Natural and stable solutions of alpha-acids and their use for the improvement of foam quality of beer

Abstract

This invention describes natural hop products that are stable solutions of alpha-acids, as well as their preparation and their use for post-fermentation addition to beer for the purpose of improving the foam quality, as measured by foam stability and foam cling. Improvements in the stability (both physical and chemical) of the alpha-acids are described by incorporation of iso-alpha-acids and tetrahydroiso-alpha-acids as well as various brewery-acceptable co-solvents. The alpha-acids also improve the physical stability of the reduced iso-alpha-acids, including tetrahydroiso-alpha-acids.

Claims

1. A process for making a stable solution of alpha-acids, which comprises the steps in sequence of: a) adding an acid to an aqueous extract of alpha-acids from a carbon dioxide extract of hops in a first acidification step to lower the pH of the aqueous extract to a pH in the range of 7.5 to 8.2, whereby to produce a first resin phase and an aqueous phase; b) removing the first resin phase; c) adding an acid to the aqueous phase in a second acidification step to further lower the pH of the aqueous phase to form purified alpha-acids in free-acid form in a second resin phase; d) isolating the second resin phase; and e) mixing or blending the second resin phase with a solvent to form a stable solution containing alpha-acids in free-acid form; f) adding an iso-alpha-acid to the solution, wherein said solvent reduces the alpha-acids' susceptibility to degradation and/or precipitation upon storage of the solution, and wherein said solution, when added to a malt-based beverage, improves the foam stability of the beverage.

2. The process of claim 1, wherein the solvent is selected from the group consisting of ethanol, propylene glycol, ethyl acetate, a higher alcohol, and a mixture of two or more thereof.

3. The process of claim 1, wherein one or more additional hop acids selected from the group consisting of a tetrahydroiso-alpha-acid, a hexahydroiso-alpha-acid and a dihydroiso-alpha-acid is added to the solution.

4. A process for making a stable solution of alpha-acids, which comprises the steps in sequence of: a) adding an acid to an aqueous extract of alpha-acids from a carbon dioxide extract of hops in a first acidification step to lower the pH of the aqueous extract to a pH in the range of 7.5 to 8.2, whereby to produce a first resin phase and an aqueous phase; b) removing the first resin phase; c) adding an acid to the aqueous phase in a second acidification step to further lower the pH of the aqueous phase to form purified alpha-acids in a second resin phase; d) isolating the second resin phase; e) mixing or blending the second resin phase with water and a co-solvent; f) adding an iso-alpha acid to the solution; and g) adding an alkali to raise the pH at least until the alpha-acids are dissolved in alkaline form, thereby forming a stable solution that contains alpha-acids, wherein the solution, when added to a malt-based beverage, improves the foam stability of the beverage.

5. The process of claim 4, wherein the co-solvent is selected from the group consisting of ethanol, propylene glycol, glycerol, a higher alcohol, and a mixture of two or more thereof.

6. The process of claim 4, wherein one or more additional hop acids selected from the group consisting of a tetrahydroiso-alpha-acid, a hexahydroiso-alpha-acid and a dihydroiso-alpha-acid is added to the solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of Free Acid Form of Alpha-Acids and Stable Solutions in Propylene Glycol

(1) This example demonstrates the basic process for making a free acid form of alpha-acids, as well as the ease of preparation of solutions of alpha-acids in a solvent such as propylene glycol and its good physical and chemical stability.

