WO2020102170A1

WO2020102170A1 - Enzymatic process for production of modified hop products

Info

Publication number: WO2020102170A1
Application number: PCT/US2019/060882
Authority: WO
Inventors: Jane GUIDO; James REDWINE; Katie Whalen; Donald Richard BERDAHL; Brian Patrick Buffin; Matthew Blake JONES; Katrina Williams
Original assignee: Kalamazoo Holdings, Inc.
Priority date: 2018-11-13
Filing date: 2019-11-12
Publication date: 2020-05-22

Abstract

The present invention relates to a process for producing a beer bittering agent via yeast derived enzyme catalyzed bioconversion of hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids and to the novel yeast derived enzyme catalysts which may be employed in such a process.

Description

ENZYMATIC PROCESS FOR PRODUCTION OF MODIFIED HOP PRODUCTS

FIELD OF THE INVENTION

[0001 ] The present invention relates to a process for producing a beer bittering agent via enzyme catalyzed bioconversion of hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids and to the novel enzyme catalysts which may be employed in such a process. Dihydro-(rho)-isoalpha acids have superior characteristics which improve utility as a beverage additive. Consumers may prefer dihydro-(rho)-isoalpha acids produced via enzyme catalyzed bioconversion, a process which does not require the use of harsh chemical reagents and may utilize enzymes which may be naturally occurring.

BACKGROUND OF THE INVENTION

[0002] Traditional methods of bittering beer use whole fresh hops, whole dried hops, or hop pellets added during the kettle boil. Hop extracts made by extracting hops with supercritical carbon dioxide, or isomerized hop pellets, made by heating hops in the presence of a catalyst are more recent bittering innovations that have also been adopted by brewers. Hop pellets can also be added later in the brewing process and in the case of dry hopping, hops are added to the finished beer prior to filtration. These methods suffer from a poor utilization of the bittering compounds present in the hops, which impacts the cost unfavorably. Beer or other malt beverages produced in this manner are unstable to light and must be packaged in dark brown bottles or cans or placed to avoid the light induced formation of 3-methyl-2-butene-1 - thiol (3-MBT) which gives a pronounced light-struck or skunky aroma. Placing bottles in cardboard boxes or completely wrapping them in light-proof or light-filtering paper, foil, or plastic coverings is another expensive method of protecting these beverages from light-struck flavor and aroma.

[0003] Bitterness in traditionally brewed beer is primarily derived from isoalpha acids. These compounds are formed during the brewing process by the

isomerization of the humulones, which are naturally occurring compounds in the lupulin glands of the hop plant. A consequence of this is, given the natural instability of the isoalpha acids towards photochemical reactions in beer, a beverage prone to the formation of light-struck or skunky flavor and aroma. [0004] Fully light stable beers or other malt beverages can be prepared using so- called advanced or modified hop acids. Beers made using these bittering agents can be packaged in non-colored flint glass bottles without fear of forming skunky aromas. Dihydro-(rho)-isoalpha acids are reduction products of isoalpha acids which are light stable. To date, these compounds have not been found in nature.

Traditionally, the portion of the isoalpha acids which is responsible for the

photochemistry has been altered by reduction of a carbonyl group using sodium borohydride.

[0005] Sodium borohydride is an inorganic compound that can be utilized for the reduction of ketones. It is extremely hazardous in case of skin contact, eye contact, inhalation, or ingestion, with an oral LD50 of 160 mg/kg (rat). Sodium borohydride is also flammable, corrosive, and extremely reactive with oxidizing agents, acids, alkalis, and moisture ( Sodium Borohydride ; MSDS No. S9125; Sigma-Aldrich Co.: Saint Louis, MO November 01 , 2015.

http://www.sigmaaidnch.com/catalog/product/siama/s9125)·

[0006] Consumers are increasingly expressing a preference for natural materials over synthetic or semi-synthetic ones. Thus, a need exists not only to provide compositions employing natural materials as bittering agents for beer and other beverages, but also processes for more natural production of said materials.

[0007] Biocatalytic production is an emerging technology which provides highly selective, safe, clean, and scalable production of high value compounds. Biocatalytic production relies on naturally occurring enzymes to replace chemical catalysts.

