WO2006065131A1

WO2006065131A1 - Method of improving the stability of hop extracts and hop flavoured beverages

Info

Publication number: WO2006065131A1
Application number: PCT/NL2005/050047
Authority: WO
Inventors: Erica Georgina Wilson; Alfi Khatib; Hai-Rong Zhang; Robert Verpoorte
Original assignee: Universiteit Leiden, Faculteit Van Wiskunde En Natuurwetenschappen
Priority date: 2004-11-22
Filing date: 2005-11-22
Publication date: 2006-06-22

Abstract

The present invention relates to a method of improving the stability of hop extracts and hop flavoured beverages as well as to isomerised hop extracts and hop flavoured beverages that exhibit improved stability. More particularly, the present invention relates to hop flavoured beverages containing iso-α-acids in a cis/trans ratio of at least 19:1 or containing added β-cyclodextrin. Furthermore the invention concerns an isomerised hop extract containing iso-α-acids in a cis/trans ratio of at least 9:1. The invention also concerns isomerised hop extracts containing iso-α-acids in a cis/trans ratio of less than 1:1. The invention encompasses methods of manufacturing the aforementioned beverages and extracts as well as the use of β-cyclodextrin for separating cis-iso-α-acids and trans-iso-α-acids, for separating α-acids and β-acids and for enhancing the stability of trans-iso-α-acids in hop flavoured beverages.

Description

METHOD OF IMPROVING THE STABILITY OF HOP EXTRACTS AND HOP

FLAVOURED BEVERAGES

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of improving the stability of hop extracts and hop flavoured beverages as well as to isomerised hop extracts and hop flavoured beverages that exhibit improved stability.

BACKGROUND OF THE INVENTION

Hop plants belong to the Cannabinaceae family. Hop materials used in the production of beer and other brewed beverages are primarily derived from a plant in the genus Humulus known as Humulus lupulus L. The key flavouring ingredients obtained from hop plants reside within cone- like structures which are harvested and used in manufacturing brewed beverages. The primary flavouring ingredients in hop cones involve materials known as "α-acids" or "humulones". Hop cones are subjected to various extraction technologies, such as solvent extraction and supercritical extraction, to produce hop resins. These hop resins are widely used as a flavour imparting ingredient in the brewing industry and mainly consist of the aforementioned α-acids and the structurally related, but flavour- wise less interesting β-acids. The chemical structures of α-acids and β-acids can be depicted as follows:

α-acids p-acids

R = -CHCH₃CH₃ Coliumulone / Colupulone

R = -CH₂CHCH₃CH₃ Humulone / Lupulone R = -CHCH₃CH₂CH₃ Adhumulone / Adlupulone

Typical examples of α-acids naturally occurring in hop plants residue include humulone, cohumulone, adhumulone, prehumulone and posthumulone. Of the total amount of α-acids present, tests have indicated that the foregoing ingredients are present in the following approximate proportions (% by weight): humulone (35-70%), coliumulone (20-65%), adhumulone (10-15%), prehumulone (1-10%), and posthumulone (1-3%).

Traditionally, hops are added to sweet wort during the boiling stage of the .. brewing process. This facilitates the extraction of hop resins such as the aforementioned α-acids (humulones) which are subsequently isomerised to "iso-α- acids" which are much more soluble and bitter than the original compounds. The term "iso-α-acids" encompasses the cis and trans isomerised forms of the humulones. A few typical examples of cis- and trans-iso-α-acids are present below:

CIS trans R = CH₂CH(CH₃)₂ : cis- and trans-isohumulone

R - CH(CH₃)CH₂CH₃ : cis- and trans-isoadhumulone R - CH(CH₃)₂ : cis- and trans-isocohumulone

The aforementioned iso-α-acids or isohumulones are largely responsible for the characteristic bitterness associated with beer. The extraction and isomerisation of humulones in boiling wort, however, are inefficient and typically only about 30% of these compounds are utilised in the brewing process. It has long been established that isomerisation of humulones can take place more efficiently outside the brewing process. According to the prior art, there are two main methods of isomerising humulones either by action of heat on aqueous solutions of potassium or sodium humulate or so called solid state isomerisation using alkaline or earth alkaline metallic isomerisation materials. Typically, the isomerised hop extracts so obtained contain at least 10 wt.% iso-α-acids by weight of the combined amount of humulones and iso-humulones.

Hop bittering substances, especially iso-α-acids, are subject to alterations during the aging of beer. Iso-α-acids degrade in the absence of light and in the presence of light. It is widely accepted that iso-α-acids are the prime source of so called "sunstruck" flavour formation in beer. This flavour defect develops particularly rapidly in beer that is being exposed to UV-light. 3-Methyl-2-butene-l -thiol (MBT), an important degradation product of iso-α-acids, has been identified as the substance that is largely responsible for this highly undesirable off-flavour.