Example 1a. Preparation of Free Acid Resin of Alpha-Acid

(2) To 323.9 g of CO.sub.2 extract of hops (56.3% alpha-acids by HPLC) was added 1620 g of deionized water, warmed to 53° C. and with mixing 48.5 ml of 45% potassium hydroxide was added; pH was 8.49 at 56° C. After settling for 1 hour in a separatory funnel in a 54° C. oven, the lower aqueous phase containing most of the alpha-acids was transferred to a beaker. To 117.7 g of the upper resin phase was added 972 g of deionized water and 1.0 ml of 45% KOH was added to bring the pH to 8.51 at 53° C. After settling for 1 hour in a separatory funnel in a 55° C., this second, lower aqueous phase (containing most of the residual alpha-acids) was combined with the first aqueous phase. The 107.4 g of resin phase had only 0.8% of the initial alpha-acids. To the combined aqueous phases was added 1.0 ml of 50% sulfuric acid to drop the pH to 8.14 and after settling for 1 hour in an oven at 55° C., 6.2 g of lower resin phase was removed. The remaining aqueous phase was warmed to 57° C. and 67 ml of 50% sulfuric acid was added to bring the pH to about 1.7 (Merck pH strip) and then allowed to settle for 1.5 hours in a separatory funnel in a 60-65° C. oven. 202.1 g of lower, alpha-acids resin was obtained at an overall recovery of alpha-acids of 97.5%. The hplc concentration of alpha-acids was 88.0%.

Example 1b. Preparation of Solutions of the Free Acid Form of Alpha-Acids in Propylene Glycol

(3) To make 20% alpha-acids in propylene glycol, 13.44 g of propylene glycol (FCC grade) was added to 3.98 g alpha-acids resin of Example 1a. With mixing and warming to about 50° C., all of the alpha-acids dissolved. The yellow-colored, alpha-acids solution remained transparent even upon refrigeration. To make 30% alpha-acids in propylene glycol, 13.54 g of propylene glycol (FCC grade) was added to 7.006 g of alpha-acids resin of Example 1a. Upon mixing and warming to about 50° C., all of the alpha-acids were dissolved to form a transparent, yellow-colored solution that remained transparent even upon refrigeration.

Example 1c. Stability of the Free Acid Form of Alpha-Acids in Propylene Glycol

(4) This example demonstrates that alpha-acids have good physical and chemical stability in propylene glycol. Samples of Example 1b were stored for 6 months in freezer or at room temperature. The freezer and room temperature samples did not have any visible precipitate. There was only a 2% decrease in concentration of alpha-acids (relative to the freezer samples) as shown in Table 1.

(5) TABLE-US-00001 TABLE 1 HPLC analyses of samples of α-acids in propylene glycol after 6 months storage glass vials and in freezer or at room temperature (about 20-24° C.). % Loss of % α- % Iso- Sample Storage Temp. α-acids acids α-acids 30% α-acids in Freezer — 29.6 0.6 propylene glycol Ambient 2.0 29.0 0.4 20% α-acids in Freezer — 19.8 0.3 propylene glycol Ambient 2.0 19.4 0.25

Example 2

Co-Solvents can Improve the Stability of Free Acid Form of Alpha-Acids in Propylene Glycol

(6) To 225.0 g of free acid form of alpha-acids (84.1% alpha-acids by HPLC) was added 132.0 g of propylene glycol (USP grade) and mixed while being warmed on a hot plate. When the temperature reached 41° C., all of the resin was dissolved. The concentration of alpha-acids was 54% by HPLC. To aliquots of this stock solution were added co-solvents to make solutions with 45% alpha-acids and 15.5 weight percent co-solvent. Samples were stored in either 1 oz. PETG bottles (glycol-modified, polyethylene terephthalate) at room temperature or in glass vials in a freezer for 6 months and then analyzed by HPLC.

(7) The co-solvents ethyl acetate and iso-amyl alcohol, but not ethanol, further improved the stability of the free acid form of alpha-acids in propylene glycol as shown in Table 2.

(8) TABLE-US-00002 TABLE 2 Effect of co-solvents on the stability of the free acid form of alpha-acids in propylene glycol stored in 1 oz. PETG bottles at room temperature (about 20-22° C.) for 6 months. *% Loss of % Alpha- % Iso- Co-solvent Alpha-acids acids alpha-acids None 2.0 44.1 0.45 Ethyl acetate 1.4 44.4 0.50 Iso-amyl alcohol 1.4 44.4 0.48 Ethanol 2.0 44.1 0.50 *Loss of alpha-acids from solutions stored in freezer.

