a particular polymeric amino acid is to evaluate a selected polyrneric amino acid in the presence of an isohumulone extract. The evaluation process simply involves either addition of the selected polymeric amino acid to a poor foaming beer (pouring test) or by mixture alone (shaker (HRV) test). By this method, certain polymeric amino acids are found to be more effective than others, as some are more reactive to isohumulone without significantly increasing haze in the beer. These include polymeric argilune, glu~amic acid, glycine, leucine, Iysine and tyrosine.
Thus, there are several polymeric amino acids which have worked well in combination with isohumulone extracts in beer, while there are also some polymeric amino acids which did not work well. At present, the selection of the speci~lc polymeric amino acid to be used with a particular isohumulone extract is best made by experimentation~ which experimentation is not undue and is clearly within the ordinary skill of one in the art.

EX~MPLES
The following examples illustrate procedures for carrying out the invention, but it will be understood that they are presented purely for purposes of illustration and not as indicating the limits of the invention.
i E2camDle 1 Test Methods The advantages of adding an isohumulone extract and foaming proteins to a fermented malt beverage were evaluated generally by using a calculated Sigma Foarn Value, Percentage of Cling, and Collapse Rate for each sample ~ested. The Sigma Foam Value reflects the general stability of SJJB~I~UrE SHEET

WO 93/15181 PCI`/US93/009:~2 ~,~ ?.~ 4 a particular beer foam was de~erlruned in accordance ~nth procedures set forth by the American Society of Brewing Chemists. A detailed description of these procedures can be found at Mçthods ofA~ly~ the A~rican Societv Qf Brewin~ Chemists (E. Kneen ed. 1976), American Society of Brewing Chemists; St. Paul, Minn.
In addition, the Percentage of Cling and Collapse Rate for beer foam was determined under the following conditions. Bottled beer was equilibrated at 20C and poured into a 200 ml graduated glass recep-tacle in such a manner so as to generate approximately lS0 ml of foam in the receptacle within a few seconds. Standardized pouring conditions were achieved through the use of an acrylic bottle holder which allowed for simultaneous pouring of multiple bottles of beer at identical rates and angles. During pouring, the bottle mouths were maintained two inches above the tops of the glass receptacles. Each bottle was poured into three lS different glass receptacles and the volume of foam (Total Foam (ml)) in each receptacle was measured irnmediately. Thereafter, the time for the beer foam to collapse (Foam Collapse Time (sec)) was determined. Foam Collapse Time was recorded when a beer ring of at least 0.5 cm in diameter could be obsened at the bottom of the foam. Total Foam and Foam Collapse Time were recorded for each of the three samples taken from each bottle of beer, and average values were determined. From these averages, the Collapse Rate was determined as follows:

Total Foam (ml~ x 60 Collapse Rate (rnl/min) = ~ - --Foam Collapse nme (sec) The Collapse Rate measures the rate at which the beer foam disappears.
The larger the value, the poorer the quality of the foam.
Throughout this specification, the terms 'tcling" and ~ace" are used interchangeably and essentially refer to the foam's ability to adhere to the glass receptacle. To determine the Percentage of Cling, an estimate of residual foam was made throu~h obseIvation of the foam remaining on the Su8~Tr~lE SHEET

wo 93/15181 212 7 ~ 4 0 Pcr/uss3/oos32 .

inner surface of the glass receptacle at the end of the Foam Collapse Time.
Percentage of Cling measures the stability of a beer foam. The greater the value, the more enhanced the faam stability of the beer.
In most instances, the efficacy of the isohumulone extract and S the foaming proteins was evaluated in the following beers: CLASS I -nSTROH IlGHr' and CLASS II - "PABST BLUE RIBBON" and '~IAMMS." TABLE 1 sets fonh general ranges for various characteristics of these beers.

Descn~tion of Test Beçrs Sigma Foam ProteinBitterness Beer ' Value (~:) (%) (%) CLASS I ' 102-110 0.25-0.3510.~14.0 CLASS II ' 85-97 0.20-0.309.5-11.5 Examole 2 Polymelic ~nino Acids Example 2 was performed in order to assess the efficacy of various commerciaily-available polymeric amino acids as foaming proteins for combination with an isohumulone extract. As noted earlier, a poly-meric amino acid is a polypeptide or protein consisting entirely of an number of a single, repeating amino acid unit. For example, polymeric Iysine consists entirely of repeating units of lysine. Samples of different polymeric amino acids were obtained from Sigma Chemical Company.
To prepare polymeric amino acid stock solutions, all samples were dissolved in a 4.75~o ethanol solution. Some of the less soluble (or insoluble in water) samples needed to be first dissolved in a small amount of acid or solvent are as follows:

~ TUrE SHEET

wo 93/15181 Pcr/us93/oo932 ~1 4 40 1. Poly-kArginine (sulfate) - 43.600 MW
? ~ 50 mg dissolved in 2 ml of 6M (molar) HC1 (hydrochloric acid) and volume raised to 25 ml with 4~75% ethanol solution.
2. Poly-L-Arginine (hydrochloride) - 11,600 MW
50 mg dissolved in 25 ml of 4.75~o ethanol solution.
3. Poly-~Lysine (hydrobromide) - 43,700 MW
100 mg dissolved in S0 ml of 4.75% ethanol solution.

4. Poly-L,Methionine - 36,000 MW
50 mg dissolved in 1.0 ml of chloroform and volume raised to 25 ml with 4.75% ethanol solution.