[0008] Enzymes are naturally occurring proteins capable of catalyzing specific chemical reactions. Enzymes exist in nature that are currently capable of replacing chemical catalysts in the production of modified hop bittering compounds (Robinson, P. K., Enzymes: principles and biotechnological applications. Essays Biochem 2015, 59, 1 -41 .).

[0009] Abundant precedence exists for the utility of enzymes in brewing and their favorable influence on the final character of beer (Pozen, M., Enzymes in Brewing. Ind. Eng. Chem, 1934, 26 (1 1 ), 1 127-1 133.). The presence of yeast enzymes in the natural fermentation of beer is known to produce compounds that affect the flavor and aroma of the final beverage (Praet, T.; Opstaele, F.; Jaskula-Goiris, B.; Aerts,

G.; De Cooman, L, Biotransformations of hop-derived aroma compounds by

Saccharomyces cerevisiae upon fermentation. Cerevisia, 2012, 36, 125-132.).

Exogenously added enzymes provide a variety of improvements to the brewing process, such as reduced viscosity, increased fermentable sugars, chill-proofing and clarification (Wallerstein, L. (1947) Bentonite and Proteolytic Enzyme Treatment of Beer, US Patent 2,433,41 1.; Ghionno, L.; Marconi, O.; Sileoni, V.; De Francesco, G.; Perretti, G., Brewing with prolyl endopeptidase from Aspergillus niger the impact of enzymatic treatment on gluten levels, quality attributes, and sensory profile. Int. J. Food Sci. Technol, 2017, 52 (6), 1367-1374.). Additionally, hop extracts have been specifically pretreated with enzymes for modifying hop-derived aroma compounds (Gros, J.; Tran, T. T. H.; Collin, S., Enzymatic release of odourant polyfunctional thiols from cysteine conjugates in hop. J. Inst. Brew. 2013, 1 19 (4), 221 -227.).

[0010] Prior to the present invention, however, enzymatic conversion of hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids employing yeast derived enzymes has not been observed or described.

OBJECT OF THE INVENTION

[001 1 ] It is an object of the present invention to provide a process for converting hop- derived compounds, for example, alpha and/or isoalpha acids, to dihydro-(rho)- isoalpha acids, the process comprising the steps of contacting the hop-derived compounds with yeast derived enzymes which are ultimately capable of reducing hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids, and optionally cofactors for the enzymes, and incubating the mixture for a period of time sufficient to convert the hop-derived alpha and/or isoalpha acids to dihydro-(rho)- isoalpha acids employing such enzymes.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a process for converting hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids, the process comprising isolating and/or modifying yeast derived enzymes such that the yeast derived enzymes are capable of being used in a process for reducing hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids, and contacting the hop-derived alpha and/or isoalpha acids, yeast derived enzymes and, optionally cofactors for the enzymes, for a period of time sufficient to convert hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structures of co- and n-Rho showing the source of the fragment ions monitored in the MRM experiment.

Figure 2 shows the Rho MRM chromatogram recorded for a Rho standard solution.

Figure 3 shows the Rho MRM chromatogram recorded for the FV1 "beer" sample demonstrating the presence of Rho.

Figure 4 shows the Rho MRM chromatogram recorded for the FV1 yeast sample demonstrating the presence of Rho.

Figure 5 shows the Rho MRM chromatogram recorded for the FV2 "beer" sample demonstrating the absence of Rho.

Figure 6 shows the Rho MRM chromatogram recorded for the FV2 yeast sample demonstrating the absence of Rho.

Figure 7 shows the Rho MRM chromatogram recorded for the Homebrew beer - "blow off" sample demonstrating the presence of Rho.

Figure 8 shows the Rho MRM chromatogram recorded for the Homebrew yeast - "blow off" sample demonstrating the presence of Rho.

Figure 9 shows the Rho MRM chromatogram recorded for the Homebrew beer - "final" sample demonstrating the presence of Rho. Figure 10 shows the Rho MRM chromatogram recorded for the Homebrew yeast - "final" sample demonstrating the presence of Rho.

DETAILED DESCRIPTION OF THE INVENTION

[0013] In this invention, a yeast derived enzyme replaces the function of sodium borohydride and allows a more natural production method for the beverage additive, dihydro-(rho)-isoalpha acids.

[0014] Suitable enzymes include any yeast derived enzyme specifically reducing a ketone group to a hydroxy group of any or all isomers of isoalpha acid (co-, n- ad-, and cis/trans-). The yeast derived enzymes may be cofactor dependent (NADH or NADPH) or independent.