Example 3

Potassium Salt of Alpha-Acids in Propylene Glycol and Glycerol Preparation and Stability

(9) To 36.3 g of free acid form of alpha-acids (89.1% alpha-acids by HPLC) was added 271.7 g of propylene glycol and all of the alpha-acids were dissolved upon warming and mixing. The concentration of alpha-acids was 10.5%. To stirred aliquots of this stock solution were added 45% potassium hydroxide (at room temperature) or potassium carbonate (required 53° C. to dissolve the potassium carbonate) such that more than one equivalent of potassium is added since lower equivalents were found to be not as stable. For the alpha-acids in glycerol, alpha-acids resin (89.1% alpha-acids by HPLC) and glycerine were mixed at 53-55° C. and 45% KOH or potassium carbonate were added; mixing was continued until all of the resin (or potassium carbonate) was dissolved. Aliquots of each sample were put in glass vials and stored at either 40° C. or at room temperature for stability tests.

(10) Alpha-acids stored at room temperature for 9 months were similarly stable in propylene glycol whether in the free acid form or the potassium salt form, as long as the pH was in the range of 7.6 to 8.6, at 2% alpha-acids, as shown in Table 3. If the pH is too high, in the region of 9 (at 2% alpha-acids), the alpha-acids in propylene glycol are quite unstable, forming mostly iso-alpha-acids. At elevated temperature of 40° C. the free acid form of the alpha-acids was more stable than the potassium salt form in propylene glycol due to the formation of the iso-alpha-acids at elevated temperatures. The potassium salt of the alpha-acids had relatively good stability in glycerin, especially if stored at about 20° C.

(11) TABLE-US-00003 TABLE 3 Stability of solutions of alpha-acids (10-20%) in propylene glycol and glycerin after 9 months at room temperature and after 5 weeks at 40° C. pH at 2% % Loss of Alpha-acids (average) Solvent Alkali Alpha-acids 20° C. for 9 months 40° C. for 5 weeks Propylene glycol None 3.3-3.5 3.0, 4.9 (3.9)  2.4, 3.0 (2.8)  Propylene glycol 45% KOH 7.6-8.6 3.6-4.6 (4.0) 4.6-5.3 (4.9) Propylene glycol Potassium 7.4-8.2 3.6-7.6 (5.3) 3.5-7.1 (5.4) carbonate Propylene glycol Potassium 9.1 25.5 47.5 carbonate Glycerin 45% KOH 8.2  3.4  6.2 Glycerin Potassium 7.9-8.4 3.1-4.1 (3.5) 5.4-7.1 (6.5) carbonate

Example 4

Preparation and Stability of Alpha-Acids in Water

(12) This example demonstrates that aqueous solutions are physically more stable at higher concentrations of alpha-acids and that co-solvent such as propylene glycol can also improve stability.

(13) The samples made in water were prepared as follows: 23.9 g of alpha-acids free acid resin (89.2% alpha-acids, 1.2% beta-acids and 1.0% iso-alpha-acids) was warmed to 55° C. and 15.4 g of reverse osmosis water was added and then 45% potassium hydroxide was added with mixing; the maximum temperature was 60° C. and the sample was quickly cooled to prevent isomerization of the alpha-acids. A total of 7.9 g of 45% KOH was require to bring the pH to 8.45 (when diluted to 2% alpha-acids). This sample had a concentration of alpha-acids of 46.3% and was transparent (brown-amber in color) upon cooling to room temperature. To aliquots of this stock solution were added varying amounts of reverse osmosis water to make samples of concentration of alpha-acids in the range of 10-45%. The three samples with added propylene glycol were prepared by making 33.0% alpha-acids in water at a pH of 8.3 (at 2% alpha-acids) and then aliquots were diluted with water and propylene glycol. Samples were stored in glass vials at room temperature.

(14) Samples with concentrations of alpha-acids in water in the range of 20-45% had no precipitate after storage for 3 months as shown in Table 4. In fact they did not have any precipitate even after storage for 6 months at room temperature, followed by overnight refrigeration. A 3 month storage solution of alpha-acids of 10% had a resinous precipitate at room temperature and required addition of some co-solvent such as propylene glycol to maintain long-term physical stability.