5. Poly-D-Tyrosine - 66,700 MW
100 mg dissolved in 50 ml of 4.75% ethanol solution.

6. Poly-L-Alanine - 20,000 MW
S0 mg dissolved in 1 ml of DCA (dichloroacetic acid) and volume raised to 25 rnl with 4.75~o ethanol solution.

7. Poly Glycine - 15,600 MW
50 mg dissolved in S0 ml of 4.75% ethanol solution.

8. Poly-L-Glutamic acid (sodium salt) - S0,740 MW
50 mg dissolved in 25 ml of 4.75% ethanol solution.

9. Poly-L Aspartic acid (sodium salt) - 42,500 MW
50 mg dissolved in 25 ml of 4.75% ethanol solution.

10. Poly-k,Leucine - 21,700 MW
10 mg dissolved in 1 ml of TFA (trifluoro acetic acid) and volume raised to 25 ml with 4.75~ ethanol solution.

11. Poly-L-Leucine - 80,000 MW
10 mg dissolved in 0.5 rnl of TFA (trifluoro acetic acid) and volume raised to 25 ml with 4.75~o ethanol solution.

The ~mal concentrations of the stock solutions were in the range of 1600 to 2000 ppm of polyrneric amino acid per ml of stock solution. Tbese polymeric amino acid concentrations were deterrnined to avoid significant increases in turbidity. All of the polymeric arnino acid solutions were adjusted to a pH of 4.2 using sodium hydroxide, and one ml SUE~TITI~ SHEET
1~
.. .. . . . ,~, . .

W O 93/15181 2 1 2 7 4 ~ ~ PC~r/US93/00932 .

of each stock solution was injected into a 12 ounce bottled beer previously chilled at 32F for 24 hours.
To prepare mixed stock solutions, each polymeric amino acid stock solution was mixed with an isohumulone (marked '1-~amîno acid]") or S a reduced isohumulone (marked "larnino acid] +"). The concentration of isohumulone or reduced isohumulone extract in the mixed stock solution was 2000 ppm. All of the polymeric aIIuno acid solutions were adjusted to a pH of 4.2 using sodium hydroxide, and one ml of the mixed stock solution was injected into a 12 ounce bottled beer previously chilled at 32F for 24 hours. All treated beers were equilibrated at room temperature for one day before determining Sigma Foam Value, Percent Cling and Collapse Rate.
Under the above-described conditions, not all polymeric amino acids were found to combine wlth isohumulone or reduced isohumu-lone extract to enhance foam stability. However~ the foam stability of a Class I beer did improve with the addition of combined mixtures of iso-humulone or reduced isohumulone extract and a polyrneric amino acid chosen from the following group: arginine, glycine, glutamic acid, leucine and tyrosine. TABLE 2 below summarizes the effect on the foam proper-ties of a Class I beer through the addition of these foam positive polvmeric amino acids in combination with an isohumulone or reduced isohumulone extract.

sus~rnurE SHE~ET

WO 93/1~181 PC~/US93/00932 44~ - 18-Pol~neric Amino Acids Showin~ Favora~le Results - Class 1 Beer POLYMERIC MW DOSE CL~NG COLW.R~TE SIGMA FOAM
AMINO ACIDS fDaltons~(mg/12 oz! % !ml/n~in~ VALUE
Control 18 50 91 Isohumulone 2.0 39 44 103 Red. Isobum. 2.0 45 47 101 Arginine 11,600 1.61 32 47 80 I-Arg. 45 42 132 Arg. + 80 44 10 Glutamic Acid 50,740 1.68 27 Y ~1 I-GIu. 55 43 92 Glu. + 70 49 111 Glycine 15~600 2.0 37 41 107 I-Gly. 57 40 125 Gly. + 67 44 113 Leucine 21,700 1.94 40 44 84 Leu. 48 36 112 Leu. + 80 41 75 Tyrosine 66,700 1.93 65 45 83 I-Tyr. 50 42 102 Tyr. + 80 4~ 86 .
1- = A mix of isohumulone and a polymeric amino acid.
+ = A mix of reduced isohumulone and a polymeric amino acid.

On the other hand, several polymeric amino acids demon-strated less favorable results when tested in a Class I beer. These polymeric amiDo acids are listed below in TABLE 3.

SuBsTm~ SJ~IEET

WO 93/15181 2 1 2 7 4 4 O PCl'/l lS93/00932 PolYme~ Ami~o ~cids Showi~ Less FaY~r~ble Results - Class I Beer s POLYMERIC MW DOSE CLING COLLAP.RATE SIGMA FOAM
AMINQ ACIDS (Daltons)(m~/12 oz! (mllmin) VALUE
Alanine 20,0001.95 I-Alanine 40 36 111 Alanine + 35 43 88 Asparlic Acid 42,500 1.67 I-Aspartic Acid 45 39 104 Aspa~ic Acid + 48 40 92 Lysi~e 43,7001.90 I-Lysine 27 39 100 Lysine + 47 44 97 Methionine 36,0001.98 I-Methionine . 60 43 97 Methionine ~ 30 47 107 I he polymeric amino acids were similarly tested in a Class II
beer. TABI.E 4 below summarizes the effect on the foam properties of a Class II beer through the addition of foam positive polymeric amino acids in combination with an isohumulone or reduced isohumulone extract.