[0015] Herein, "isoalpha acids", "hop isoalpha acids", and "hop-derived isoalpha acids" may be used interchangeably.

[0016] An alpha acid and/or isoalpha acid solution is subjected to enzymatic treatment using a purified yeast derived enzyme or a mixture containing yeast derived enzymes, and optionally a cofactor for the enzyme, and optionally additional enzymes for cofactor recycling. The amount of enzyme depends on the incubation parameters including duration, temperature, amount and concentration of substrate.

[0017] Alternatively, an alpha acid and/or isoaipha acid solution is subjected to enzymatic treatment using a mixture containing a microorganism expressing said yeast derived enzyme.

[0018] Alpha add and/or isoalpha acid mixture may be subjected to an enzymatic reaction using a yeast derived enzyme, for example a reductase enzyme, in addition to enzymes for catalyzing additional desired modifications, such as but not limited to, dehydrogenases, isomerases, hydratases and lyases. Yeast derived enzymes of varying activity may be combined in one reaction pot or added sequentially to the reaction. [0019] A suitable solvent to use in the enzyme incubation inciudes water and mixtures of water with another solvent compatible with the yeast derived enzyme, such as ethanol. Enzymatic activity benefits from buffering of aqueous solutions. Buffering agents include, but are not limited to: tris(hydroxymethyl)aminomethane (aka. Tris), 4-(2-hydroxyethyl)piperazine-1 -ethanesulfonic acid (aka. HEPES), sodium phosphate, and potassium phosphate.

[0020] The yeast derived enzyme(s) and alpha acids and/or isoaipha acids, and optionally cofactors for the enzyme(s), are incubated within a suitable pH range, for example pH 6 to 10, and temperature range, for example 10 to 90 °C, and held at this temperature for a sufficient time to convert alpha acids and/or isoalpha acids to the desired dihydro-irhoHsoalpha acids yield. Continuous stirring will ensure a constant temperature and exposure of substrate to enzyme. The reaction duration, typically 24 to 48 hours, will depend on the amount and concentration of the enzyme and substrate, solvent present, and temperature chosen.

[0021 ] The yeast derived enzyme may be free in solution, immobilized onto beads or similar mixable scaffolds, or immobilized onto a film or resin over which a solution of alpha acid and/or isoaipha acids is passed. The purity level of the yeast derived enzyme may vary from 30 to 90+% depending on the purification method.

[0022] The yeast derived enzyme may be removed from the final product via physical filtering or centrifugation. The yeast derived enzyme may also be rendered inactive by extreme temperature or pH and remain in the final product.

[0023] The yeast derived enzymes suitable for the process of the present invention include reductases, such as ketoreductase enzymes.

EXAMPLES

[0024] The following examples illustrate the invention without limiting its scope.

EXAMPLE 1 - Production of Rho from Hop Acids in Whole Hops by Yeast Under Normal Fermentation Conditions [0025] The following example demonstrates that yeast converts the hop-derived compounds, for example alpha and/or isoalpha acids, present in wort after the brew kettle boil into dihydro-(rho)-isoalpha acids (Rho). This is an enzymatic conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids.

Sample Preparation - Lab Produced Minimal "Beer"

Materials

Two (2) New, Unused 1 L HPLC Solvent Bottles (Fermentation Vessels)

Corn Sugar (Glucose) (LD Carlson Company)

Yeast Nutrient (Urea and diammonium phosphate, LD Carlson Company) Yeast Energizer (diammonium phosphate, Springcell, MgS04, LD Carlson Company)

Safale US-05 Yeast (lot 38210-0846-047, Fermentis)

Type-90 Hops pellets, Columbus cultivar (Alpha acid 17.3%, lot P91 - AJUCBS0079, Yakima Chief-HopUnion)

Milli-Q water (Millipore Direct-Q 3 UV Water Purification System)

Analytical balance (Ohaus Pioneer)

Two hotplates

Sanitizer (50/50 by volume EtOH/H20)

Procedure

[0026] After the two fermentation vessels were tared, corn sugar and water were added to a target pre-boil gravity of 7.9 Brix (1 Brix = 1 g sugar + 99 g water). Final weights were 56.60 g sugar in 700.3 g of solution (8.1 Brix) for fermentation vessel 1 (“FV1”) and 54.98 g sugar in 701 .64 g of solution (7.8 Brix) for fermentation vessel 2 (“FV2”). The two vessels were brought to a boil and boiled for 60 minutes. To FV1 , hop pellets were added according to the following schedule: 0.391 g at start of boil, 0.187 g at 55 minutes into the boil, and 0.286 g at the end of the 60 minute boil (“flameout”). Hop pellets were not added to FV2. At 55 minutes into the boil, both vessels were dosed with yeast nutrient and energizer at the rates shown in Table 1.