In a publication by Walters et al. (J. Am. Soc. Brew. Chem. (1997) 55(3); 91- 98) it is concluded that trans-iso-α-acids are much more prone to degradation than are cis-iso-α-acids. Hence, in order to improve the stability of isomerised hop extracts and hop flavoured beverages, it would be desirable to selectively remove trans-iso-α-acids from such extracts and/or to protect these trans-isomers from degradation.

SUMMARY OF THE INVENTION

The inventors have discovered that the aforementioned objectives may be realised by treating isomerised hop extracts or hop flavoured beverages with β- cyclodextrin. More particularly, the inventors have found that is feasible to selectively remove trans-iso-α-acids from a solution containing cis- and trans-iso-α-acids by contacting such a solution with β-cyclodextrin and removing a solution containing a significantly reduced level of trans-iso-α-acids relative to cis-iso-α-acids. Provided a suitable solvent system is employed, β-cyclodextrin will exhibit a surprisingly high affinity for trans-iso-α-acids and a much lower affinity for cis-iso-α-acids. Thus, β- cyclodextrin may advantageously be employed to complex trans-iso-α-acids present in a solution, following which the complex of β-cyclodextrin and trans-iso-α-acids may be separated from the solution. Thus, the concentration of trans-iso-α-acids in the starting solution can be lowered effectively whilst retaining most of the cis-iso-α-acids originally contained in the solution. In other words, the present invention makes it possible to increase the cis/trans ratio of products, such as isomerised hop extracts and hop flavoured beverages, that contain appreciable amount of cis-iso-α-acids and trans- iso-α-acids.

Following separation of the β-cyclodextrin/trans-iso-α-acid complex from the solution, the entrained trans-iso-α-acids may be released from the β-cyclodextrin, e.g. by contacting the complex with a suitable organic solvent. The released trans-iso-α- acid may advantageously be used as a starting material for the production of reduced iso-α-acids. In addition, trans-iso-α-acids may suitably be employed as antibacterial agents, e.g. in food applications (J. App. Bacteriol (1992) 72(4), 327-324). Another embodiment of the present invention relates to the use of β- cyclodextrin as a stabilising agent in hop flavoured beverages that contain significant levels of trans-iso-α-acids. In accordance with this embodiment, β-cyclodextrin is added to a hop flavoured beverage or to an isomerised hop extract that is intended for use in such a beverage. In the hop flavoured beverage the β-cyclodextrin will form a complex with the trans-iso-α-acids that is much more stable than the trans-iso-α-acids per se. Thus, the stability of the hop flavoured beverage may be enhanced significantly.

Finally, the inventors have found that cyclodextrins can advantageously be used to selectively remove α-acids from a solution containing α- as well as β-acids by contacting such a solution with α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or combinations thereof, allowing the acids to form complexes with the cyclodextrin and selectively removing the α-acids from the cyclodextrin complex. Unlike α-acids, β- acids as such are not bitter, β-acids are prone to oxidation during storage resulting in a typical oxidised β-acid bitterness. The development of such undesired bitterness may be prevented or at least minimised by selectively removing α-acids from hop extracts and applying the isolated α-acids. Furthermore, the remaining residue, which is enriched in β-acids, may advantageously be utilised as a starting material for the production of e.g. tetrahydro-α-acids, which substances are light stable and may be used to improve foam quality, as well as the cling and mouth feel of beer, hi addition, β-acids may advantageously be employed as antibacterial agents against dangerous pathogens such as Clostridium botulinum, Clostridium difficile, and Helicobacter pylori as described in US 6,251,461.

DEFINITIONS

The term "α-acids" as used herein refers to humulone, cohumulone, adhumulone, prehumulone, posthumulone and to combinations of these humulones. The term "β-acids" as used herein refers to lupulone, colupulone, adlupulone and combinations of these lupulones.

The term "iso-α-acids" as used herein refers to isohumulone, isocohumulone, isoadhumulone, isoprehumulone, isoposthumulone and to combinations of these isohumulones. Unless indicated otherwise, the term iso-α-acids encompasses both the cis- and trans isomerised forms of the humulones mentioned above. The term "hop derived acids" as used herein encompasses the group of acids consisting of α-acids, β-acids, iso-α-acids, reduced iso-α-acids and combinations thereof. The term "β-cyclodextrin" as used herein refers to a cyclic macromolecule composed of 7 D(+)-glucose residues bonded through α-(l-4) glycosidic linkage (cycloheptaamylo se) .

The term "hop extract" as used herein refers to any composition containing appreciable amounts, e.g. at least 3% by weight of dry matter, of components, especially hop derived acids, that have been isolated from hops. Hop extracts according to the present invention may obtained from hops in many different ways well-known in the art, such as solvent extraction, supercritical extraction, (steam) distillation, pressing etc. The term "isomerised hop extract" as used herein refers to a hop extract in which the ratio iso-α-acids to α-acids exceeds 5%.

