STABILIZATION OF WINE

Abstract: The present invention relates to stabilized wine and to a process for the production thereof that comprises the addition of an oligonucleotide or polynucleotide containing preparation.

Description: STABILIZATION OF WINE Field of the invention The present invention relates to stabilized wines and to a process to prepare these stabilized wines. Background The presence of tartaric salts, potassium hydrogen tartrate ( KHT) and calcium tartrate (CaT) is one of the major causes of instability of wines. Tartaric acid is the main organic acid produced by the grape berry during its development. It is solubilised as potassium and calcium salts into grape musts during the processing of berries. During the fermentation, tartrate salts are preserved by the yeast but their solubility decreases with the increase of ethanol concentration due to the fermentation of sugars. In young wines, potassium hydrogen tartrate is always present in supersaturating concentrations and crystallises spontaneously. After bottling of wines, the KHT instability may become a major commercial problem due to the unpredictable character of crystallisation and the presence of unattractive crystals in a bottle is often considered as an inferior quality by consumers. Physical treatments can be used prior to bottling of the wine to prevent crystallisation of tartrate salts. These treatments consist in promoting the crystallisation by cooling the wine at -4° C or in elimination of the potassium and tartaric ions by electrodialysis or by the use of ion- exchange resins. However, these time and energy consuming processes are supposed to alter the colloidal equilibrium of wines. The alternative to physical treatments of wines is to use additives which prevent the nucleation and/or the growth of KHT crystals. An example of additives that prevent the nucleation of crystals are mannoproteins, as described in WO 96/1 3571 and in WO 97/49794. However, since they have no effect on the growth of added crystals, they cannot guarantee the complete stabilization of wine. Consequently, treated wines are in fact unstable. In addition, the average mannoprotein concentration in wine is not sufficient to guarantee complete prevention of crystallisation. Carboxymethyl cellulose and meta-tartaric acid belong to the group of additives which inhibit the growth of KHT crystals. Unfortunately, carboxymethyl cellulose has not been accepted by the wine community due to its presumed negative organoleptic effect on treated wines and meta-tartaric acid is unstable at the pH of wine and at the temperature at which wine is stored. Over time, the meta-tartaric acid will hydrolyse and its protective effect disappears. Therefore, its use is restricted to low quality wines for quick consumption. Another drawback is that ideally an additive should be a natural component of wine. This is definitely not the case with meta-tartaric acid or carboxymethyl cellulose. Natural additives which are active on both nucleation and growth rate of crystals are by far the best guarantee for long-term stability. Until now, no such additives have been available. Summary The present invention relates to the use of an oligonucleotide or polynucleotide containing preparation to produce stabilized wine, to oligonucleotide or polynucleotide containing preparation for stabilizing wine, to a process for preparing such preparations and to stabilized wines. Detailed description The present invention relates to a process for the production of stabilized wine. This process comprises the addition of an oligonucleotide or polynucleotide containing preparation to wine. In the present context, stabilized wine is wine in which the crystallization of salts of tartaric acid is prevented or retarded, either as a result of the prevention or retardation of the nucleation process or the inhibition or retardation of the growth of said crystals or both. Unstable wine is characterized by rapid crystallization of salts of tartaric acid, which is usually of an unpredictable behaviour. Surprisingly, it was found that the addition of an oligonucleotide or polynucleotide containing preparation has a stabilizing effect on wine by preventing both the nucleation as well as the growth of KHT crystals. In the present context, an oligonucleotide consists of 2-10 nucleotides, a polynucleotide consists of more than 1 0 nucleotides The sugar moiety in the oligonucleotide or polynucleotide may be ribose, as in RNA, or deoxyπbose, as