COMPOSITION AND ITS USE

Abstract: A novel composition derivable from yeast cells comprises proteinaceous material having an isoelectric point in the range 3 to 6. The composition is prepared by a selective pH extraction method, and is useful as a flocculant in a variety of industrial applications, and in particular in the clarification of beer.

Description: COMPOSITION AND ITS USE Field of the Invention The present invention relates to a novel composition and its use in clarification processes, and in particular its use in clarifying beer. Backcrround of the Invention Beer is produced by the fermentation of wort by yeast. Wort is a hot water extract of malted barley, which, after boiling with hops, is cooled, to allow the addition of yeast. The yeast converts carbohydrate in the wort to alcohol. The yeast cells used to ferment the wort ideally clump together, a process known as fiocculation, and the majority of them sediment out towards the end of fermentation or, in the case of top cropping yeast, rise to the surface. The fermented beer including non- flocculent yeast is generally moved into a maturation vessel in which maturation takes place. After maturation the beer is usually filtered before being packaged into bottles, cans or kegs. Alternatively, unfiltered beer may be packaged in casks. A certain amount of suspended yeast is required to aid the beer maturation. At the end of maturation, however, it is desirable to have as little yeast and other insoluble material eg. high molecular weight proteins, present in the beer as possible, as an excess of such materials can cause a reduction in filtration efficiency. In addition, and most importantly for cask beer, removal of excess yeast and other insoluble material is essential if the beer is to be served bright on dispense. A large number of studies have been carried out with a view to determining the fiocculation mechanism of yeast and attempts have been made to isolate components thought to play a part in the fiocculation process, and which have been termed "flocculins". The result has been the elucidation of numerous types of different putative flocculins, none of which have been applied to a commercial flocculating process. Examples of such work include that reported in Stewart et al, Kemia-Kemi (1976) 10:465-479; Straver et ^1., Applied and Environmental Microbiology (1994) 6.0:2754-2758; Straver et al, Yeast (1994) .10:1183- 1193; Smit et al. Applied and Environmental Microbiology (1992) 11:3709-3714; Holmberg, Carlsberg Res. Commun. (1978) 43.:401-413; Shankar et ai. Microbiology (1994) ___ :1097-1101; Fernandes et aj., Biochimica et Biophysica Acta (1992) 1159:67-73; and Saito et al, Argric. Biol. Chem. (1990) 54(6) .-1425-1432. At present the clarification of beer destined for filter or cask is usually accelerated by the addition of isinglass finings which is a collagen-based fining agent produced by the acid hydrolysis of swim bladders of certain fish species. The mechanism of isinglass action is not fully understood, but it is believed that it carries a net positive charge at beer pH. Since yeast cells tend to carry a net negative charge at beer pH they clump together bridged by the isinglass macromolecule. Isinglass also removes other particulate matter which, along with the yeast, sediments. In some instances it is also known to add auxiliary finings in conjunction with isinglass. There are two main types of auxiliary finings, acidified silicates and acidic polysaccharides. It is thought that these net negatively charged molecules complex with net positively charged matter suspended in the beer, providing more negatively charged sites for the action of isinglass. Conventionally, isinglass is added to the beer in-line before the maturation vessel allowing for optimum mixing. For cask beer, the isinglass is generally added at racking when the casks are filled with beer. Any auxiliary finings are typically added before isinglass. While the correct combination of isinglass and auxiliary finings will greatly enhance filtration efficiency and in the case of cask beer produce a bright product, there are disadvantages associated with the use of isinglass. For example, any excess of isinglass added may impede downstream filtration. Excess isinglass may also carry through to the finished product where it is then detectable by chemical analysis, and, as isinglass is an animal-based product, its use is not acceptable to all consumers. Further, due to differences in beer, the performance of isinglass is not always predictable. Therefore, continual optimisation of the amount and addition rate of isinglass is necessary. Summary of the Invention According to a first aspect of the present invention, a novel composition is derivable from yeast, eg. yeast cells, and comprises proteinaceous material having an isoelectric