The term "hop flavoured beverage" as used herein refers to a beverage containing at least 2 mg/kg hop derived acids.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a hop flavoured beverage containing at least 1 mg/kg iso-α-acids in, said iso-α-acids being present in an elevated cis/trans ratio of at least 19:1. Such a hop flavoured beverage containing iso-α-acids in a cis/trans ratio that is significantly higher than the cis/trans ratio encountered in hop flavoured beverages of the prior art can be obtained by using β-cyclodextrin to selectively remove trans- iso-α-acids from the hop flavoured beverage or, alternatively, from the isomerised hop extract employed in the manufacture of such a beverage. Consequently, the present invention also encompasses an isomerised hop extract containing at least 1% iso-α-acids, calculated on the total amount of hop derived acids contained in the extract, said iso-α-acids being present in a cis/trans ratio of at least 9:1, preferably of at least 14:1, more preferably of at least 19:1. Preferably, the hop flavoured beverages and isomerised hop extracts according to the present invention contain detectable amounts of isohumulone, isocohumulone, isoadhumulone.

The present invention also encompasses isomerised hop extracts containing iso-α-acids in a cis/trans ratio that is significantly lower than the cis/trans ratio encountered in conventional extracts. As explained herein before, such extracts may suitably be used as a starting material in the production of e.g. tetrahydro-α-acids or as antibacterial agents. Accordingly, a further aspect of the invention concerns an isomerised hop extract containing at least 1% iso-α-acids, calculated on the total amount of hop derived acids contained in the extract, said iso-α-acids being present in a cis/trans ratio of less than 1:1, preferably of less than 1:2, more preferably of less than 1:4.

Typically, the hop flavoured beverages and isomerised hop extracts as described above exhibit a weight ratio α-acids to β-acids exceeding 3:1, preferably exceeding 5:1. The benefits of the present invention are particularly pronounced in hop flavoured beverages containing at least 5 mg/kg, more preferably at least 10 mg/kg iso-α-acids.

Another embodiment of the invention concerns a hop flavoured beverage containing at least 1 mg/kg iso-α-acids and at least 1 mg/kg β-cyclodextrin, said β- cyclodextrin and iso-α-acids being present in a molar ratio of at least than 1:3. In accordance with this embodiment the β-cyclodextrin is incorporated into a hop flavoured beverage to act as a stabilising agent. The β-cyclodextrin may be added as such during any stage of the manufacturing process or, alternatively, it may be combined with another product ingredient, e.g. an isomerised hop extract, prior to application in the beverage.

The incorporation of β-cyclodextrin in an isomerised hop extract may improve the stability of the hop extract and provides convenience. Hence, another embodiment of the invention relates to an isomerised hop extract containing at least 1% iso-α- acids, calculated on the total amount of hop derived acids contained in the extract, and at least 4 g/kg β-cyclodextrin, said β-cyclodextrin and iso-α-acids being present in a molar ratio of at least than 1:3. Typically, the total amount of hop acids contained in the isomerised extract according to the present invention exceeds 5% by weight of dry matter, preferably it exceeds 20% by weight of dry matter.

The benefits of the present invention may be realised in any hop flavoured beverage. Most preferably, the hop flavoured beverage of the present invention is beer. Another aspect of the present invention relates to the use of β-cyclodextrin for separating cis-iso -α-acids and trans-iso-α-acids. This particular use of β-cyclodextrin typically involves addition of β-cyclodextrin to a solution containing cis-iso-α-acids and trans-iso-α-acids, followed by separating a solution containing a substantially decreased level of trans-iso-α-acids relative to cis-iso-α-acids. Typically, the cis/trans ratio of the iso-α-acids contained in the solution is increased by at least a factor 3, preferably by at least a factor 5 as a result of the contacting with β-cyclodextrin.

A further aspect of the invention concerns the use of cyclodextrin for separating α-acids and β-acids. Typically, such use involves contacting a solution containing α-acids and β-acids with α-cyclodextrin, β-cyclodextrin γ-cyclodextrin or combinations thereof, allowing the cyclodextrin to form complexes with the α-acids and β-acids, isolating the cyclodextrin complexes and selectively removing the α- acids from the isolated cyclodextrin complexes by solvent addition. Typically, the α/β ratio in the removed α-acid fraction is increased by at least a factor 3, preferably by at least a factor 5 relative to the starting solution.

Yet another aspect of the invention relates to the use of β-cyclodextrin for enhancing the stability of trans-iso-α-acids in hop flavoured beverages. Preferably, such application of β-cyclodextrin comprises incorporating β-cyclodextrin in the beverage in a concentration of at least at least 1 mg/kg, more preferably of at least 2 mg/kg and most preferably of at least 5 mg/kg.

The present invention also concerns a method of separating cis-iso-α-acids and trans-iso-α-acids, said method comprising contacting a starting solution containing cis-iso-α-acids and trans-iso-α-acids with β-cyclodextrin and removing a solution in which the ratio of cis-iso-α-acids to trans-iso-α-acids is a least twice as high as in the starting solution. Preferably, the present method comprises removing a solution in which the ratio of cis-iso-α-acids to trans-iso-α-acids is a least thrice, preferably at least five times as high as in the starting solution.