SuBE~lTurE SHEET

WO 93/15181 PCl`/US93/0093 Po!vmer;c Amino Acids Showin~ Favoral~l~esul~ - Clas~ 11 Beer POLYMERIC MW DOSE CLING COLLAP.RATE
AMINO ACIDS (Daltons) (me/12 oz) % (ml/min) Corltrol 7 67 Isohumulone 2.0 27 62 Red. Isohum. 2.0 13 59 Argi~ine 43,600 1.57 7 55 I-Arg. 30 48 Arg. + 45 57 Glutamic Acid 50,740 1.68 3 S4 I-GIu. 5 33 Glu. + 28 51 Glycine 15,600 2.0 6 57 I-Gly. 25 41 Gly. l 4S 48 Leucine 80,000 1.94 4 62 I-Leu. 10 47 Leu. + 45 56 :
Lysine 43,700 1.90 2 60 I-Lys. 43 5 Lys. + 58 61 1- - A mLl~ of isohumulone and polymeric amino acids.
+ = A mD~ of reduced isohumulone and polymeric amino acid.

As in the case ~,vith the Class 1 beer, several polymeric amino acids similarly demonstrated less favorable results when tested in a Class II
beer. These polymeric amino acids are listed below in TABLE 5.
~0 :
SUB8TITUl-E SHEET
:

WO 93/15181 2 1 2 7 4 ~ O PCl`/US93/00932 TABLE S
Polymeric Amino Adds Sho~nng L~ss Favorable Results - (~la~ !I Beer POLYMERIC MW DOSE CLING COLLAP.RATE
AMINO ACIDSLDaltons~(m~/12 oz) ~ml/min) Alanine 20,000 1.95 I-Alanine 13 58 Alanine ~ 17 50 Aspartic Aad42,500 1.67 I-Aspartic Aad 7 48 AsparticAcid + 38 54 Methionine36,000 1.98 I-Methio~ne 4 69 Met~ionine + 17 62 Tyrosine 66,70~ 1.93 I-Tyrosine 1 63 Tyrosine + 23 60 Surprisingly, polymerie tyrosine, in combination ~Anth regular and reduced isohumulone extract, was not found to improve the foam stabili~ of a Class I~ beer significantly. On the other hand, polymeric Iysine, which was not a veIy active foarn stabilizer for a c!ass I beer was found to show great improvement for cling in a Class Il beer after mLlcing with regular or reduced isohumulone extract.
These results show that Sigma Foarn Values were not uni-formly improved by all polymeric amino acids tested. However, signi~lcant increases in Sigma Foam Values resulted from addition of an isohumulone extract in combination with arginine, glycine or leucine in a Class I beer.
On the other hand, only a reduced isohumulone extract in combination with arginine, glycine or glutamic acid increased the Sigma Foam Values for a Class I beer~

Exam~le 3 Studv of Role of Molecular Weight on Ef~lcac~ Or Polymeric Amino Acids Example 3 was performed in order to assess the role of molecular weight on a polvmeric amino acid's capacity, in combination with SU~TUIE SHEFr wo 93/1~181 Pcr/uss3/oo932 ~,~ Z l 44 22 -an isohumulone extract, to enhance beer foam. ln this regard, various sizes of pol~meric arginine and leucine were tested alone and in combination with the reduced isohumulone extract, '~IEXALONE," in both a Class 1 and Class II beer. The results are summarized in TABLES 6 and 7 below.

Variability of Molecular Weight of Polymeric Amino Acids and Their Effects Q~ Beer Foamin~2erties in a Class I Beer POLYMERIC MW SIGMA ~OAM COLLAP. RATE CLING TURBIDITY
AMlNO ACIDS (DaltOns! VALUE (ml/min~.~ (1 TU!
Arginine11,600 80 41 32 150 Argilune43,600 94 47 63 110 Arginine100,000 97 44 38 170 Arginine ~11,600 105 44 80 111 Arginine +43,600 94 S1 75 147 Arginine ~100,000 101 40 20 120 Leucine 21,700 84 44 40 12S
L~:ucine80,000 103 44 57 115 2S Leucine ~21,700 75 41 80 123 Leucine ~80,000 82 41 48 158 + = with the addition of '~EXALONE."

aJESrl~UrE SHEET

wo 93/15181 2 1 2 7 4 4 0 Pcr/uss3/00932 " . ~

Variability of Molecular Weight of Polymeric Amino Acids and Their Effecls of Beer Foamin~ Pro~enies in a Class II Beer POLYMERIC MW COLLAP. RATE CLING
AMINO ACIDS (DaltnS! (ml/min! %
Arginine 11,600 44 17 Arginine 43,600 55 7 Arginine + 11,600 58 20 Arginine + 43,600 57 45 Leucine 21,700 63 5 Leucine 8Q000 63 4 Leucine + 21,700 50 5 Leucine + 80,000 57 45 = with the addition of ~IEXALONE.'`
At present, ~he data in TABLES 6 and 7 do not appear to establish a general pattern regarding efficacy when the molecular weight of the polyrneric amino acids are changed, at least with respect to polymeric arginine or leucine. Wbile a given polymeric amino acid alay have an optimal molecular weight for enhancing and stabilizing the foam for a Class I beer, that same polymeric amino acid may have a different optimal molecular weight for use in a Class II beer. Similarly, the efficacy based on molecular weight may also vary depending on the presence of an isohumu-lone extract. Accordingly, because the above date suggests that each particular polymeric amino acid may have an optimal molecular weight for a given beer, it is suggested that each particular beer be tested separately to deterrnine the optimal molecular weight for a given polymeric amino acid. Such experimentation is not undue and can be readily performed by one skilled in the art according to the procedures set forth in Example 8.