in DNA. The oligonucleotide or polynucleotide in the preparation, or the complete preparation, may be obtained from organisms, including plants, or be synthesized, e.g. by a nucleotide synthesizer. Suitable organisms include but are not limited to microorganisms, fish and mammals Preferably, the oligonucleotide or polynucleotide is obtained from a microorganism. This includes but is not limited to bacteria, fungi and yeast. Examples of suitable yeasts are Brettanomyces, Saccharomyces and Kluyveromyces, such as for example Brettanomyces bruxellensis, Saccharomyces cerevisiae and Kluyveromyces lactis. For instance, heat- inactivated yeast may be used to prepare the ohgonucleotides or polynucleotides. Ohgonucleotides and polynucleotides may be prepared according to methods known in the art. These methods comprise the cultuπng and harvesting of the cells, disruption of the cells in order to liberate the cell contents and isolation of the oligonucleotide or polynucleotide containing fraction by precipitation techniques, chromatography and the like. Both RNA and DNA may be used in oligonucleotide or polynucleotide containing preparations. The RNA and DNA oligonucleotides or polynucleotides may be in any form, be it single stranded or double stranded. Suitable RNA and DNA molecules have a molecular weight between 0.4 and 500 kDa. If RNA is used, preferably RNA with a molecular weight between 3 and 200 kDa, more preferably between 1 0 and 1 00 kDa is used. If DNA is used, preferably DNA with a molecular weight between 1 and 340 kDa, more preferably DNA with a molecular weight between 1 and 1 00 kDa is used. RNA and DNA may be used separately, or in combination. A well known method in the art for determining the molecular weight of the oligonucleotide or polynucleotide fraction is size exclusion chromatography using a column that is calibrated with protein molecular weight standards. The oligonucleotide or polynucleotide containing preparation according to the invention may consist exclusively or almost exclusively of nucleotides, e.g. for more than 80%. However, it is not necessary to use completely purified or almost completely purified nucleotides, any proportion of nucleotides between 0.1 and 1 00% of the preparation may be used in a preparation according to the invention. For instance, it is also possible to use a crude preparation obtained from e.g. yeast, which may contain 0.1 to 50% nucleic acids, or even 0.1 to 20% nucleic acids. If the nucleotides are not completely or almost completely purified, the nucleotide-containing preparation preferably contains 0.1 -1 5% nucleotides, more preferably 1 -1 5% nucleotides (all % on a dry matter weight basis). Other suitable constituents of the preparation apart from the oligonucleotides or polynucleotides are e.g. yeast cell wall components, such as for instance mannoproteins, or parts thereof. The oligonucleotide or polynucleotide containing preparation may be added to the grape must or to wine. It is preferably added to the wine during ageing, i.e. after fermentation but before bottling. The invention is extremely suitable for white wines and rose wines. The oligonucleotide or polynucleotide containing preparation is added in such amounts that a stabilizing effect is achieved. In general, one will try to work with as low as possible amounts. Good results are possible if so much preparation is added that a final nucleic acid concentration in the wine is reached of 1 to 400 mg per liter wine. Preferably, so much is added that a concentration of 1 to 200 mg per liter wine is reached, most preferably 1 to 1 00 mg per liter wine is added. Any insolubles which develop may subsequently be removed using standard techniques. The skilled person will understand that the amount added will also depend on the addition or presence of e.g. other stabilizers. The nucleation and crystal growth can be measured and quantified by the following methods (Moutounet et al. In : Actualites CEnologiques 1 999 Vieme Symposium International d'Oenologie de Bordeaux (Lonvaud- Funel ed.) Method 1 , indicative of crystal nucleation, measures the time of appearance of crystals in the wine when stored at -4° C. A visual inspection is performed daily and the time necessary to detect the appearance of crystals (T crγs ) is expressed in number of days. Unstable wine has a T crγs that can vary between 0.5 and 1 5 days. Stabilized wine is characterized by a τ crys sta l zed w,n T crys unstable w,ne of > 2, preferably > 5, more