The benefits of the present invention are particularly pronounced when β- cyclodextrin is used to selectively remove trans-iso-α-acids from isomerised hop extracts having a weight ratio α-acids to β-acids exceeding 3:1, preferably exceeding 5:1.

The solvent employed in the present method should be capable of dissolving adequate amounts of the cis-iso-α-acids and trans-iso-α-acids. Typically, each of cis- iso-α-acids and trans-iso-α-acids should exhibit a solubility in said solvent at the temperature at which the method is conducted of at least at least 0.1 wt.%, preferably of at least 0.5% wt.% and most preferably at least 1 wt.%. Furthermore, the solvent should favour the formation of trans-iso-α-acid/β-cyclodextrin complexes. Suitable solvents include water, ethanol and mixtures thereof. If the present method is conducted as a precipitation method, it is important that also the β-cyclodextrin dissolves well in the solvent employed in the starting solution. Typically, the β- cyclodextrin should exhibit a solubility of at least 0.1 wt%, preferably at least 0.5 wt.% and more preferably at least 1 wt.% in said solvent.

In a particularly preferred embodiment of the invention, the starting solution is a solution of isomerised hop extract in a polar solvent selected from the group consisting of water, ethanol and mixtures thereof. An example of such a starting solution is a hop flavoured beverage that largely consist of water and/or ethanol. Preferably, the starting solution is a relatively concentrated solution containing at least 0.5 wt.%, more preferably at least 1 wt.% of the combination of iso- and trans-iso-α- acids.

Another embodiment of the present invention relates to a method of separating α-acids and β-acids, said method comprising contacting a starting solution containing α-acids and β-acids with α-cycodextrin, β-cyclodextrin, γ-cyclodextrin or a combination of these cyclodextrins, allowing the cyclodextrin to form complexes with the α-acids and β-acids, isolating the cyclodextrin complexes and selectively removing the α-acids from the isolated cyclodextrin complexes by solvent addition. Preferably the the ratio of α-acids to β-acids in the removed fraction is at least three times, more preferably at least five times as high as in the starting solution.

Typically, each of α-acids and β-acids should exhibit a solubility in the starting solution at the temperature at which the method is conducted of at least at least 0.1 wt.%, preferably of at least 0.5% wt.% and most preferably at least 1 wt.%. Suitable solvents for the starting solution include water, ethanol, methanol, acetone and mixtures thereof. If the present method is conducted as a precipitation method, it is important that also the cyclodextrin dissolves well in the solvent employed in the starting solution. Typically, the cyclodextrin should exhibit a solubility of at least 0.1 wt%, preferably at least 0.5 wt.% and more preferably at least 1 wt.% in said solvent. In the aforementioned method the combination of α- and β-acids is typically contained in the starting solution in a concentration of at least 0.1 wt.%, preferably of at least 0.5 wt.% and more preferably of at least 1.0 wt.%. The solvent employed to selectively remove the α-acids from the isolated cyclodextrin complexes in accordance with the embodiment described above, preferably is a mixture of water and a C₂-C₄ alcohol, more preferably a mixture of ethanol and water, especially a mixture containing ethanol and water in a weight ration within the range of 1:5 to 5:1. In a particularly preferred embodiment of the methods described above, the starting solution is a solution of a hop extract in a polar solvent selected from the group consisting of water, ethanol and mixtures thereof. The hop extract employed in the present method may be an isomerised or a non-isomerised hop extract. An example of such a starting solution is a hop flavoured beverage that largely consists of water and/or ethanol.

The separation methods described above may be practised in different ways, e.g. as precipitation methods, adsorption methods or chromatographic methods. In precipitation methods the iso-acids and β-cyclodextrin are dissolved in the starting solution following which conditions are employed that favour precipitation of the trans-iso-α-acid/β-cyclodextrin complex or the β-acid/cyclodextrin and α- acid/cyclodextrin complexes. The precipitate may be removed from the supernatant by means of techniques well-known in the art, such as decanting, filtration, centrifugation etc. In absorption methods, the cyclodextrin is employed in undissolved form, preferably immobilised onto a carrier material. Typically, said carrier material will exhibit a very high surface/volume ratio so as to create a large cyclodextrin supporting surface area.

Chromatographic methods may be regarded as an adsorption method with proviso that the starting solution is passed through a stationary bed (or passed over a stationary surface) of immobilised cyclodextrin. The trans-iso-α-acids contained in the starting solution will bind to the β-cyclodextrin whilst the cis-iso-α-acids remain dissolved. Thus, the eluent obtained will contain significantly reduced levels of trans- iso-α-acids. The chromatographic method offers the advantage that it can be carried out in a semi-continuous fashion, especially if two or more columns are used interchangeably, allowing one column to be regenerated while another is in use.

Accordingly, in a particularly preferred embodiment, the starting solution is subjected to chromatographic separation using immobilised β-cyclodextrin as the stationary phase.