:~

aJB~IlJ~ SHEET

WO 93/15181 PCr/l~S93/00932 ?. Example 4 PreDaration of Whe~lut~n Foamin~ Protein Example 4 was performed in order to test wheat gluten as a source of foaming proteins for combination with an isohumulone extract.
5 . A wheat gluten was partially hydrolyzed using papain, a proteolytic er~ne, as follows:

Step 1: 200g of gluten was mixed thoroughly with æog of enzyme, and the rnL~ture was allowed to digest at room temperature for three hours.
Step 2: 200g of water was added and mixed. The mixture was allowed to digest further for one hour.
lS Step 3: The solution was diluted three-fold with water and ~lltered.
Step 4: The pH of the ~lltrate was adjusted to 3.0 with phosphoric acid.
Step 5: The filtrate was boiled for one rninute (pasteurization).
Step 6: 0.15% Sodium Benzoate (a preservative) was added.
-~ 25 Step 7: The final mLxture was concentrated.

The hydrolyzed protein solution was adjusted to pH 4.2 from its original pH of about 2.2 and kept at ODC for 24 hours to allow for precipitation of proteins ha~ing an is~lec~ic point at pH 4.2. ln order to remove haze-causing particles, the precipitated protein was removed by Slltration through a 0.45 ,~ membrane. Accordingly, the protein molecules that could cause ha~e in beer were significantly reduced. The treated gluten stock solution, with its remaining protein, was then adjusted to pH
8.5 and coded as HGluten Foamer." The molecular weight of this Gluten Foamer was determined to be approximately 15,000 Daltons. The concentration of wheat gluten protein in the stock solution was approximately 4300 ppm.

SUB~mUTE SHEET

Wo 93/15181 2 1 2 7 4 4 O Pcr/usg3/00932 To prepare a new beer foam composition, a portion of the Gluten Foamer was m~xed with isohumulone or hexahydro isohumulone extract to form a salt complex. The amount of regular or reduced isohumulone extract in the stock solution was now 2000 ppm.
To test the efficiency of the Gluten Foamer, both alone and in combination wtth an isohumulone or a reduced isohumulone extract, one rnilliliter of the test sarnple was injected into a 12 ounce bottled beer previ-ously chilled at 32F for 24 hours. Accordingly, the amount of wheat gluten protein in the beer was approximately 12.0 ppm, and the concentration of isohumulone or reduced isohumulone extract in the beer was 5.6 ppm.
After injection, the beer was irnmediately recrowned (i.e., bottle cap was replaced). A similar injection of one ml of 4.75% ethanol into a 12 ounce bottled beer was performed as a control.
The test results are shown in TABLES 8 and 9 below.
TABLE 8 tabulates the results regarding the effect of Gluten Foamer on foam properties of a Class I beer.
:

GLUTEN FOAMER - CLASS I Bl~ER
FOAM STABllIZE~ CLII~ COLI~P. RATE (ml/rnin~ SIGMA FOAM VALUE
Control 28 S3 96 IsoJlumulone 43 46 105 Red. Isohum. 43 47 104 Gluten Foamer 31 S4 I00 I-GIIlt. Foamer 73 43 lm Glut. Foamer ~ 75 44 116 1- = A mut ol isohurnulone and Glutcn Foamer.
+ = A mix of Gluten Foamer and reduced isohumulone.

Similarly, TABLE 9 tabulates the results regarding the effect of Gluten Foamer on foam properties of a Class II beer.
.

aJE16TlTUrE SHEET

WO 93/1~181 PCI/US93/00932 ~, ... .
4 4~ 26 -GLUTEN FOA~ER - CLASS 11 Bl~R
S FOAM STABILIZER CLINGI%~ COLLAP. RATE (ml/min~ SIGMA FOAM VALUE
Control 11 ~V 91 Isohumulone 27 62 100 Rcd. Isohum. 18 59 95 Gluten Foamer 14 6S 100 l-Glul. Foamer 63 41 110 Glut. Foamer ~ 30 47 103 1- = A mLl~ of isohumulone and Gluten Foa~ner.
+ = A mo~ of Gluten Foamer and reduced isohumulone.

It is clear that the addition of Gluten Foamer alone improved foam stability only slightly in both a Class l and Class II beer. However, the combination of Gluten Foamer with an isohumulone (I Gluten Foamer) or reduced isohumulone extract (Gluten Foamer + ) greatly increased Percent Cling and Sigma Foam Values. The combination of isohumulone extract aIld Gluten Foamer reduced the foam's Collapse Rate to 81%
(43/53 x 100) of the control in a Class l beer, and to 51~ (41/80 x 100) of the control in a Class II beer. The combination of hexahydro (reduced) isohumulone extract and Gluten Foamer also reduced Collapse Rates significantly.
These results clearly indicate the advantage of using the combination of Gluten Foamer with an isohumulone or reduced isohumulone extract for foam improvement.

~ample 5 PreDaration of Ale and er Yeast Foaming Proteins Example S was performed in order to test yeast as a source of foaming proteins for combination with an isohumulone extract. Active dry ale and lager yeasts were used to prepare foarning protein stock solutions.
Ten grams of an active dry yeast (ADY) were dispersed into 100 ml of a .