(15) TABLE-US-00004 TABLE 4 Effect of concentration of alpha-acids on the physical stability of aqueous solutions of alpha-acids stored at room temperature for 3 months. Physical Stability Concentration of pH (at 2% after 3 months alpha-acids Wt % PG α-acids) Room temp. *Refrigerated 45% 0 8.45 Transparent Transparent 30% 0 8.45 Transparent Transparent 20% 0 8.45 Transparent Transparent 15% 0 8.45 Transparent Thin layer of resin 10% 0 8.45 Layer of Layer of resin resin 15% 15 8.28 Transparent Transparent 10% 15 8.28 Transparent Precipitate, no resin 10% 30 8.28 Transparent Transparent *The room temperature samples were stored overnight in a refrigerator.

Example 5

Improvement in Stability of Alpha-Acids in Water with a Co-Solvent

(16) To 290.7 g of alpha-acids free acid (84.2% alpha-acids, 2.2% beta-acids) was added 291 g of reverse osmosis water at 54° C. and with mixing at 500 rpm, 94.6 g of 45% (w/w) potassium hydroxide was added and the temperature was kept below 51° C.; the pH was 8.9 (measured as-is at 26° C.); the concentrations of alpha-acids and iso-alpha-acids were 36.3% and 0.5%, respectively. To aliquots of this stock solution were added water and/or 95% ethanol (Everclear) or propylene glycol such that the concentration of alpha-acids was 20%. Samples were stored in 1 oz, PETG bottles and analyzed by HPLC.

(17) The aqueous solution of 20% α-acids in water had poor chemical stability when stored at room temperature due to formation of the iso-alpha-acids as shown in Table 5. For such a product refrigeration is needed, though only down to about 10° C. since at this temperature there was only a 1% loss of alpha-acids after 6 months; though lower temperatures would result in even lower losses of alpha-acids. This result demonstrates how sensitive the isomerization of the potassium salt of alpha-acids is to temperature. Relatively low concentrations of the co-solvents ethanol and propylene glycol dramatically improved the chemical stability of the potassium salt of the alpha-acids in water by decreasing the formation of the iso-alpha-acids. The co-solvents also prevented the formation of beta-acids crystals that formed in this particular batch of 20% alpha-acids. Concentrations of alpha-acids of greater than 30% also prevented the formation of crystals of beta-acids; further demonstrating the improved physical stability of aqueous solutions of alpha-acids with increasing concentration.

(18) TABLE-US-00005 TABLE 5 Effect of a co-solvent on the stability of aqueous solutions of 20% Alpha-acids stored in 1 oz. PETG bottles for 6 months at about −3° C., about 10° C. or at room temperature (about 20-22° C.). Wt % Co- Storage % Loss of *% Increase in Co-Solvent Solvent Temp. Alpha-acids % Iso-Alpha None 0   10° C. 1.3 0.5 None 0 20-22° C. 9.1 7.9 Ethanol 10 20-22° C. 4.1 4.3 Ethanol 15   10° C. 0.8 0.3 Ethanol 15 20-22° C. 3.4 3.2 Ethanol 25 20-22° C. 1.3 2.1 Propylene Glycol 15 20-22° C. 5.0 4.5 Propylene Glycol 30 20-22° C. 2.3 2.6 *100 × (Increase in % Iso-alpha-acids)/% Alpha-acids (at −3° C.)

Example 6

Improvement in Stability of Alpha-Acids with Iso-Alpha-Acids

(19) The 20-30% solutions of alpha-acids were prepared as described in Example 5, though using a free acid form of alpha-acids with 87.7% alpha-acids (+0.5% beta-acids). The solutions of alpha-acids with iso-alpha-acids were prepared by blending the free acid forms of the alpha-acids and the iso-alpha-acids (typically about 88-90% iso-alpha-acids) and mixing with reverse osmosis water in a water bath at about 50° C., followed by addition of 45% potassium hydroxide to bring the pH to 8.9 (as-is at 23-26° C.).