[0027] Due to differing boil intensities between the two hotplates, the final weights of the wort for FV1 and FV2 differed. The final weight of the wort for FV1 was 622.6 g (initial gravity of 9.1 Brix) and FV2 was 531 .2 g (initial gravity of 10.4 Brix). These weights are not corrected for hops, energizer, or nutrient. The vessels were sealed with a sanitized top and allowed to cool to 25 °C before pitching yeast. Pitch rates were 0.2907 g yeast in FV1 and 0.2918 g yeast in FV2. Fermentation proceeded for 20 days with occasional swirling. The final density of both vessels was measured to be 0.992 g/mL via high-resolution hydrometer. To collect yeast and“beer” samples, the liquid (“beer”) was decanted until approximately 100 ml_ of volume remained.

This volume was swirled into a slurry (yeast) and emptied into a sanitized 4 oz (1 18 ml_) glass sample jar.

Sample Preparation - Homebrew Beer

Materials

5 gallons of unhopped, un-boiled wort (Bell’s Brewing, via Homebrew

Competition)

Cablecar California Common Lager yeast (Imperial Yeast)

Fresh Homegrown hops (Chinook and Cascade varietals)

Yeast Nutrient (JD Carlson)

Procedure

[0028] The 5 gallons of wort was transferred to the boil kettle and 0.5 oz of Columbus hops pellets were added ("first wort hopping"). The wort was brought to a boil and boiled for a total of 60 minutes. At 45 minutes into the boil, 1 .5 tbsp of yeast nutrient and 1 .5 oz of fresh Chinook hop cones were added. At 50 minutes into the boil a stainless steel immersion chiller was placed in the kettle to sanitize. At 55 minutes into the boil 1 .5 oz of fresh Chinook and 1 .5 oz of fresh Cascade hop cones were added. Upon completion of the 60 minute boil, 2 oz of fresh Cascade hop cones were added and the burner was turned off. Tap water was run through the immersion chiller for 1 hour to bring the wort to 76 °F. The wort was transferred to a plastic carboy. Using a scale, 7 lbs. of water were added to account for excessive evaporation and absorption losses to bring the initial gravity to 1 .060 g/mL. The package of liquid yeast ("Cablecar" strain of California Common Lager yeast,

Imperial Yeast) was introduced into the carboy and a 3-piece airlock installed.

[0029] No appreciable lag phase in fermentation was observed, and the fermentation proceeded vigorously enough to fill the airlock with yeast and wort during day 1 of fermentation. The yeast and partially fermented wort from the airlock was collected in a sanitized glass vessel and submitted for mass spectral analysis ("blow off" sample). A blow off tube was fitted to the center portion of the airlock and routed into a bowl of sanitizer (Star-san) for the duration of primary fermentation.

[0030] After 4 days of primary fermentation, 1 .25 oz of fresh Cascade hop cones were added to the fermenter. After 8 days of primary fermentation, the beer was racked into a glass carboy for secondary fermentation, gravity at this point was 1 .012 g/mL. After 3 days of secondary fermentation the beer was racked into a 5 gallon ball-lock“corny” style keg for carbonation and conditioning. A yeast slurry was collected and submitted for mass spectral analysis (“final” sample). The final gravity of the beer was 1 .009 g/mL, indicating that a small amount of fermentation took place during the 3 day secondary fermentation.

Preparation of Samples for Analysis

Beer Samples

[0031 ] The beer samples were filtered through 0.45 pm PTFE syringe filters prior to injection.