SW~~ rE SHEET

WO 93/15181 PCl'/US93/00932 -` 21274~Q

4.755'o ethanol solution and agitated by a magnetic stirrer for one hour at room temperature. The pH of this suspension was determined to be in the range of 6.5 + 0.2.
` Alternatively, the pH of the suspension was adjusted to pH2 ; ~ S (labeled "ADY extract 2N) with hydrochloric acid, and to pH 10 (labeled ADY extract 10") with sodium hydroxide after agitadng for fivc minutes.
~; Each of these suspensions was agitated for an additional 55 n~inutes.
After agitation, each of the above ADY suspensions was ~ centrifuged and the yeast cells were removed. Each supernatant was -~ 10 adjusted to pH 4.2, mixed with 5,000 ppm of filter aid (JOHNS-MANVILLE OELITE 512, a form of diatomaceous earth) and 5,000 ppm , of silica hydrogel, and kept at 0C for 24 hours. After cooling, each was subsequently filtered through a coarse filter to remove the filter aid and silica hydrogel in the suspension. The filtrates were then mixed with an lS~ addîtional 5,000 ppm of filter aid and refiltered through a 0.45 1~ (micron) memblane. l~ne filtrates obtained were diluted to 100 ml with distilled water ~and designated as yeast extract. Thc molecular weights of the lager s~ yeast extracts at pH 10 and pH 2 were esdmated to be 10,300 and 10,S00 respectively.
`. ~ 2 0~ To prepare a new foam composition, one ml of a reduced isohumulone extract was added to each of the above filtrates and diluted to 100 ml with distilled water. To evaluate the foam stabilizing effect, one ml of each test sample was injected into a chilled beer and recrowned. The test beer was pre-chilled at 32F for 24 hours prior to use. A similar 2S injection of one ml of 4.75~o ethanol was perfor~ned as a control.
~, The test results for both a Class I and Class II beer are shown in TABLES 10 and 11 below. Ale yeast extracts at pH 6.5 and pH 2.0 injected in the absence of reduced isohumulone actually reduced the cling capacity of a Class I beer in comparison to the control. Sigma Foam Values for the treated beers were also reduced.
In addition, all ale and lager yeast extracts were mixed with a , ~ reduced isohumulone e~nract. Upon tesdng~ the foam stability was found to 1~

wo 93/15181 Pcr/uss3/oo932 - 2~ -~ 4~ be significantly improved in a Class I beer, both with regard to foam cling'~? and Collapse Rate. TABLE 10 tabulates the results on the effect of yeast extract at various pH values on the foa~ing properties of a Class I beer.

YEAST EXTRA~ - CI~AS.~i I BEER
FO~M STABILIæR CLING (%~ COLLAP. ~AT~ (ml/min~ SIGMA FO~M VAl UE
Co~trol 28 S0 8g Red. Isohum. 43 47 103 Ale, pH 6.5 23 50 83 Ale +, pH 65 68 42 92 Alc, pH 2.0 17 4S 86 Alc +, pH 2.0 78 42 104 Ale, pH 10 12 44 77 ~; Ale +, pH 10 57 45 83 Lager, pH 6.5 57 51 89 ~ger +, pH 63 73 44 91 ~ Lager, pH2.0 33 46 87 : ~ Lager +, pH 2.0 53 50 97 Lager, pH 10 43 44 , 96 Lager +, pH 10 72 43 83 + = A mD~ of yeast e~ract and reduced isohumulone.

TABLE 11 tabulates the results on the effect of yeast extract at various pH values on the foaming properties of a Class II beer. The yeast extract samples obtained from an active dry ale yeast showed some degree of inhibition to foam cling in a Class II beer.

L
SUB~TUrE SHEET

WO ~3/15181 2 1 2 7 4 ~ O PCI`/US93/00932 ~T EXTRAcr - (~LASS I BE~ER
FOAM STABILIZERCLING (/c) CQLL.AP. RATE (ml/min~
Control 13 70 Red. Isohum. 18 51 Lagcr, pH 6~ 20 70 Lager +, pH 65 60 47 ~ger, pH 2.0 27 62 Lager +, pH ~0 65 56 Lager, pH 10 18 79 Lager ~, pH 10 73 59 Ale, pH 65 33 5~
Ale + pH 6.5 17 50 Ale, pH 2.0 23 60 Ale +, pH 2.0 47 52 Ale, pH 10 38 52 Ale ~, pH 10 45 46 : + = A mo~ of yeast e~nract and reduced isohumulone.

When each yeast extract was combined with a reduced isohumulone ex~ract, the foam stability was significantly improved. The results clearly show that the rnixture of yeast extract and a reduced isohumulone extract improved fo~n stability.