The separation methods according to the present invention may suitably be operated at temperatures within the range of -10 to 100⁰C. Preferably, the present methods are operated at temperatures between 0 and 65 ⁰C, more preferably at temperatures between 10 and 55 ⁰C.

Finally, the present invention concerns a method of stabilising trans-iso-α- acids in a hop flavoured beverage, said method comprising incorporating into said beverage β-cyclodextrin in an amount of at least 1 mg/kg. In order to stabilise a significant fraction of the trans-iso-α-acids β-cyclodextrin should be incorporated in a molar concentration that is not much lower than the molar concentration of trans-iso- α-acids in the beverage. Preferably, β-cyclodextrin is incorporated in a molar concentration of at least 30%, more preferably of at least 50% and most preferably of at least 80% of the molar concentration in which the trans-iso-α-acids are present in the hop flavoured beverage.

The invention is further illustrated by means of the following examples.

EXAMPLES

Example 1

Preparation and isolation of pure iso-a-acids

A liquid carbon dioxide hop extract (ex Botanix, Paddock Wood, Kent, UK) was subjected to centrifugal partition chromatography using the procedure described by Hermans-Lokkerbol et al. (Preparative separation and isolation of three α bitter acids from hop, Humulus lupulus L., by centrifugal partition chromatography. J. Chromatogr. A. (1994) 664, 45-53).

The isolated α-acids (cohumulone, humulone and adhumulone) were subsequently isomerized according to the method described by Koller (Magnesium ion catalysed isomerization of humulone: a new route to pure isohumulones, J. Inst. Brew. (1969) 75, 175-179) with a small modification: MgSO₄.7H₂O ( 2.15 g.) was dissolved in 25 ml water and 30 ml methanol in a 300 ml dark bottle. This solution was heated to 7O⁰C with stirring. A solution of purified α-acids (1.8 g) in 50 ml methanol and 5.35 ml NaOH 1 N was poured slowly into the reaction dark bottle. The reaction mixture was heated to 7O⁰C for 45 minutes with continued stirring. After cooling in an ice bath, the reaction mixture was acidified with 20 ml sulphuric acid 30 % and extracted 3 times with 100 ml n-hexane. After washing the n-hexane phase 2 times with water and drying with sodium sulphate, the solvent was removed on a rotating evaporator. Separation ofcis and trans-iso-a-acids

A β-cyclodextrin solution was prepared by adding 3.17 g of β-cyclodextrin to 40 ml ethanol-water (1 :2) and heating to 5O⁰C in order to dissolve all β-cyclodextrin. The guest compound solution, which was prepared by dissolving 500 mg of the previously prepared iso-α-acids in 6.5 ml ethanol, was added drop- wise to 21 ml β- cyclodextrin solution while continually stirring and heating to 5O⁰C during 30 minutes. The mixture was stored at 4⁰C for several days, the β-cyclodextrin inclusion complex precipitated as a white-yellow crystalline powder. The precipitate was filtered through a Bϋchner funnel and then washed several times with 50 ml EtOH:H₂O (1:2) and n-hexane.

In order to release trans- iso-α-acid from the β-cyclodextrin complex, the precipitates were eluted with different organic solvents, i.e. ethanol, ethyl acetate, dichloromethane, n-heptane, n-hexane and methanol using 50 ml of each solvent 2 times. Each supernatant was collected in a separate Erlenmeyer. Non ethanol/methano I/water supernatants were evaporated using rotary evaporator and dissolved in 100 ml ethanol. AU of the supernatants were subsequently analysed by HPLC. The eluent system used in the HPLC analysis was a gradient elution consisting of two solvent systems, system A and system B. System A was a mixture of water- phosphoric acid (100:1), and system B was a mixture of acetonitrile- water-phosphoric acid (81:19:1). The gradient was created by gradually increasing the percentage of system B (85% to 91%) relative to system A during 10 minutes with a flow rate of 1.0 ml/min. After 10 minutes elution, the system returned to 85% system B for column equilibration during 5 minutes. The total analysis for 1 measurement was 15 minutes. An iso-α-acid mix was used as a reference mix.

The supernatants that were found to contain essentially pure trans- or cis-iso- α-acid were pooled. As much as 50 ml water was added to each 50 ml pooled supernatant and followed by addition of HCl 6M to reach pH 1 while continuously being stirred. The acidified supernatant was extracted 2 times with 100 ml n-hexane using a separatory funnel. The n-hexane phase was washed 2 times with water in separatory funnel in order to remove all dissolved β-cyclodextrin and HCl. The excess water in n-hexane phase was removed by sodium sulfate and evaporated on rotary evaporator. The concentrates were dissolved in ethanol and stored in a dark bottle under -2O⁰C.

Affinity ofoc-.β-, and γ-cyclodextrins for cis- and trans-iso-a-acids

Table 1 shows that the ability of α-.β-, and γ-cyclodextrins to bind the isolated trans-iso-α-acids is very different. The results show that unlike α- and γ-cyclodextrin, β-cyclodextrin has a high affinity for trans-iso-α-acids. Since, under identical conditions the affinity of β-cyclodextrin for cis-iso-oc-acids is considerably lower, β- cyclodextrin can advantageously be employed to selectively complex trans-iso-α- acids in a solution that contains both cis- and trans-iso-α-acids.