(20) The stability of the alpha-acids was improved with increasing concentrations of iso-alpha-acids or increasing ratio of iso-alpha-acids to (alpha-acids+iso-alpha-acids) such that inclusion of about 13 weight % of iso-alpha-acids caused a 50% improvement in the stability of the alpha-acids as shown in Table 6. The improved stability was due to a decrease in the formation of iso-alpha-acids. The iso-alpha-acids also improved the physical stability of the alpha-acids. The sample of 10% alpha-acids solution with 20% iso-alpha-acids stored at room temperature did not have any chill precipitate, while a similarly stored solution of 10% alpha-acids had chill resin.

(21) TABLE-US-00006 TABLE 6 Effect of iso-alpha-acids on the stability of alpha- acids, as determined by HPLC, of samples stored at room temperature for 6 months in PETG bottles. *Ratio of Iso- Conc. Of Conc. Of % Loss of #% Increase in % alpha-acids α-acids Iso-α-acids α-acids Iso-α 0.024 20.1 0.5 10.1 9.1 0.026 29.9% 0.8% 10.4 9.1 0.332 20.1 10.0 5.3 4.9 0.402 18.0 12.1 5.4 3.9 0.498 15.1 15.0 4.3 3.9 0.662 10.2 20.0 3.1 3.6 All of the 6-month storage samples were transparent after standing overnight in a 0-1° C. refrigerator. *Ratio of iso-alpha-acids = % Iso-alpha-acids/(% alpha-acids + % Iso-alpha-acids) #100 × (Increase in % Iso-alpha-acids)/% Alpha-acids (at −3° C.)

Example 7

Co-Solvents Improve the Stability of Alpha-Acids in Solutions Containing Iso-Alpha-Acids

(22) This example shows how a co-solvent further improves the stability of alpha-acids in a solution of alpha-acids containing iso-alpha-acids.

(23) To 214.3 g of alpha-acids and 205.7 g of iso-alpha-acids, both in free acid forms, were added 424 g of reverse osmosis water at 45° C. and mixed at 450 rpm. A total of 140.9 g of 45% potassium hydroxide was added to bring the pH to 8.9 (as-is at 23° C.). To aliquots of this stock solution were added co-solvents and water such that the final concentrations of alpha-acids and iso-alpha-acids were 15.0% and 15.3%, respectively by HPLC and stored in 1 oz. PETG bottles for 6 months.

(24) Both the co-solvents ethanol and isopropyl alcohol caused a similar and significant improvement in the stability of the alpha-acids as shown in Table 7. There was a 50% decrease in the loss of alpha-acids with about 9 weight % of ethanol. Nearly all of the loss of alpha-acids was due to the formation of iso-alpha-acids. The sample of 10 wt % ethanol+15% alpha-acids+15% iso-alpha-acids had about 20% of the loss of alpha-acids as solutions of 15-30% alpha-acids in water. Propylene glycol also improved the stability of the alpha-acids, though not as effectively as ethanol.

(25) TABLE-US-00007 TABLE 7 Effect of co-solvents on the stability of samples of 15% Alpha- acids + 15% iso-alpha-acids stored in 1 oz. PETG bottles for 6 months at about −3° C. or at room temperature (about 20-22° C.). Wt % Co- % Loss of *% Increase in % Co-solvent solvent α-acids Iso-α None 0.0 4.2 4.2 Ethanol 5 3.1 3.0 Ethanol 10 1.8 1.8 2-Propanol 5 2.9 2.6 Propylene Glycol 15 2.2 1.9 *100 × (Increase in % Iso-alpha-acids)/% Alpha-acids (at −3° C.)

Example 8

Improvement of Stability of Alpha-Acids with Tetrahydroiso-Alpha-Acids

(26) To free acid form of alpha-acids were added the free acids of either iso-alpha-acids, tetrahydroiso-alpha-acids or hexahydroiso-alpha-acids and mixed with water and 45% potassium hydroxide to bring the pH to 8.9 (as-is at 24-26° C.); the concentrations of alpha-acids and iso-alpha-acids or reduced iso-alpha-acids were all 15% by HPLC. Samples were stored in 1 oz PETG bottles for 6 months at either—3° C. or at room temperature (20-22° C.).