Example ~
Prepara~ion of ~alted Barlev Foamin~ I~rotein Example 6 was performed in order to test malted barley as a sourse of foaming proteins for combination with an isobumulone extract.
A brewely malt was rnilled and added to a 45C brewing water containing O.OB% calcium sulfate. The mash bill contained high protein 2 and 6 row malts. It should be noted that the calcium sulfate concentration was fle~ible and could be lowered or increased as desired. The mash was stirred at 45C for fifteen minutes to allow larger malt protein molecules to be hydrolyzed and form shorter amino acid chained molecules having mole-SUBSrlTUTE SHEET

cular weights benveen S,000 to 100,000 Daltons. Notably, this protein hydrolyzing time was shortened from the usual thirty minutes in a normal brewing process in order to reduce the production of small amino acid molecules having a molecular weight of less tharl S000 Daltons.
S After fifteen rninutes resting time, the mash temperature was raised to 65C and maintained at that temperature for appro~mately thirty rninutes to allow the malt starch to be actively hydrolyzed. Most starch molecules were hydrolyzed to sugars within this time period. The mash temperature was finally raised to 80C for about 5 to 10 rninutes (depending on complete conversion) to destroy all malt enzymes, and the mash was transferred to a lauter tub. The mash was then filtered through the lauter tub to obtain a clear filtered mash. ln order to preserve partially hydrolyzed proteins, kettle boiling was avoided. For the sarne reason, no ; ~ hops were added.
lS In accordance with traditional methods, the clear filteredmash was ferrnented to a desired attenuation with a brewer's yeast in the presence of anti-foarning agents. AT1Y type of food grade anti-foamer approved by the brewing industry is acceptable. The ferrnented mash was then centrifuged to remove suspended yeast cells and filtered through a coarse filter bed to remove small particles. The resulting non-hopped, fermented beer was chilled to 28-32F for 1 to 2 days. This fermented, non-hopped mash was reffltered with the aid of 400 to 800 ppm silica hydrogel and 400 ppm of filter aid (JOHNS-MANVIT ~ F CELITE 512) to remove chill-ha~e causing materials.
To isolate the protein complex, the non-hopped, fermented beer was then extracted in a preset~ hollow ~lber cartridge using an ultrafiltration system with a molecular weight cut off limit of 30,000 ~altons. During this concentration step, a number of small molecular weight mat~rials were removed including water, alcohol, amino acids, non-fermented sugars, and fatty acids. This ultrafiltration step extracted the beer to 10% of its original volume. This beer extract was designated as Malt Foamer I.

SU~llIITE SHEET

Wo 93/15181 2 1 2 7 ~ 4 0 Pcr/US93/00932 To test the efficiency of Malt Foamer I as a foam enhancing composition, Mal~ Foamer I was blended with 1.0 ppm to 18 ppm of either isohumulone (coded as I-Malt Foam) or hexahydro isohumulone extract (coded as Malt Foam + ) and added to a Class II beer according to the same procedures described in Example 2 above.
As shown below in TAB~-F- 12, isohumulone extract, reduced isohumulone extract, or Malt Foamer I alone could not improve foam stability signi~cantly. However, the foam stabilizing effect was significantly improved if Malt Foamer I was blended with either isohumulone or reduced isohumulone ex~ract. Notably, for those test beers treated with this new combination, a significant increase in the beer's abili~r to cling to the glass receptacle was demonstrated.

-~ MALT FOAMERl- ÇLA~S II BEER
FOA~ STABILIZER CU~G (~ COLLAP. RATE fml/min! SIGMA FOA~ VALUE
Cont~ol 14 57 101 Isohumulone 22 48 105 Red.Isohum. 19 45 Malt Foamer 1 35 46 102 I-Malt Foamer 1 45 38 110 Malt Foarmer l+ 63 39 110 I- - A mLl~ isohumulone a~d Malt Foamer.
+ = A mix of Malt Foamer and reduced isohumulone.

S~ l ITF .~IFI~T

WO 93/15181 PCI'/US93/00932 4~ - 32 --- Example 7 Prevaration of a~Lted ~Yh~at Foamin~ PrQ~in Example 7 was perfonned in order to test malted wheat as a source of foarning proteins for combination with an isohumulone extract.
S A clear filtered mash was obtained according to the procedure set forth in Example 6, with tbe exception of the use of a 6-row malt and wheat malt (10%) combination as the original mash bill (rather tban a 2-row and 6-row combination). Fermentation was carried out in bioreactor-containing, immobilized yeast cells. Ferrnentation proceeded rapidly and resulted in low levels of free-flowing yeast cells in the finished, non-hopped beer.
Accordingly, no centrifugation was required to separate the yeast cells, and only a primary filtration was required to remove particulates before going on to the ultrafiltration step.
Ultrafiltradon was accomplished using a hollow fiber cartridge having a molecular weigbt cut-off limit of 30,000 Daltons to separate higher molecular weight proteins from those witb molecular weights less than 30,000 Daltons. These smaller proteins were removed along with unhopped beer, leaving the high molecular weight protein fraction in the ultrafiltration system~ Approximately 40 to 80 ppm of tannic acid and 400 ppm of filter aid (JOHNS-MANVILLE OELITE S12) were added to the extracted, higb molecular weight protein fraction for chillproofing, and the ex~ract (designated MF3T) was kept at 2~-32F for 1 day before filtration.
Alternatively, 400 to 8~ ppm of silica hydrogel was used in place of tannic acid to chillproof the extract (designated MF3C~. Both of the chillproofed, high molecular weight protein fractions were separately combined with 1.0 ppm to 18 ppm isohumulone or reduced isohumulone extract to produce alternative foam stabilizers MF3T and MF3C. The new compositions were tested on a Class II beer according to the procedures described in Example 2 above. The results are summarized in TABLE 13 below.