preferably > 1 0 and most preferably > 40. Method 2, indicative of crystal growth, measures the Degree of Tartaric Inhibition (DTI) of the wine. Hereto, wines are stirred at -4° C and the initial conductivity is measured. Subsequently, calibrated crystals of KHT are added and the conductivity is then measured after a stable value has been reached. The DTI is defined as the percentage decrease of the initial conductivity. Stabilized wine is characterized by a DTI s,ablh2ed w,ne /DT |uns t a bi e w ,ne of < 0 .8, preferably < 0.5 and more preferably < 0.30. Method 3, indicative of KHT crystal nucleation, measures the true, dissolved tartaric acid concentration. An accurate volume of the wine is transferred into a glass vial, and mixed with the same accurate volume of D 2 0 containing a precisely known concentration of maleic acid. The 1 H NMR spectrum is run with conditions of full relaxation, and the integral of the internal standard (maliec acid) is compared with the integral of tartaric acid. In this way the dissolved tartaric acid concentration can be determined with very high precision and accuracy. Stabilized wines according to the invention are characterized by a T crγs stab " ιzed w ™7T- rv5 unstable w,ne of > 2, preferably > 5, more preferably > 1 0 and most preferably > 40 and by a D τi sta lilzed wιne /DTI unsτable w,πe of < 0.8, preferably < 0.5 and more preferably < 0.3 and are biologically stabilized. In this context, biologically stabilized means that the stabilization is achieved by the addition of an additive which is generally considered as a natural component of wine, e.g. because it is derived from yeast or grapes. Examples of such biological additives are mannoprotein, DNA and RNA. EXAMPLES The time of the appearance of crystals (T crys ), T crys stab " ιzed w,n 7T crys uns,ablβ w,ne , the degree of tartaric inhibition (DTI) of the wine and tartaric acid concentrations were measured as described above. The concentration of nucleic acids was determined by dissolving a well known amount of the sample in D 2 O, the nucleic acids were hydrolyzed by means of RNase and DNase, and the concentration of mononucleotides was measured by 600 MHz NMR after the addition of a suitable internal standard, such as maleic acid. In all experiments an unstable white wine, Chardonnay from harvest 2000, was used. The concentration of tartaric acid at the onset of this study was 1 .92 g/l. All quantitative experiments were carried out by means of NMR as described above. 0.500 ml of wine was added to 0.500 ml of a stock solution of D 2 0, containing approximately 5 g/l EDTA and 5.0637 g/l maleic acid disodium salt as an internal standard for NMR measurements. Example 1 a Effect of DNA on the crystallization of potassium hydrogen tartrate in wine. To test the effect of DNA on the stabilization of wine, herring sperm DNA (Sigma) was purchased. Small volumes were added to 1 0 ml of unstable white wine, to achieve final concentrations of 0.4, 0.2, 0. 1 and 0.05 g/l. The samples were stored for 1 hour at + 4° C. After the removal of insolubles by centrifugation at 3000 rpm for 30 min. , the samples were stored at -4°C. Results are presented below and show that DNA has a stabilizing effect on this unstable white wine, which wine showed crystals after only 1 6 hours at -4°C. The DNA was also used for tests with DNA fractions of different molecular weight. 250 mg of the DNA were dissolved in H 2 0 and passed over an ultra-filtration membrane (MW cut-off 1 0 kDa), to the retentate water was added twice for complete removal of lower molecular weight components. The retentate was freeze dried, and a yield of 1 65 mg was obtained, this fraction was labeled DNA-LS (large size). The permeate was passed over a membrane with MW cut-off of 1 kDa To the retentate water was added two times, and the retentate was freeze-dried. The yield was 76 mg. This product was labeled DNA-MS (medium size) . The permeate was also freeze-dried, and a yield of 9 mg was obtained. This product was labeled DNA-SS (small size) . The low molecular weight fraction was not used for these experiments, because the yield was too low. Example 1 b DNA MS fraction ( 1 kDa < MW < 10kDa) Freeze-dried DNA with a molecular weight between 1 and 1 0 kDa was dissolved in water in a concentration of 1 0 mg/ml. Small volumes were added to wine as described in Example 1 a. Example 1 c DNA-LS fraction (10kDa < Mw) Freeze-dried DNA with a molecular weight > 1 0 kDa was dissolved in water in a