(27) This example shows that not only the iso-alpha-acids but also tetrahydroiso-alpha-acids, but not hexahydroiso-alpha-acids, improves the chemical stability of alpha-acids by decreasing the formation of iso-alpha-acids; see results in Table 8.

(28) TABLE-US-00008 TABLE 8 Effect of hop acids, at 15%, on the stability of aqueous solutions of 15% alpha-acids stored at room temperature for 6 months in PETG bottles. *% Loss of #% Increase in % Type of Hop acid Humulone Iso-α None, 30% alpha-acids 7.6 7.6 Iso-alpha-acids 4.1 3.8 Tetrahydroiso-alpha-acids 5.0 5.0 Hexahydroiso-alpha-acids 7.1 6.1 *The alpha-acids humulone (+adhumulone) were only quantified because cohumulone co-eluted with some of the HPLC peaks of the reduced iso-alpha-acids. #100 × (Increase in % Iso-alpha-acids)/% Alpha-acids (initial values)

Example 9

Improvement in the Physical Stability of Tetrahydroiso-Alpha-Acids with Alpha-Acids

(29) Aqueous solutions of tetrahydroiso-alpha-acids are commercially sold at concentrations of 9-10% by HPLC. Though initially the solutions do not contain precipitate, over time upon storage at ambient temperatures and with subsequent refrigeration, a chill precipitate may form (due to a drop in pH upon storage), resulting in loss of tetrahydroiso-alpha-acids in a resin at the bottom of the container. Also with tetrahydroiso-alpha-acids made from certain hop varieties with low cohumulone content (such as Nugget, Zeus and Apollo), crystals can form that consist mostly of the cis-form of tetrahydroisohumulone (+adhumulone). Either of these two solids requires heating and shaking of the container to redissolve. Thus it is advantageous for the brewer never to have either of these solids occur during the shelf life of the solution of tetrahydroiso-alpha-acids.

(30) In the absence of any alpha-acids, cis-tetrahydroiso-alpha-acids crystals formed during a 6 months storage at room temperature of a 9% tetrahydroiso-alpha-acids solution made from the variety Apollo via the solvent-free process of Wilson and Smith (US 2008 0160146 A1). After cooling in a refrigerator to about 0° C., there was nearly a total loss of 10% of the initial tetrahydroiso-alpha-acids in a chill precipitate and the crystals; see Table 9. By comparison, inclusion of 10% alpha-acids with the 10% tetrahydroiso-alpha-acids resulted in a physically stable product in which there were no crystals and almost no loss of tetrahydroiso-alpha-acids upon chilling to 0° C. If stored for 6 months at about 10° C., there was almost no loss of alpha-acids and there were no solids formed upon cooling to 0° C.

(31) TABLE-US-00009 TABLE 9 Storage stability of solutions of tetrahydroiso-alpha-acids (from the hop variety Apollo; tetrahydroiso-cohumulone ratio of 0.27) in 1 oz PETG bottles for 6 months. % % Tetra- # Loss of Alpha- hydroiso- Storage * Loss of Crystals Tetra in acids alpha-acids Temp. Humulone Formed? Solids at 0° C. 0 9.1 20° C. — Yes 9.9 9.8 10.2 10° C. 0.1 No 0.0 9.8 10.2 20° C. 4.2 No 0.2 * The alpha-acids humulone (+adhumulone) were only quantified because cohumulone co-eluted with some of the HPLC peaks of the tetrahydroiso-alpha-acids. # % loss of tetrahydroiso-alpha-acids in both the crystals and the resin that formed upon chilling overnight to about 0° C.