point in the range 3 to 6, preferably 4 to 5.5, and most preferably about 5. The composition of the present invention is useful as a flocculant, and effectively fines both yeast cells and other particulate material in beer, eg. high molecular weight proteins. This results in sedimentation of the flocculated material either in the maturation vessel, where they can be drawn-off before the matured beer is filtered, or in the cask, where surprisingly good beer clarity is achieved. Further, the composition of the present invention is capable of re-fining beer, for instance when, after initial fining, dispersal of flocculated material occurs on transport, e.g. from the brewery to the public house. Again, this is particularly beneficial in the context of cask beer. In addition to its application in brewing, the composition of the present invention has widespread application as a general bioflocculant, in particular in the clarification of other alcoholic beverages eg. wine, or other fermented products, water treatment and in the removal of food and cloth dyes from water. The flocculant has been shown to be highly active in flocculating suspensions of flour, cellulose, kaolin clay and microorganisms such as streptomyces griseus, Lactobacillus , Streptococcus lactis, Bacillus subtilis, Escherichia coli and Proteus vulgaris . According to a second aspect of the present invention, a process for preparing a composition as defined above comprises the steps of: a) preparing a slurry of yeast having a pH of at least 6.5; b) removing insoluble material from the slurry, eg. by centrifugation, to leave a supernatant comprising soluble material; and c) adjusting the pH of the supernatant to be in the range 3 to 6, and collecting the precipitate. According to a third aspect of the present invention an aqueous dispersion, ie. a solution or suspension, comprises a composition of the type described above. Further aspects of the present invention include different uses of the above-described composition or dispersion thereof, including a method of clarifying beer, either for filtration or a cask beer. Description of the Invention As is apparent from the above, the composition, or flocculant/fining agent, of the present invention is made by a selective pH extraction from yeast cells. In this Application, all quoted isoelectric points are measured either by determining the range of pH at which the composition is insoluble, or using a Brookhaven Instruments Corporation Zetaplus Zeta Potential Analyser, with a flocculant concentration of 0.006% weight protein/volume and having a solution ionic strength equating to a conductance of about 150 μS, and an applied current of 1 mA at 20°C. The composition of the present invention can be extracted from brewer's yeast, and also other yeasts suitable for use in producing alcoholic beverages, such as wine and cider. It may also be extracted from baker's yeast, and possibly from a number of other yeasts. The composition of the present invention comprises primarily proteinaceous material comprising a mixture of proteins, the amount depending upon its purity. For best results it should comprise at least 50% by weight, typically at least 60% by weight protein, and most preferably at least 80 or 90% by weight protein. Making up this proteinaceous material is a relatively high proportion of hydrophobic amino acids and the amino acids asparagine/aspartic acid, glutamine/glutamic acid, leucine and lysine. While the amounts of the individual amino acids may vary from preparation to preparation, their relative proportions remain reasonably uniform. The remainder of the composition is typically made up of a number of constituents, including up to 20% by weight carbohydrate. This tends to comprise predominantly mannose and glucose, in amounts of up to 5% by weight and 20% by weight, respectively, but more typically both below 5% by weight. The constituents of the composition are of varying molecular weight, with some having a molecular weight as low as 30 kDa as characterised by ultra-filtration using a Millipore Minitan Unit. However, the majority of its constituents, and typically at least 75% by weight thereof, pass through a 300 kDa cut-off ultrafiltration plate. At present, the mechanism of action of the composition as a flocculant is not yet fully understood. However, the composition does not appear to be, or include, a lectin (a protein thought to be involved in yeast fiocculation, and which has been the subject of much study of some of the authors listed above) , as it shows no characteristics of conventional lectin-type binding mechanism such as calcium dependency and mannose sensitivity. What is believed to be key, however, is its hydrophobic nature, and, particularly in brewing, its slight positive charge and insolubility at beer pH (below pH 