:
:

SU~ITUrE SHEET

WO 93/15181 2 1 2 7 4 ~ O PCr/US93/00932 Malt FQamers MF~T and MF3C - Class II Beer FOAM STABILIZER CUNG (Yo?COLLAP RATE rmllmin! SIGM~ FOAM Y~Ll.~E
Control æ 44 lol Isohumulone 45 38 105 Red. Isohum. 53 38 103 Malt Foamcr 3C 18 41 103 Malt Foamer 3T 22 43 105 I-Malt Foamcr 3C 70 37 105 I-Malt Foamer 3T 68 38 103 Malt Foamer 3C+ T7 34 107 Malt Foamer 3T+ 70 35 106 1- = A mL~ isohumulone and Malt Foamer 3C or 3T.
+ = A mK of Malt Foamer 3C or 3T and reduced isohumulone.

2S Exam~le ~
Sh~lke~RV) Tçst In order to determine the optimal levels of isohumulone extract and foaming proteins to be added to a given malt beverage, shaker (HRV) tests were performed. To perform the shaker (~RV) test, a series of 50 ml graduated cylinders containing 10 ml of a particular foaming protein solution were prepared~ The foaming protein solutions were obtained according to the methods and techniques set forth in Examples 2-7 above. Various increments of isohumulone or reduced isohumulone extract were then added to the cylinders. Each grlinder was stoppered, shaken vigorously for five seconds, and the foam height recorded in rnilliliters~ After five minu~es, the ~mal foam and liquid heights were recorded. From tbese values, the head retention value (i~e~ the difference between the final foam and liquid height, in milliliters) for each sample was determined.
TABLE 14 provides an example of data compiled using a 20%
solution of reduced isohumulone extract and Malt Foamer 1 (MF1), a , SUE~llUrE SHEET

wo 93/15181 PCr/uss3/00932 ..~..

foaming protein derived from malt ~ccording to the methods and techniques set forth in Example 6 above.

SHAKER (HR~ TEST: Hop E~aract -- 20% Reduced Isohumulone E~racl Pure ~itial Fmal FIDal Addition Reduced Foam Foam Liquid Head (ml) to Isohumulone Height Height HeightRetentio~
10ml MF1 (e/L MFl! (ml! (ml~ (ml~ Valvc Clin~
0 0 27 27 7 20 Fair 0.015 03 33 32 7 2s Fair O.Q3 0.6 36 37 7 30 Good 0.06 1.2 36 37 6 31 Good ; 0.09 1.8 44 43 6 37 Exc.
0.12 2A 40 40 ~ 35 Exc.
0.15 3.0 41 42 6 36 Exc.
Q.18 3.6 42 42 ~ 37 Exc.

Cling Key: 0-1096 = Poor, 10-3096 s Fair 30-609~ = Good, 609~+ = Exc.

ln this example, as in most cases, the head retention value ceases to increase after an optimum level of isohumulone extract has been - 30 reached. This value is considered to be the saturation point of foaming proteins ~,vith isohumulone extract. Further addition has no effect, as a plateau is reached where additional isohumulone extract fails to react with the foaming proteins.
Thus, it can be seen from each of the foregoing examples that the addition of a single component alone of either isohumulone extract, reduced isohumulone extract, or a partially hydrolyzed protein is ineffective above certain concentrations. However, a combination of either isohumulone or reduced isohumulone extract, together with a partially hydrolyzed protein solution, enhances foam stability without undesirable side effects such as increased turbidity or haze formation. In order to achieve these positive benefits, the components are added in the later stages of the brewing process, usually after filtration or during ~mishing.

SUB~TITUIE SHEET
:

Claims (41)

WHAT IS CLAIMED IS:

1. A method of making a fermented malt beverage with enhanced foam properties comprising the steps of:
obtaining malt beverage ingredients;
processing said malt beverage ingredients to make a fermented malt beverage;
obtaining an isohumulone extract in a process independent of the malt beverage making process;
adding said isohumulone extract in an amount of between about 0.1 ppm and about 20 ppm by weight of the beverage;
obtaining foaming proteins in a process independent of the malt beverage making process; and adding said foaming proteins in an amount of between about 2 ppm and about 250 ppm by weight of the beverage.

2. The method according to claim 1 wherein the isohumulone extract and foaming proteins are added after maturation of the malt beverage.

3. The method according to claim 1 wherein the isohumulone extract comprises non-reduced isohumulone.

4. The method according to claim 1 wherein the isohumulone extract comprises reduced isohumulone.

5. The method according to claim 2 wherein the isohumulone extract comprises hexahydro isohumulone.

6. The method according to claims 1, 2, 3, 4 or 5 wherein the foaming proteins are polymeric amino acids.

7. The method according to claim 6 wherein the polymeric amino acids are selected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

8. The method according to claims 1, 2, 3, 4 or 5 wherein the foaming proteins are derived from a source selected from the group consisting of wheat gluten, malted barley, malted wheat, ale yeast, and lager yeast.

9. The method according to claim 8 wherein the foaming proteins have a molecular weight in the range of about 10,300 to about 100,000 Daltons.

10. The method according to claim 9 wherein isohumulone extract in an amount of between about 0.8 ppm and about 17 ppm by weight of the beverage and foaming proteins in an amount of between about 4 ppm and about 100 ppm by weight of the beverage are added.

11. The method according to claim 1 wherein about 12 ppm of foaming proteins derived from wheat gluten and having a molecular weight of about 15,000 Daltons and about 5.6 ppm of hexahydro isohumulone extract are added.