Example 10

Utilization of Alpha-Acids Added to Cold Beer

(32) Cold beer (brand A) was degassed by addition of 8 drops of octanol to four 12 oz bottles of beer, followed by bath sonication. 460 g aliquots were transferred to 600 ml glass beakers, cooled to about 1° C. in a refrigerator and then the 10% (w/w) alpha-acids solutions were added to the stirred (Teflon-lined stirring bar) beer to make a 5 ppm alpha-acids. After a few seconds of mixing, about 10 ml aliquot was filtered through a 0.45μ sintered glass filter (Whatman GMF media) and 5 ml of the filtrate was diluted to 10 ml with acidic methanol (0.5 ml of 85% phosphoric acid in 1 L of methanol); 20μ was injected onto HPLC column.

(33) This example demonstrates that the utilization (% of the initial alpha-acids in solution in cold beer) of the free acid form of the alpha-acids (in propylene glycol) in cold beer was not as good as the potassium salt of the alpha-acids added to beer. The utilization of the potassium salt of alpha-acids in beer was essentially identical whether the solvent was propylene glycol or water.

(34) TABLE-US-00010 TABLE 10 HPLC results of filtrate of beer to which about 5-ppm of alpha-acids were added. Utilization of Concentration of Alpha-acids in alpha-acids added Alpha-acids Product cold filtrate to beer, ppm Free acid in propylene glycol 67 4.9 Potassium salt in Propylene glycol 86 4.9 Potassium salt in water 86 5.0

Example 11

Alpha-Acids Improves the Foam Stability of Beer in Concentration-Dependent Manner

(35) A solution of 10% (w/w) alpha-acids (free acid form in propylene glycol) was prepared as of in Example 1b. This solution was diluted with water and dilute potassium hydroxide was added to bring the pH to 7.2. After chilling, the haze was removed by filtration through a 0.45 μm filter; concentration of alpha-acids was 0.17%. From this stock solution of alpha-acids were prepared various dilutions with water and 1.0 g aliquots were added to 12 oz bottles of beer (Brand A), then foamed to top of each bottle in order to remove most of the air and then capped. The bottles were inverted 10 times during a period of about one hour and then stored for 2 weeks at room temperature. The foam stability of each bottle was determined using a Haffmans foam stability tester model Nibem-T. The foam stability is measured as the time it takes for the layer of foam to collapse 30 mm in a glass container.

(36) Addition of a solution of purified alpha-acids directly into beer resulted in a concentration-dependent increase in the foam stability of beer as demonstrated in Table 11. Even 1 ppm of alpha-acids caused a significant improvement in the foam stability of this beer.

(37) TABLE-US-00011 TABLE 11 Foam stability (Nibem-30) of beer (brand A had 6.7 ppm iso-alpha- acids and 0.5 ppm alpha-acids) as affected by alpha-acids. Amount of Alpha- *Concentration of Nibem-30, seconds acids added in ppm Alpha-acids in ppm (Average ± std. dev.) 0 0.49 184 ± 3 1.00 1.35 199 ± 2 1.99 2.17 208 ± 3 2.99 3.18 214 ± 2 4.00 3.93 221 ± 2 *Concentration of alpha-acids in beer were determined by HPLC.

Example 12

Alpha-Acids Improve Foam Stability and Foam Cling (or Lacing) Better than Iso-Alpha-Acids

(38) To 12 oz bottles of beer (Brand A) were added weighed amounts of aqueous solutions of 0.12% of either iso-α-acids, α-acids or tetrahydroiso-alpha-acids to make 3-ppm of each compound in beer. After fobbing to top and recapping, the bottles were shook at 120 oscillations for 1 hour and then allowed to stand at room temperature for 4 days. The foam stability of each bottle was determined using a Haffmans foam stability tester model Nibem-T. The foam stability is measured as the time it takes for the layer of foam to collapse 30 mm in a glass container. After foaming stability measurements were taken, the foam cling (or lacing) of beer was determined by modification of the method described by Kunimune and Shellhammer in J. Agric. Food Chem. 56, 8629-8634 (2008). Quantification of the amount of cling (or lacing) was done by absorbance at 240 nm in water as described by G. Jackson and C. W. Banforth in J. Inst. Brew. 88, 378-81 (1982). The wavelength of 240 nm was chosen instead of 230 nm because at that wavelength the alpha-acids, iso-alpha-acids, and tetrahydroiso-alpha-acids have nearly equal absorbance in water at pH of 5.4 (the approximate pH of the foam cling in water). The foam cling (or lacing), between top of container to 4 cm below the top, was dissolved in 50 ml of water and was quantified by measuring the absorbance at 240 nm in water.