5) . Consequently, on addition of a soluble form of the composition to beer it precipitates. and attracts and entraps particulate material, thereby removing this from the body of the beer. One process for preparing the composition, or flocculant, of the invention involves raising the pH of a yeast slurry to a pH of at least 6.5, preferably alkaline pH eg. at least pH 9, and more preferably to pH 11 or 12, or above. Alternatively, pressed yeast may be re-suspended in an alkaline medium. The slurry can be stirred to facilitate extraction of the flocculant material from the yeast cells. Stirring can be carried out for only a short time, for example 30 seconds, or it can be carried out for longer, for example overnight where this is convenient. The time of stirring is not critical, although greater amounts of the flocculant material may be extracted with longer stirring. Stirring can be carried out at any suitable temperature, for instance from ambient temperature up to boiling or reflux at about 95°C, the fining agent of the invention being relatively heat-stable. It is generally preferred to extract at ambient temperature and to monitor extracted protein until this reaches a maximum. Optionally, before or after the adjusting the pH of the yeast slurry to be at least 6.5, the slurry can be disrupted in order to break open the yeast cells. It is preferred, however, for method simplicity and commercial reasons, not to disrupt the yeast cells. However, the yeast cell slurry may be disrupted mechanically by subjecting it to maceration, attrition or abrasion in any suitable mixer or agitator, e.g. a DYNOMILL (trade mark) , or by passage through a French Press in which the slurry is passed under pressure through a very small aperture with the result that the yeast cells are disrupted and break open, or by ultrasonic vibration. In a DYNOMILL the slurry is agitated with beads which are typically made of glass. The insoluble material is removed from the supernatant by, for example, using a centrifuge. The supernatant remaining, which is believed to contain a whole host of materials, is then adjusted to a pH in the range 3 to 6, and preferably 4 to 5.5, to precipitate the composition of the invention (or "H-Factor", as the Applicant terms it). The composition can then be subjected to one or a number of purification steps, preferably until the protein content of the composition is optimised to at least 90% by weight, and preferably greater. Each purification step comprises re¬ suspending the composition at at least pH 6.5 and then re- precipitating it by dropping the pH to 3 to 6, preferably 4 to 5.5, the closer to pH 5 the better. In order to reduce the number of purification steps, and prior to adjusting to the precipitation pH, the supernatant can be ultrafiltered to remove impurities, eg. using 30 or 100 kDa cut-off plates, preferably a 30 kDa cut-off plate. The composition may then be spray-dried to form a powder or left as an aqueous extract. However, for use it will typically be re-suspended to be in liquid form, preferably of a pH so as not to adversely affect the environment to which it is to be added, see below. While freshly cultivated yeast can be used in the process of the invention, it is preferred to use yeast that has been stored as, surprisingly, this results in a significantly-improved yield of effective flocculant. The conditions for storage of the yeast varies from yeast to yeast and from batch to batch. Typically, the yeast is stored for at least 5 days, and more typically 10 to 20 days, at a temperature of upto 65°C. Preferably, however, the yeast is stored at a temperature from 4°C to 10°C, for around 10 days. Pressed yeasts generally require less storage than yeast slurries. An alternative process for preparing the composition of the present invention comprises the steps of: a) preparing a slurry of yeast; b) adjusting the pH of the slurry to be less than 3; c) removing insoluble material from the slurry, eg. by centrifugation, to leave a supernatant comprising soluble material; and d) adjusting the pH of the supernatant to be in the range 3 to 6, and collecting the precipitate. As described above, the precipitate is then preferably subjected to one or more purification steps, by solubilising and then re-precipitating. It is the solubility profile of the composition that allows it to be extracted from the yeast cells at either pH 6.5 or higher, or at very low pH as defined above. This process is least preferred as it tends to result in lower yield than the above-described alkaline extraction. The amount of composition obtained is typically between 5-20%, often between 5 to 10%, by weight of the dry weight of yeast cells used, and varies from yeast to yeast, independent of whether the yeast is flocculent or non- flocculent. In use, the composition, or