Table 1. Ability of α-.β-, and γ-cyclodextrins to bind cis- and trans-iso-α-acids

^a' % binding was derived from HPLC analysis on concentration of trans- or cis-iso-α- acids in starting solution and supernatant. Thus % binding = 100- (area after treatment/area before treatment)* 100%

It can be concluded from the above results that β-cyclodextrin can also be used to selectively bind trans-iso-α-acids in a solution containing cis- and trans-isomers of isocohumulone, isohumulone and isoadhumulone. Rate of complexation

Table 2 shows a graph depicting the ratio of the concentrations in which trans- and cis-iso-α-acids were found in the supernatant as a function of time (precipitation at 4⁰C).

Table 2. Ratio of trans- to cis-iso-α-acids in the supernatant during precipitation

^a analysed using HPLC, % trans = (area of trans/area of cis)* 100%

Recovery of trαns-iso-α-αcids

The binding of guest molecules to cyclodextrins is a dynamic equilibrium. Thus, guest molecules can be released by decreasing the stability of the complex. There are four industrial methods of releasing guest molecules from inclusion complexes: heating, acid resolution, hydrolytic enzyme decomposition, and extraction with organic solvent. The latter method was applied in the following experiment.

After repeatedly washing the precipitates with the same solvent used in inclusion experiments (ethanol/water, 1 :2), the inclusion complexes were eluted with several organic solvents, i.e.: ethanol, ethyl acetate, dichloromethane, n-heptane, n- hexane and methanol (figure 2). It was found that methanol showed the highest potency to release trans-iso-α-acid from the complex. Ethanol showed a potency that was much lower than that of methanol but significantly higher than those of the non- polar solvents.

1 2 3 4 5 6 1 2 3 4 5 6

Trans Isocohumulone Trans Isohumulone

1 2 3 4 5 6

Trans-isoadhumulone

Figure 2. Release of trans-iso-α-acids from β-cyclodextrin complex with organic solvents : ethanol (1), ethyl acetate (2), dichloromethane (3), 11-heptane (4), n-hexane (5) and methanol (6).

The purity of the isolated trans-iso-α-acids fractions obtained by elution with methanol was checked by means of HPLC. It was found that all of the isolated trans- iso-α-acid fractions had a purity above 95.0%. The impurities observed are believed to originate from degradation reactions that occur during the inclusion process and from the ability of β-cyclodextrin to also bind cis-iso-α-acids. Example 2

Procedure for Separation

Solutions (70 mM) of α-, β- and γ-cyclodextrin were prepared in water, ethanol-water (1 :2) and acetone-water by heating the solvent to 5O⁰C and stirring until all cyclodextrin was dissolved. The acetone/water ratios employed were 1 :2, 1 : 14 and 1:6 respectively for -α-, -β- and γ-cyclodextrin. A hop extract solution (7.5 %, w/v, in ethanol or acetone) was added drop-wise to the cyclodextrin solutions at 5O⁰C and continuous stirring during 30 minutes. The ratio of hop extract solution/cyclodextrin solution in the resulting mixture was 1:3.

The mixture was stored at 4⁰C for 2 days. During storage, the cyclodextrin inclusion complex precipitated as a white-yellow crystalline powder. The precipitate was filtered through a Buchner funnel and then washed several times with n-hexane. In order to release the guest compound from cyclodextrin complex, the precipitate was extracted with water, ethanol-water (1:2), ethanol, methanol, acetone-water (1:2) and acetone. Supernatants were directly injected to HPLC, except acetone containing samples which were first dried and dissolved in methanol prior to injection.

Chromatography The chromatographic system used was a HPLC consisting of a pump type 626

(Waters) with pump controller type 600 S (Waters), an auto sampler, type 717 plus (Waters), a photodiode array detector type 2996 (Waters). The column used was Hypersil 5 Cl 8, 250 x 4.6 mm (Phenomenex, Torrance, CA, USA). Mobile phases were filtered over a 0.2 μm hydrophilic polypropylene membrane filters 47 mm type GH Polypro ( Pall Corporation, Michigan, USA) and degassed by means of helium.

The HPLC eluent for isochratic elution consisted of 0.05 M triethanolamine in methanol-water (65:35, v/v), pH brought to 6.0 with H₃PO₄; and flow rate 1.1 ml/min. The total analysis for 1 measurement was 35 minutes.

Complexation using water

Figure 3 shows the results of the HPLC analysis of the supernatants obtained after complexation with the aqueous cyclodextrin solutions. Also shown are the chromatograms obtained after the isolated cyclodextrin complexes had been extracted with an ethanol-water (1 :2) mixture. The results show that the ethanol-water (1 :2) mixture selectivity removes α-acids from cyclodextrin complexes. Furthermore, it was found that the resulting extract was enriched with cohumulone in comparison to the starting hop extract. The results depicted in Table 3 show that the ethano I/water extracts of the cyclodextrin complexes were enriched in cohumulone relatively to the starting hop extract.