Yeast Samples

[0032] Approximately 1 g of each of the yeast samples was weighed into individual 30 mL Omni Bead Ruptor tubes and 10.0 mL of 1 % formic acid in acetonitrile was added, the samples were then homogenized on the Omni Bead Ruptor for 30 seconds at 3.55 m/sec. The Bead Ruptor tubes were centrifuged for 5 minutes at 4000 rpm and the supernate transferred to a scintillation vial. The supernate was dried under a stream of N2 and the residue was reconstituted with 0.5 ml_ of 1 % formic acid in acetonitrile. The vial was shaken and sonicated to ensure that all soluble material was dissolved; the resulting solution was filtered through a 0.45 pm PTFE syringe filter prior to injection.

UPLC Conditions

Instrument: Waters Acquity UPLC (binary pump)

Mobile Phase A: 50 mM ammonium acetate in 95:2.5:2.5 water:methanol:acetonitrile Mobile Phase B: 0.1 % ammonium hydroxide in methanol

Flow Rate: 0.7 mL/min

UPLC Column: Waters Acquity UPLC BEH C18 (2.1 x 100 mm, 1 .7 pm) column Column Temperature: 75 °C

Injection Volume: 1 pL

[0033] The mobile phase gradient is shown in Table 2.

Mass Spectral Detection

[0034] Mass spectrometry was used to detect low levels of Rho in beer and yeast produced both in the laboratory and in homebrew samples.

co-Rho ad-Rho n-Rho

dihydro-isocohumulone dihydro-isoadhumulone dihydro-isohumulone

p-isocohumulone p-isoadhumulone p-isohumulone

Chemical Formula: C2oH₃₀0₅ Chemical Formula: C21H32O5 Chemical Formula: C21 FI32O5

Exact Mass: 350.2093 Exact Mass: 364.2250 Exact Mass: 364.2250

[0035] Mass spectral detection was performed in the multiple reaction monitoring (MRM) mode on the Waters Acquity TQD triple quadrupole mass spectrometer; this is the most sensitive and selective mode of mass spectral detection. The selectivity is achieved by double filtering of the species of interest prior to detection; in the first quadrupole, the molecular ion of the species of interest is selected (filtered away from all of the other ions entering the quadrupole), it is then transmitted to the second quadrupole where fragmentation occurs to form diagnostic fragment ions, these fragment ions are transmitted to the third quadrupole where only the fragment ions of interest are allowed to exit the quadrupole (filtered away from all of the other ions that entered the quadrupole with it) and enter the detector. This mode of detection results in a low noise floor due to the filtering out of unwanted ions and very selective detection of the species of interest due to the filtering which occurs in the first and third quadrupoles.

[0036] The MRM transitions monitored for the detection of Rho were: co-Rho: m/z 349.2 m/z 182.0

m/z 349.2 m/z 233.1

m/z 349.2 m/z 251 .1 n/ad-Rho: m/z 363.2 m/z 196.0

m/z 363.2 m/z 247.1

m/z 363.2 m/z 265.1

[0037] The source of these fragment ions is shown in Figure 1 .

[0038] Figure 2 shows the MRM chromatogram recorded for a Rho standard; the peak pattern can be used as a fingerprint for the identification of the presence of Rho in other samples.

[0039] Figures 3 through 10 show the MRM chromatograms recorded for the beer and yeast samples described above. The mass spectral responses are labeled in each chromatogram. As can be seen, all samples containing both hops and yeast have responses consistent with the presence of Rho while the two samples that contain yeast but no hops, FV2 "beer" and FV2 yeast, do not have responses consistent with the presence of Rho.

[0040] These data demonstrate that under normal fermentation conditions typical of a moderately hopped American Pale Ale style beer, Rho is produced. Mass spectral analysis of these samples shows that the Rho produced under these fermentation conditions is consistent with Rho produced upon standard borohydride reduction of purified Iso-alpha acids. Based upon the data present herein, it is speculated that the Rho produced during fermentation is through an enzymatic reduction of Iso-alpha acids present in the wort by the yeast.

[0041 ] In order to understand the source of the yeast produced Rho, experiments will investigate, individually, the use of Alpha acids and Iso-alpha acids as the starting material; these would be added post kettle boil so as not to isomerize the Alpha acids into Iso-alpha acids before the yeast has a chance to be in the presence of the Alpha acids. This experiment will help to determine if yeast can produce Rho directly from Alpha or if it must be pre-isomerized to Iso-alpha for the yeast to perform the reduction.