flocculant, of the present invention is added to beer, preferably fermented beer, or other alcoholic beverage, in the form of an aqueous dispersion i.e. as a solution or suspension. Preferably, the composition is added to beer as a solution, having a pH in the range 6.5 to 11, as more effective results are achieved in this form. However, solutions of other pHs can be used if so desired, but tend to have reduced fining efficiency. The amount of aqueous liquid used to re- suspend the composition is not essential. Typically, however, the composition will be added to beer as a solution containing 0.5 to 2% w/w, preferably 0.5 to 1% w/w, based on the protein content of the flocculant. The amount of composition added to clarify beer effectively is typically in the range 20 to 50 g/hl, preferably 40 to 50 g/hl (dry weight of protein/volume of beer) . The precise amount of composition necessary will depend upon the turbidity of beer or beverage to which it is to be added, and possibly also on the yeast from which it has been derived. The composition is typically added in-line on transfer of beer from vessel to vessel or vessel to cask, preferably throughout the transfer process, to ensure good dispersal thereof. The present invention is now further illustrated by the following examples. Example 1 A flocculant was prepared according to the following procedure: 1. To a 1 litre Duran flask add approximately 500 g of yeast slurry. Remove a small portion of slurry to assess % yeast dry wt. 2. To the yeast add ultrapure water to provide a total volume of approximately 1 litre. Give the flask a good shake. 3. Using demineralised water, delivered using the peristaltic pump and with the Dynomill running, rinse the Dynomill chamber. After approximately 1 litre of demineralised water has passed through the chamber run through with air to remove the internal water volume. 4. With the stopper removed from the top of the chamber and with the Dynomill turned off, fill the chamber with the prepared yeast slurry using the peristaltic pump. Once full, turn off the pump and re-stopper the chamber. Start the Dynomill, turn on the peristaltic pump and set to a flow rate of 300 ml/min. Place the outlet from the chamber in the Duran flask containing the yeast slurry, effectively recirculating the disrupted yeast slurry. Agitate the slurry using a magnetic stirrer. 5. Disrupt the yeast for 1.5 hrs. After this time remove the inlet tube from the yeast slurry and place in ultrapure water. Wash the glass beads until the volume of the yeast slurry reaches approximately 1400-2000 ml. 6. Adjust the pH of the disrupted yeast to 11-12 using 40% NaOH and leave stirring overnight on a magnetic stirrer. 7. Centrifuge the alkali slurry at 12,000 g for 30 min. 8. Using a Millipore Minitan unit, ultrafilter the supernatant through 0.45 μm cut-off plates, followed by the ultrafiltrate (<0.45 μm) through a 300 kDa cut-off plate and again the ultrafiltrate (<300 kDa) through a 100 kDa cut-off plate. The pump should be run at setting 9 and a back pressure of 6 psi maintained through the system. During all stages attempt to reduce the retentate to approximately 300 mis, thereby, maximising the overall extraction. The 300-100 kDa fraction should be reduced using the 100 kDa plates to between 150 and 300 mis and a protein content of 0.5% w/v (monitor using the Bradford Assay for protein determination, as described by Bradford, in M.M. Anal. Biochem. (1976) 7_2:248). 9. The 300-100 Kda fraction should be stored at 4°C. 10. 15 mϋ of the alkaline, 300-100 kDa fraction produced above is self-flocculated by addition to 100 m£ of 50 mM succinate buffer adjusted to pH 4 (approx. beer pH) . Once it has self-flocculated and sedimented, the fraction is removed, dialysed and freeze-dried for analysis. As a result of some fouling of the ultrafiltration plates in this preparation, the efficiency of those plates was reduced and consequently the molecular weight of some flocculant constituents was characterised as being higher than now observed. Analysis of cell extract I. Protein - Protein content was approximately 60% w/w and was composed of the amino acids shown in Table l. The amounts of amino acids quoted were after acid hydrolysis, and the analysis was performed in accordance with the method described in "Chemistry and Biochemistry of the Amino acids", (1985) pages 376-398, Chapman and Hall, by Alta Bioscience, The University of Birmingham. Asparagine and glutamine were converted to aspartic acid and glutamic acid, respectively. The values for threonine and serine have been corrected for hydrolysis losses of 5% and 10% respectively. Tryptophan usually suffers complete loss during acid hydrolysis and the recovery of cysteine is variable. Reference is now also made to the accompanying drawing, Figure 1, which shows a Radar Chart of the amino acid composition shown in Table 1. II. Carbohydrate - contained approximately 2% w/w mannose and 10% w/w glucose. c. Performance. The results in Table 2 show fining of a lager which has a rather powdery (poorly flocculent) yeast. A control was performed with rough fermented lager only and two tests, one with the addition of isinglass finings and the other with the addition of the flocculant, or fining agent, of the invention. The experiments were performed as follows: 100 ml of rough beer was placed in a 150 ml screw-cap glass bottle. 