12. A fermented malt beverage foam-enhancing composition for adding to a quantity of malt beverage ingredients comprising isohumulone extract and foaming proteins, wherein the foaming proteins are selected from the group consisting of isolated, naturally-occurring foaming proteins and amino acids, and wherein the ratio of isohumulone extract to foaming proteins is between about 1 to about 1 and about 1 to about 2500.

13. The composition according to claim 12 wherein the isohumulone extract comprises non-reduced isohumulone.

14. The composition according to claim 12 wherein the isohumulone extract comprises reduced isohumulone.

15. The composition according to claim 14 wherein the isohumulone extract comprises hexahydro isohumulone.

16. The composition according to claims 12, 13, 14 or 15 wherein the foaming proteins are polymeric amino acids.

17. The composition according to claim 16 wherein the polymeric amino acids are selected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

18. The composition according to claims 12, 13, 14 or 15 wherein the foaming proteins are derived from a source selected from the group consisting of wheat gluten, malted barley, malted wheat, ale yeast, and lager yeast.

19. The composition according to claim 16 wherein the foaming proteins have a molecular weight in the range of about 10,300 to about 100,000 Daltons.

20. The composition according to claim 19 wherein the ratio of isohumulone extract to foaming proteins is between about 1 to about 10 and about 1 to about 250.

21. The composition according to claim 12 wherein the isohumulone extract comprises hexahydro isohumulone extract, the foaming proteins are derived from wheat gluten and have a molecular weight of about 15,000 Daltons, and wherein the weight ratio of said hexahydro isohumulone extract to said foaming proteins is about 1 to about 2.15.

22. A beverage composition with enhanced foam properties comprising:
(a) a fermented malt beverage;
(b) an isohumulone extract, the extract comprising between about 0.1 ppm and about 20 ppm by weight of the fermented malt beverage; and (c) foaming proteins, the foaming proteins comprising between about 2 ppm and about 250 ppm by weight of the fermented malt beverage, wherein the foaming proteins are selected from the group consisting of isolated, naturally-occurring foaming proteins and polymeric amino acids.

23. The composition according to claim 22 wherein the isohumulone extract comprises non-reduced isohumulone.

24. The composition according to claim 22 wherein the isohumulone extract comprises reduced isohumulone.

25. The composition according to claim 24 wherein the isohumulone extract comprises hexahydro isohumulone.

26 The composition according to claims 22, 23, 24 or 25 wherein the foaming proteins are polymeric amino acids.

27. The composition according to claim 26 wherein the polymeric amino acids are selected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

28. The composition according to claims 22, 23, 24 or 25 wherein the foaming proteins are derived from a source selected from the group consisting of wheat gluten, malted wheat, malted barley, ale yeast, and lager yeast.

29. The beverage composition according to claim 22 wherein the isohumulone extract comprises about 5.6 ppm by weight of the beverage and the foaming proteins comprise about 12 ppm by weight of the beverage, the foaming proteins being derived from wheat gluten and having a molecular weight of about 15,000 Daltons.

30. A method of enhancing the foam properties of a fermented malt beverage comprising adding to the beverage:
an isohumulone extract in an amount of between about 0.1 ppm and about 20 ppm by weight of the beverage; and polymeric amino acids in an amount of between about 7 ppm and about 250 ppm by weight of the beverage.

31. The method of claim 30 wherein the isohumulone extract is added in an amountof between about 0.8 ppm and about 17 ppm by weight of the beverage and the polymeric amino acids are added in an amount of between about 4 ppm and about 100 ppm by weight of the beverage.

32. The method according to claim 30 wherein the polymeric amino acids are selected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

33. The method according to claims 30, 31 or 32 wherein the polymeric amino acids have a molecular weight in the range of about 10,300 to about 100,000 Daltons.

34. A fermented malt beverage foam-enhancing composition comprising an isohumulone extract and polymeric amino acids, wherein the ratio of isohumulone extract to polymeric amino acids is between about 1 to 1 and about 1 to 2500.

35. The composition according to claim 34 wherein the ratio of isohumulone extract to polymeric amino acids is between about 1 to 10 and about 1 to 250.

36. The composition according to claim 34 wherein the polymeric amino acids are selected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

37. The composition according to claims 34, 35 or 36 wherein the polymeric amino acids have a molecular weight in the range of about 10,300 to about 100,000 Daltons.

38. A beverage composition with enhanced foam properties comprising:
(a) a fermented malt beverage;
(b) an isohumulone extract, the extract comprising between about 0.1 ppm and about 20 ppm by weight of the fermented malt beverage; and (c) polymeric amino acids, the polymeric amino acids comprising between about 2 ppm and about 250 ppm by weight of the fermented malt beverage.

39. The composition according to claim 38, wherein the polymeric amino acids areselected from the group consisting of polymeric arginine, glutamic acid, glycine, leucine, lysine and tyrosine.

40. The composition according to claims 38 or 39 wherein the polymeric amino acids have a molecular weight in the range of about 10,300 to about 100,000 Daltons.

41. A method of enhancing the foam properties of a fermented malt beverage comprising adding an isohumulone extract in an amount of between about 0.1 ppm and about 20 ppm by weight of the beverage and foaming proteins in an amount of between about 2 ppm and about 250 ppm by weight of the beverage, wherein the foaming proteins are selected from the group consisting of isolated, naturally-occurring foaming proteins and polymeric amino acids.