EXAMPLE 2 - Enzyme Candidate Identification, Enzyme Expression and Purification

[0042] Enzymes derived from yeast which catalyze the conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids are obtained directly from brewer's yeast by means conventional in the art of protein isolation and purification.

[0043] In an embodiment, enzymes derived from yeast which catalyze the conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids are obtained by means of recombinant DNA techniques.

[0044] Yeast derived enzyme candidates which catalyze the conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids are selected after bioinformatic mining of public protein sequence databases, for example, UniProt

(http://www.uniprot.org/), Pfam (http://pfam.xfam.org/), and InterPro

(www.ebi . ac. uk/i nterpro/) to identify yeast derived enzymes selected from

reductases, isomerases, dehydrogenases, hydratases and lyases. Bioinformatics rely on BLASTP sequence alignments ( h it ps ://bl as )

[0045] Synthetic genes and/or genes capable of expressing the yeast derived enzymes are cloned into expression cassettes and/or expression vectors (e.g.

plasmids), according to known techniques in the art of molecular biology, for expression of the yeast derived enzymes in prokaryotic and/or eukaryotic cells.

[0046] Yeast derived enzymes are identified as candidates for use in the conversion of hop-derived alpha and/or isoalpha acids into dihydro-(rho)-isoalpha acids. Such yeast derived enzymes are isolated and purified.

[0047] In an embodiment a yeast derived enzyme may exhibit the amino acid sequence of SEQ ID NO:1 , the amino acid sequence is as follows: MSVFVSGANGFIAQHIVDLLLKEDYKVIGSARSQEKAENLTEAFGNNPKFSM

EVVPDISKLDAFDHVFQKHGKDIKIVLHTASPFCFDITDSERDLLIPAVNGVKG

ILHSIKKYAADSVERVVLTSSYAAVFDMAKENDKSLTFNEESWNPATWESC

QSDPVNAYCGSKKFAEKAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDK

DVKKHLNTSCELVNSLMHLSPEDKIPELFGGYIDVRDVAKAHLVAFQKRETIG

QRLIVSEARFTMQDVLDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKK

SKKLLGFKFRNLKETIDDTASQILKFEGRI (SEQ ID N0:1 )

[0048] The expression vectors (e.g. plasmids) comprising the genes encoding the yeast derived enzymes are introduced into a host bacterium, for example Escherichia coli or Bacillus, for production of the yeast derived proteins.

[0049] Moreover, in an embodiment, Pichia pastoris, a eukaryotic expression system, is used for protein production/overexpression of yeast derived enzymes.

[0050] In another embodiment, the bacuiovirus expression system utilizing insect host ceils is used for protein production/overexpression of yeast derived enzymes.

[0051] The yeast derived protein which catalyzes the conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids may be modified for increased expression ievels, for enhanced refolding and/or for boosting solubility of the expressed enzyme. Furthermore, the yeast derived protein may be modified for species specific expression by codon optimization.

[0052] What is more, the yeast derived protein which catalyzes the conversion of alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids may be modified for enhanced enzymatic activity.

EXAMPLE 3 - Enzyme Treatment of Hop-Derived Isoalpha Acids with Cofactor Recycling by Isopropanol Oxidation

[0053] In a 1 .5 mL microcentrifuge tube, 10 mg yeast derived enzyme is

resuspended in 700 uL of buffered aqueous solution (eg. Sodium Phosphate pH 7.5). 290 uL of isopropanol is added. 10 uL of alkaline isoalpha acid solution (29% isoalpha acids) is added for a final concentration of 0.29% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 48 hours. The obtained reaction mixture is filtered to remove enzyme. Isoalpha acids and dihydro- (rho)-isoalpha acids are quantified by HPLC.

EXAMPLE 4 - Enzyme Treatment of Acidified Hop Derived Isoalpha Acids with Cofactor Recycling by Isopropanol Oxidation

[0054] In a 1 .5 mL microcentrifuge tube, 10 mg yeast derived enzyme is

resuspended in 700 uL of buffered aqueous solution (eg. Sodium Phosphate pH 7.5). 290 uL of isopropanol is added. 10 uL of Acid Form Isoalpha acids solution (68.9% isoalpha acids) is added for a final concentration of 0.69% isoalpha acids.

The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 48 hours. The obtained reaction mixture is filtered to remove enzyme. Isoalpha acids and dihydro- (rho)-isoalpha acids are quantified by HPLC.