20 mg of the flocculant was resuspended in alkali (pH 9) and added to the rough beer. The bottle was inverted 10 times before its contents were poured into a 100 ml burette. 100 ml of a control beer (no finings additions) was placed in a 150 ml screw-cap bottle, inverted 10 times and poured into a 100 ml burrette. To demonstrate conventional fining, another 100 ml of rough beer was placed in a 150 ml screw-cap bottle, and 200 μl of Triple X finings (produced by Savilles Clarification) , diluted 1 in 3 to provide a working strength, was added. Again the bottle was inverted 10 times and its contents poured into a 100 ml burette. All experiments were left to fine, and sampled periodically to assess beer clarity. Fining performance was determined by measuring the absorbance of the beer at 600 nm, a measure of turbidity, against a blank of 0.45 μm filtered beer, using a Shimadzu UV-2101PC UV-VIS scanning spectrophotometer. A reduction in absorbance at 600 nm indicated the beer fined. The flocculant of the invention, as with isinglass, seemed to act on both yeast and particulate matter in the beer. Table 1 Amino Acid % w/w of Total Amino Acids Asparagine/Aspartic Acid 11.5 Threonine 5.0 Serine 5.3 Glutamine/Glutamic acid 13.7 Proline 6.4 Glycine 5.3 Alanine 6.8 Cysteine 0.5 Valine 5.5 Methionine 1.9 Isoleucine 5.0 Leucine 7.7 Tyrosine 4.2 Phenylalanine 4.7 Histidine 3.9 Lysine 7.8 Arginine 4.8 Table 2 Time (hr) A600nm Control Isinglass Invention (200μl) (20mg in alkali) 0 0.42 0.39 0.41 0.75 0.45 0.44 0.32 1.75 0.43 0.25 0.19 3 0.42 0.14 0.15 4.3 0.41 0.11 0.16 6 0.29 0.09 0.09 18 0.22 0.06 0.03 Example 2 The procedure described in Example 1 has now been optimised. A flocculant was produced on a pilot-scale using two 400 litre stainless steel vessels. The ratio of vessel height to width was approximately one and both tanks were fitted with stirrers and a pH probe. An inlet for acid and alkali was also available as well as a sample point. The centrifuge used was a Westfalia Separator (Model SA7-06- 076) , generally used for beer clarification. The time between discharges was reduced to accommodate the higher solids loading. The procedure carried out was as follows: 1. Sterilise both tanks. 2. Fill one tank with 200 litres of demineralised brewing liquor. 3. Whilst stirring the liquor, slowly add the yeast (stored for 14 days at 4°c prior to use) . 4. When the slurry is homogeneous, add demineralised brewing liquor to provide a final volume of 400 litres. 5. Continuing to stir, adjust the slurry to pH ll using 5N sodium hydroxide, delivering the alkali via an inlet port using a peristaltic pump. 6. At fifteen minute intervals, check the slurry pH and adjust as necessary. 7. Periodically sample the slurry, centrifuge the sample at 12,000 g for 30 minutes and determining the protein content using the Bradford Assay described in Example 1. Continue to monitor the protein content until a maximum is reached. 8. Centrifuge the slurry ensuring that the centrifuge and all connecting hoses have been sterilised. 9. Whilst stirring, adjust the centrate to pH 5 using 5N hydrochloric acid. 10. Switch off the stirrer and allow the precipitating material to sediment. Once sedimented, remove the liquid above the sediment using a barrel pump. 11. Whilst stirring, fill the tank to the original level with demineralised brewing liquor. 12. Using 5N sodium hydroxide, adjust the solution to pH 11. Stir for 5 minutes before reducing back to pH5 using 5N hydrochloric acid. Switch off the stirrer and a precipitate will be formed which will sediment. 13. Repeat steps 10 to 12 until the solution above the sediment appears bright and colourless. The sediment is then adjusted to pH 11 and pasteurised to maintain microbial stability. Example 3 Using the flocculant prepared in Example 2 lab-scale (50 ml) fining experiments were conducted in a production cask beer, and compared to beer fined using an auxiliary finings and then isinglass finings (conventional fining) . 