Figure 3:

Figure 3. HPLC chromatogram of starting hop extract, supernatant (A) and separated extract (B) washed out from hop-cyclodextrin complexes using ethanol-water 1:2. HPLC chromatogram identified cohumulone (1), humulone (2), adhumulone (3), colupulone (4), lupulone (5) and adlupulone (6).

Table 3: Percentage of α-acids in starting hop extract and ethano I/water extracts obtained from different cyclodextrin complexes):

Complexation using ethanol-water (1:2)

As compared to complexation in water, this method yields a higher concentration of β-acids in the supernatant as is evident from the comparison of Figures 4 and 3.

Different from water method, methanol extract of ethanol-water method is free from β-acids and interestingly the extract is enriched with humulone compared to starting hop extract as shown in Table 4.

Figure 4:

Starting hop extract

α-CD β-CD γ-CD

Figure 4. HPLC chromatogram of starting hop extract, supernatant (A) and separated extract washed out from hop-cyclodextrin complexes using ethanol-water 1:2 (B) or methanol (C). HPLC chromatogram identified cohumulone (1), humulone (2), adhumulone (3), colupulone (4), lupulone (5) and adlupulone (6). Table 4: Percentage of α-acids in starting hop extract and extracts obtained from different cyclodextrin com lexes:

Complexation using acetone-water

The ratio of acetone/water was adjusted so as to ensure that the different cyclodextrins were fully dissolved. The ratios employed were 1:2, 1:14 and 1:6 respectively for -α-, -β- and -γ- CD. As is evident from Figure 5 and Table 5, the results obtained with complexation in acetone- water mixtures were similar to those obtained with complexation in water.

Figure 5:

Starting Hop Extract

Figure 5. HPLC chromatogram of starting hop extract, supernatant (A) and separated extract (B) washed out from hop-cyclodextrin complexes using ethanol-water 1:2. HPLC chromatogram identified cohumulone (1), humulone (2), adhumulone (3), colupulone (4), lupulone (5) and adlupulone (6). Table 5: Percentage of α-acids in starting hop extract and ethanol/water extracts obtained from different cyclodextrin complexes):

Example 3

Materials

AU organic solvents used were purchased from Biosolve Co. Ltd

(Valkenswaard, The Netherlands). Ortho-phosphoric acid 85 % was obtained from

Merck (Darmstadt, Germany), β-cyclodextrin Cavamax ^® W7 Pharma was purchased from Wacker-Chemie Co. Ltd, (Burghausen, Germany) and dimethylsulfoxide-d₆

(99.9%) were purchased from Cambridge Isotope Laboratories Inc. (Miami, FL,

USA).

Isomerized CO₂ hop extract was used. This extract is an aqueous solution of potassium salts of iso-α-acids. It contains 1.9, 3.3, 2.0, 6.8, 0.9, and 1.7 % (w/v) of trans-isocohumulone, cis-isocohumulone, trans-isohumulone,cis-isohumulone, trans- isoadhumulone and cis-isoadhumulone respectively.

Procedure for Inclusion

A β-cyclodextrin solution was prepared by dissolving 1 gram of β-CD in 13 ml ethanol- water (1 :2) and heating the solution in a shaker water bath to a temperature of 5O⁰C. The isomerized CO₂ hop extract was added to the β-CD solution in a molar ratio of 1:1 (molar ratio being calculated on the basis of the total amount of iso-α-acids), using an addition rate of 0.1 ml/min while continually shaking and keeping the temperature constant. The mixture was stored at 4⁰C for several days, and the β-CD inclusion complex precipitated as a white-yellow crystalline powder. The first supernatant was collected after filtering through a Buchner funnel and the precipitate was washed several times with EtOH:H₂O (1:2) and ethyl acetate. Next, the precipitate was mixed with methanol, vortexed and filtered through 0.20 μm filter. The composition of the supernatant was analysed directly by HPLC. HPLC analysis

The HPLC system used consisted of a pump type 626 (Waters) with pump controller type 600 S (Waters), an autosampler, type 717 plus (Waters), a photodiode array detector type 2996 (Waters). Column was a Hypersil 5 Cl 8, 250 x 4.6 mm (Phenomenex, Torrance, CA, USA). Mobile phases were filtered over a 0.2 μm hydrophilic polypropylene membrane filters 47 mm type GH Polypro ( Pall Corporation, Michigan, USA) and degassed by means of helium. An isocratic HPLC system was used and baseline separation of all 6 isomers was achieved with a total run time of 25 min. The mobile phase contained acetonitrile- water- H₃PO₄ (50:50:0.01, v/v/v) and was used with flow rate 1.5 ml/min. Pure iso-α-acids were used as an external standard.

Results and discussion

The iso-α-acids composition of the precipitate and supernatant as determined by means of HPLC is depicted in Table 6.