EXAMPLE 5 - Enzyme Treatment of Hop Derived Isoalpha Acids with Cofactor Recycling by Glucose Dehydrogenase

[0055] In a 1 .5 mL microcentrifuge tube, 10 mg yeast derived enzyme is

resuspended in 1000 uL of buffered aqueous solution (263 mM Sodium Phosphate pH 7.0, 1 .7 mM magnesium sulfate, 4.3 U/mL Glucose Dehydrogenase, 1 .1 mM NADP+, 1 .1 mM NAD+, 80 mM D-glucose). 5 uL of Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is filtered to remove enzyme.

EXAMPLE 6 - Enzyme Treatment of Hop Derived Isoalpha Acids without Cofactor Recycling

[0056] In a 1 .5 mL microcentrifuge tube, 10 mg yeast derived enzyme is

resuspended in 995 uL of buffered aqueous solution (250 mM sodium phosphate pH 7.5 or 100 mM Tris HCI pH 7.0; 0.4 mM NADPH). 5 uL of Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is filtered to remove enzyme. EXAMPLE 7 - Enzyme Treatment of Hop Derived Isoalpha Acids with

Immobilized Reductase via S1O2

[0057] A yeast derived enzyme is absorbed on S1O2 and crosslinked with

glutaraldehyde to yield an immobilized reductase material. Immobilized reductase is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is centrifuged at 10,000g to remove immobilized enzyme.

EXAMPLE 8 - Enzyme Treatment of Hop Derived Isoalpha Acids with

Immobilized Reductase via DEAE-Cellulose

[0058] A yeast derived enzyme is crosslinked with glutaraldehyde and absorbed onto DEAE-cellulose to yield an immobilized reductase material. Immobilized reductase is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is centrifuged at 10,000g to remove immobilized enzyme.

EXAMPLE 9 - Enzyme Treatment of Hop Derived Isoalpha Acids with

Immobilized Reductase via PEI-Treated Alumina

[0059] A yeast derived enzyme is crosslinked with glutaraldehyde and absorbed onto polyethylimine (PEI)-treated alumina to yield an immobilized reductase material. Immobilized reductase is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is centrifuged at 10,000g to remove immobilized enzyme. EXAMPLE 10 - Enzyme Treatment of Hop Derived Isoalpha Acids with Co- Immobilized Enzymes

[0060] A yeast derived enzyme and cofactor recycling enzyme, such as glucose dehydrogenase, are immobilized sequentially or together in one pot via any of the above-mentioned methods to yield a coimmobilized material. Coimmobilized material is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1.0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final

concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is centrifuged at 10,000g to remove immobilized enzyme.

EXAMPLE 11 - Enzyme Treatment of Hop Derived Isoalpha Acids with Living Cells

[0061 ] A microorganism (bacteria, fungus, yeast, insect cell) expressing the yeast derived enzyme is grown via fermentation to high density, harvested, washed, and pelleted to form cell paste. Cell paste is resuspended in fresh growth media containing 0.145% isoalpha acids. The cell culture is incubated at 25-37 °C with mixing for 24-72 hours. The cell culture is centrifuged at 10,000g to remove cells from spent growth media. Dihydro-(rho)-isoalpha acids are extracted from the spent growth media with ethanol.

EXAMPLE 12 - Enzyme Treatment of Hop Derived Isoalpha Acids with Cell Lysate

[0062] A microorganism (bacteria, fungus, yeast, insect cell) expressing the yeast derived enzyme is grown via fermentation to high density, harvested, washed, and lysed to yield a crude cell lysate. Isoalpha acids are added to the crude cell lysate to a final concentration of 0.145% isoalpha acids. The cell culture is incubated at 25-40 °C with mixing for 24 hours. The reaction mixture is centrifuged at 10,000g or filtered to remove cellular material from the lysate. Dihydro-(rho)-isoalpha acids are extracted from the clarified lysate with ethanol. EXAMPLE 13 - Enzyme Treatment of Hop Derived Isoalpha Acids with NADH Cofactor

[0063] Enzyme treatment where the NADPH cofactor is substituted with NADH.

Yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is filtered to remove enzyme.