1.5 ml of a 1% w/w solution of H-Factor was added to 50 ml of beer in a beaker while stirring. The pH of the beer was checked to ensure no change had occurred. The beer was gently stirred for 60 seconds before being poured into a 50 ml tube. The auxiliary finings (mixed Alginol, supplied by Savilles Clarification) and then isinglass finings Caskleer C, supplied by Savilles Clarification) were added to 50 ml beer while stirring. The dosing rates used were equivalent to 1 pint/brl of mixed Alginol followed by 4 pint/brl of Caskleer C. The Caskleer C had previously been diluted 1 in 2.25 to provide a working solution. The two experiments were then left to settle overnight at io°C before being sampled for turbidity determination (absorbance at 600 nm against a 0.45 μm filtered beer blank using the spectrophotometer of Example 1) . Once sampled, the beers were inverted ten times to resuspend and then left to settle again overnight. The process of inverting, settling and turbidity measurement was repeated to provide seven measurements. This requirement for cask beer to refine is essential unless the finings are added to a cask at the point of dispense. Table 3 Absorbence 600 nm Refine Number Auxiliary and Invention Isinglass Fining (H-Factor) 1 0.012 0.009 2 0.001 0.002 3 0.015 0.007 4 0.000 0.000 5 0.006 0.000 6 0.000 0.008 0.003 0.007 7 The absorbence values above indicate that both fining regimes provide beer with good clarity. Example 4 4% gallon cask trials were conducted using the flocculant prepared in Example 2 in production brewed beers. Beer fined with an equivalent of 42 g/hl H-Factor (added as a 1.4% w/v aqueous solution, pH 9) was compared to a beer fined with auxiliary and isinglass finings as in Example 3, and at dosing rates identical to those used in Example 3. Beers were left overnight before being resuspended and left to settle again. The resuspension and settling routine was repeated several times after which the beers were put on stillage. Periodically, samples of beer were removed from the dispense tap and assessments of clarity made. Measurements of clarity indicated that beer fined conventionally and with H-Factor were acceptable after 16 hours. In less than 48 hours both beers were bright. Both beers showed good microbial stability.

Claims: CLAIMS I. Composition derivable from yeast and which comprises proteinaceous material having an isoelectric point in the range 3 to 6. 2. Composition according to claim 1, wherein the isoelectric point is in the range 4 to 5.5. 3. Composition according to claim l or claim 2, wherein the yeast is selected from brewer's yeast, baker's yeast, wine yeast and cider yeast. 4. Composition according to any of claims 1 to 3, having a protein content of at least 60% by weight. 5. Composition according to any preceding claim, which contains amino acids whose relative proportions are substantially as depicted in the Radar Chart of Figure l. 6. A process for preparing a composition as defined in claim 1, comprising the steps of: a) preparing a slurry of yeast having a pH of at least 6.5; b) removing insoluble material from the slurry, eg. by centrifugation, to leave a supernatant comprising soluble material; and c) adjusting the pH of the supernatant to be in the range 3 to 6, and collecting the precipitate. 7. A process according to claim 6, wherein the slurry prepared in step a) has a pH of at least 9. 8. A process according to claim 6 or claim 7, wherein in step c) the pH is adjusted to be in the range 4 to 5.5. 9. A process according to any of claims 6 to 8, wherein prior to step a) the yeast cells have been stored. 10. A process according to any of claims 6 to 9, which further comprises re-suspending the precipitate in alkali, and then adjusting the pH to a value as defined in step c) , to purify it. II. A process according to any of claims 6 to 10, wherein the yeast is selected from brewer's yeast, baker's yeast, wine yeast and cider yeast. 12. A process for preparing a composition as defined in claim 1, comprising the steps of: a) preparing a slurry of yeast; b) adjusting the pH of the slurry to below 3; c) removing insoluble material from the slurry, eg. by centrifugation, to leave a supernatant comprising soluble material; and d) adjusting the pH of the supernatant to be in the range 3 to 6, and collecting the precipitate. 13. Composition obtainable by a process according to any of claims 6 to 12. 14. Aqueous dispersion, ie. a solution or suspension, of a composition as defined in any of claims 1 to 5 or 13. 15. A method of clarifying beer comprising adding to beer a composition as defined in any of claims l to 5 or 13, or a dispersion as defined in claim 14. 16. A method according to claim 15, wherein the beer is cask beer. 17. Beer containing a composition as defined in any of claims 1 to 5 or 13. 18. Use of a composition as defined in any. of claims 1 to 5 or 13 as a flocculant.

Inventors: JACKSON ANDREW CONAN, JACKSON, ANDREW, CONAN
Applicants: JACKSON ANDREW CONAN, WHITBREAD & CO LTD, JACKSON, ANDREW, CONAN, WHITBREAD PLC
Application year: 1996
Main classification: C12P21
IPC codes: C12H1, C12P21