² Recovery is percentage of the weight of each iso-α-acid obtained after separation compared to the weight of each iso-α-acid in original sample

³ not evaluated

Example 4 The procedure of example 3 was repeated, except that the β-CD solution was prepared by dissolving 1 gram of β-CD in different quantities of water (2, 4, 8, 13 and 18 ml) and heating the solution to different temperatures (24, 50, 70 and 9O⁰C). Table 7 shows the effect of temperature and the volume of solvent (V₈) for the purity and recovery of cis-iso-α-acids, using only water as the solvent.

Table 7

Measured by HPLC, which purity is percentage of CI peak area to (CI+TI) peak area

Example 5 The procedure of example 3 was repeated, except that the molar ratio of sample to β-CD was varied (1:1; 2:1; 3:1; 4:1; 5:1 and 6:1). The results obtained are presented in Table 8. Table 8

These results show that by increasing the molar ratio of sample to β-CD increases the purity of the TI- β-CD precipitate, but at the same time reduces the purity of CI supernatant.

Example 6

As shown in example 3, after precipitation, a considerable amount of β-CD remained in the supernatant. In order to remove the β-CD, a supernatant containing iso-α-acids in combination with an appreciable amount of β-CD was subjected to ultra filtration (UF). A stirred ultra filtration (UF) cell model 8400 (Amicon, USA) was used together with a regenerated cellulose YMl UF membrane disc (cut-off 1000Da) with a diameter of 76 mm (Millipore, USA). The membrane disc was cleaned to remove sodium azide by floating it with its glossy side down in a beaker with distilled water for one hour, the water was changed three times. The membrane disc was then mounted in an UF cell-glossy side toward solution, and rinsed by filtering distilled water for 5 min using N₂ gas at 55 psi while continuously stirring. The water was then replaced with the sample and the filtering process was done under 4°C and stopped until 90% of the sample volume passed the membrane. This process was repeated twice using the same membrane. The purity and recovery of the supernatant sample before and after UF were checked by HPLC, and the amount of β-CD remaining in the samples was checked by ¹H NMR.

The analyses showed that the ultrafiltered supernatant was essentially free from β-CD. Furthermore, the analyses showed that the bulk of the iso-α-acids were recovered in the ultrafiltered samples.

Claims

1. A hop flavoured beverage containing at least 1 mg/kg iso-α-acids, wherein (i) the iso-α-acids are present in a cis/trans ratio of at least 19:1 and/or (ii) at least 1 mg/kg β-cyclodextrin is present, said β-cyclodextrin and iso-α-acids being present in a molar ratio of at least than 1:3 .

2. An isomerised hop extract containing at least 1% iso-α-acids, calculated on the total amount of hop derived acids contained in the extract, said iso-α-acids being present (i) in a cis/trans ratio of at least 9:1, preferably of at least 19:1 or (ii) in a cis/trans ratio of less than 1:1, preferably of less than 1:4.

3. An isomerised hop extract containing at least 1% iso-α-acids, calculated on the total amount of hop derived acids contained in the extract, and at least 4 g/kg β- cyclodextrin, said β-cyclodextrin and iso-α-acids being present in a molar ratio of at least than 1:3.

4. Use of β-cyclodextrin for separating cis-iso-α-acids and trans-iso-α-acids or for separating α-acids and β-acids.

5. Use of β-cyclodextrin for enhancing the stability of trans-iso-α-acids in hop flavoured beverages.

6. A method of separating cis-iso-α-acids and trans-iso-α-acids, said method comprising contacting a starting solution containing cis-iso-α-acids and trans-iso- α-acids with β-cyclodextrin and removing a solution in which the ratio of cis-iso- α-acids to trans-iso-α-acids is a least twice as high as in the starting solution.

7. Method according to claim 6, wherein the starting solution is a solution of isomerised hop extract in a polar solvent selected from the group consisting of water, ethanol and mixtures thereof.

8. Method according to claim 6 or 7, comprising removing a solution in which the ratio of cis-iso-α-acids to trans-iso-α-acids is a least thrice, preferably at least five times as high as in the starting solution.

9. A method of separating α-acids and β-acids, said method comprising contacting a starting solution containing α-acids and β-acids with α-cycodextrin, β- cyclodextrin, γ-cyclodextrin or a combination of these cyclodextrins, allowing the cyclodextrin to form complexes with the α-acids and β-acids, isolating the cyclodextrin complexes and selectively removing the α-acids from the isolated cyclodextrin complexes by solvent addition.

10. Method according to any one of claims 6-9, wherein the starting solution is subjected to chromatographic separation using immobilised cyclodextrin as the stationary phase.

11. Method according to any claim 9 or 10, comprising removing a solution in which the ratio of α-acids to β-acids is a least thrice, preferably at least five times as high as in the starting solution.

12. A method of stabilising trans-iso-α-acids in a hop flavoured beverage, said method comprising incorporating into said beverage β-cyclodextrin in an amount of at least 1 mg/kg.