EXAMPLE 14 - Enzyme Treatment of Hop Derived Isoalpha Acids with

Cofactor Recycling via Ethanol Oxidation

[0064] Enzyme treatment where the isopropanol starting material is substituted with ethanol. Yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1.0 mM NADH, 10- 30% ethanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is filtered to remove enzyme.

EXAMPLE 15 - Enzyme Treatment of Hop Derived Isoalpha Acids Followed by Extraction

[0065] Enzyme treatment followed by extraction to increase final concentration of dihydro-(rho)-isoalpha acids. Immobilized yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 0-20 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is filtered to remove enzyme and extracted with food-grade solvent to achieve a desired concentration of dihydro- (rho)-isoalpha acids. EXAMPLE 16 - Enzyme Treatment of Hop Derived Isoalpha Acids Followed by Thermal Inactivation

[0066] Yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1.0 mM NADH, 10- 30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is heated at 80-100 °C for 10-30 minutes to inactivate enzyme.

EXAMPLE 17 - Enzyme Treatment of Hop Derived Isoalpha Acids Followed by Chemical Inactivation

[0067] Yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1.0 mM NADH, 10- 30% ethanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. Food-grade ethanol is added to a final concentration of >50% to inactivate enzyme.

EXAMPLE 18 - Enzyme Treatment of Hop Derived Isoalpha Acids with

Immobilized Reductase Recycling

[0068] A yeast derived enzyme is crosslinked with glutaraldehyde and absorbed onto DEAE-cellulose to yield an immobilized reductase material. Immobilized yeast derived enzyme is added to a concentration of 0.1 -10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1 -1 .0 mM NADPH, 10-30% isopropanol, pH 7-9). Isolone Isomerized Hop Extract solution (29% isoalpha acids) is added for a final concentration of 0.145% isoalpha acids. The reaction is incubated at 30 °C with orbital shaking at 180 rpm for 24 hours. The obtained reaction mixture is centrifuged at 10,000g to separate immobilized reductase from the reaction solution.

Immobilized yeast derived enzyme is recovered, washed with water or aqueous buffer, and re-used in a new reaction mixture.

Claims

1. A process for converting hop-derived compounds to dihydro-(rho)-isoalpha acids comprising contacting the hop-derived compounds with isolated yeast derived enzymes and, optionally cofactors for the enzymes, in amounts effective to convert the hop-derived compounds to dihydro-(rho)-isoalpha acids, and incubating for a period of time sufficient to convert the hop-derived compounds to dihydro-(rho)-isoalpha acids.

2. A process for converting hop-derived alpha and/or isoalpha acids to dihydro- (rho)-isoalpha acids comprising contacting the hop-derived alpha and/or isoalpha acids with an isolated yeast derived enzyme, and optionally a cofactor for the enzyme, in an amount effective to convert the hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids, and incubating for a period of time sufficient to convert the hop-derived alpha and/or isoalpha acids to dihydro-(rho)-isoalpha acids.

3. The process of Claim 1 , wherein isoalpha acids are converted to dihydro- (rho)-isoalpha acids.

4. The process of Claim 1 , wherein the isolated enzymes are derived from yeast.

5. The process of Claim 4, wherein the isolated enzymes are selected from

reductases, isomerases, dehydrogenases, hydratases and lyases.

6. The process of Claim 5, wherein the reductases, isomerases,

dehydrogenases, hydratases and lyases have been modified for enhanced activity.

7. A method of converting hop-derived alpha and/or isoalpha acids to dihydro- (rho)-isoalpha acids using yeast comprising the steps of mixing corn sugar and water in a fermentation vessel to a target pre-boil gravity of 7.9 Brix (1 Brix = 1 g sugar + 99 g water), bringing the mixture to a boil and boiling for 60 minutes, adding hop pellets, dosing with yeast nutrient and energizer, sealing the fermentation vessel and cooling to 25 °C before pitching yeast, fermenting for 20 days with occasional swirling, collecting the yeast from the fermentation liquid fraction, purifying dihydro-(rho)-isoalpha acids from the yeast and/or fermentation liquid fraction.

8. The method of Claim 7, wherein the yeast has been genetically modified to overexpress a yeast derived enzyme selected from reductases, isomerases, dehydrogenases, hydratases and lyases.

9. The method of Claim 8, wherein the reductases, isomerases,

dehydrogenases, hydratases and lyases have been modified to provide enhanced activity.