(39) Alpha-acids caused a significantly greater improvement in the foam stability of beer than iso-alpha-acids, though less than the tetrahydroiso-alpha-acids; see Table 12. Alpha-acids caused a significantly greater amount of foam cling (or lacing) of beer than iso-alpha-acids and similar to that of the tetrahydroiso-alpha-acids.

(40) TABLE-US-00012 TABLE 12 Effect of 3 ppm of alpha-acids on foam stability and foam cling (or lacing) of a beer (brand A with 4.6 ppm of iso-alpha-acids); also compared with iso-alpha-acids and tetrahydroiso-alpha- acids. Both measurements were repeated six times for each sample; average ± standard deviation are presented. Foam Stability, Foam Cling (or Lacing), Hop Acid seconds relative to control None added 181 ± 1 100  *(0.056 ± 0.002) Iso-alpha-acids 197 ± 5 121 ± 4 Alpha-acids 220 ± 3 146 ± 9 Tetrahydroiso-alpha-acids 236 ± 4 146 ± 7 *Absorbance at 240 nm in water.

Example 13

Alpha-Acids Improve the Foam Stability of a Number of Beers

(41) A solution of potassium salt of alpha-acids in propylene glycol was diluted to 1.3% alpha-acids in water (pH 7.3) and then refrigerated overnight and filtered. To each 12 oz bottle of beer was added 0.843 g of the alpha-acids solution so that the concentration of alpha-acids was 3.0 ppm. Each bottle was foamed to top and capped, shaken 10 times over a period of 1 hour and allowed to stand at room temperature for 2 weeks. The foam stability of each bottle was determined using a Haffmans foam stability tester model Nibem-T.

(42) Alpha-acids improved the foam stability of all five beers tested, though the improvement in foam stability was much better in some beers than in others.

(43) TABLE-US-00013 TABLE 13 Improvement in foam stability of various beers in which 3 ppm of alpha-acids was added. Increase in Foam Foam Stability of Brand of Beer Stability, seconds Control beer (seconds) A 30 184 B 22 159 C 19 175 D 16 209 E 13 181

Example 14

Loss of Alpha-Acids in Beer does not Affect Foam Stability

(44) To 3.23 g of 10% alpha-acids in propylene glycol were added 1.75 ml of 0.5 Molar aqueous solution of potassium hydroxide and 15.7 g of reverse osmosis water; pH was 7.5. 94 mg aliquots of this stock solution of 1.5% alpha-acids were added to 12 oz bottles of beer (brand A), then foamed to top and recapped and inverted each bottle 7 times. Bottles of beer were stored at room temperature and in the dark and were brought to 20° C. before foam stability testing using Haffmans foam stability tester model Nibem-T. Concentrations of alpha-acids and iso-alpha-acids were determined by HPLC using the international calibration extract, ICE-2 for the alpha-acids and the international calibration standard, ICS-I2 for the iso-alpha-acids.

(45) Even though the concentration of alpha-acids in beer had decreased by 47% after 18 weeks storage at room temperature, there was no decrease in foam stability. The alpha-acids treated beer had a higher concentration of iso-alpha-acids possibly due to some of the alpha-acids having been transformed to the iso-alpha-acids which have less foam-stabilizing activity than the alpha-acids. Some of the lost alpha-acids must have formed compounds that also had good foam stability.

(46) TABLE-US-00014 TABLE 13 Foam stability and concentration of alpha-acids of beer with 4-ppm alpha-acids after 18 weeks at room temperature. Storage Time Foam Stability, Alpha-acids, Iso-alpha- Sample (weeks) seconds ppm acids, ppm Control Beer 0.14 174 0.3 6.1 18 171 <0.2 5.3 4-ppm Alpha- 0.14 199 4.3 6.1 acids 18 201 2.3 5.5