concentration of 1 0 mg/ml. Small volumes were added to wine as described in Example 1 a. The results of example 1 a-c show that DNA has a stabilizing effect. Excellent results are obtained with DNA of a molecular weight between 1 and 10 kDa. Example 2 Effect of RNA on the crystallization of potassium hydrogen tartrate in wine. To further investigate the effect of RNA in the stabilization of wine, RNA was purchased from Sigma (bakers yeast). RNA fractions were prepared as described for DNA in example 1a. Yields were: fraction MW<1kDa (RNA-SS): 30mg, 1 kDa<MW< 10kDa (RNA-MS): 92 mg, 10kDa<MW (RNA-LS): 105 mg. Example 2a RNA-SS fraction (MW < 1 kDa) The freeze dried RNA fraction with molecular weight MW< 1 kDa (RNA- SS) was dissolved in water in a concentration of 10 mg/ml. The samples were added to wine as in example 1a. Example 2b RNA-MS fraction (1kDa<MW< 10kDa) The freeze dried RNA fraction with molecular weight MW< 1 kDa (RNA- SS) was dissolved in water in a concentration of 10 mg/ml. The samples were added to wine as in example 1a. Example 2c RNA-LS fraction ( 10kDa < MW) The freeze dried RNA fraction with molecular weight > 1 0 kDa was dissolved in water in a concentration of 1 0 mg/ml. The samples were added to wine as in example 1 a. The results of example 2a- c demonstrate that RNA has a stabilizing effect. The best results are obtained with RNA fractions of > 1 0 kDa. Example 3 Preparation and characterization of a nucleotide-containing preparation from yeast. Yeast cells of the species Saccharomyces cerevisiae were harvested from the fermentation broth by centrifugation. The concentrated yeast cream is autolysed by adding 2% (w/w) sodium chloride, after which the cream is heated to 52 ° C for 40 hours. During the autolysis the pH is maintained at 5.4 by using hydrochloric acid and sodium hydroxide. After the autolysis, the remaining insoluble fraction, which consists primarily of the crude cell wall fraction, is separated from the solution by use of a 4-stage disk-stack centrifuge battery. During the separation the solid-fraction is countercurrent washed in order to optimize the yield of the soluble fraction. The separated cell wall fraction with a dry matter content of 1 2-1 5% w/w was subjected to the following extraction procedure : 1 . Cell walls are suspended in water ( 1 : 1 , v/v) and centrifuged. The resulting "cake" is resuspended to a concentration of 1 2- 1 5 % (w/v) in a solution of 1 0 mM potassium metabisulfite and 20 mM citrate adjusted to pH 7.0 by the addition of NaOH . 2. The cell wall suspension thus obtained is heated at 1 20 ° C for 2 hours. 3. After cooling, the suspension is centrifuged at 1 0,000xg for 1 0 min. 4. The resulting supernatant is filtered on a filter press after addition of 2% of Clarcel CBL3 (CECA, France) as filter aid. Residual insolubles are removed by filtration on a one-plate Seitz filter press equipped with a Seitz-EKS filter (0.1 - 0.3 micron cut-off) . 5. The EKS filtrate is submitted to two successive ultra-filtration steps using two membranes of different molecular weight cut-off. First a 1 00 kDa membrane (Amicon, USA) is used to remove high molecular weight molecules, the retentate being then discarded. The filtrate obtained from the 1 00 kDa membrane is then concentrated on a membrane with a cut-off of 3 kDa (Amicon) . Diafiltration with water is used to remove salts and low molecular weight contaminants from the sample. The final concentration factor of the retentate is typically 1 0. 6. The final desalted concentrate is lyophilized to give the nucieotide preparation sample MDR 1 . This preparation is characterized by a contents of 1 - 1 .5 % RNA and 1 .5-2% DNA. Example 3a Effect of MDR1 on the crystallization of potassium hydrogen tartrate in wine. A freeze dried fraction of MDR1 was dissolved in H 2 O to obtain a stock solution of 15 mg/ml. Small volumes of this stock solution were transferred into 10 ml of unstable white wine. In this way final concentrations of MDR1 were obtained of 0.6 g/l, 0.4 g/l and 0.2 g/l. These samples were stored for approximately 1 hour at +4 °C, and all insolubles were removed by centrifugation. Next, the clear supernatants were transferred into a glass vial, and stored at -4 °C. The temperature was kept constant between -2 and -6 °C.The samples were evaluated visually for tartaric acid crystals. The dissolved tartaric acid concentration was determined as described before. These results show that also not completely purified nucleotides stabilize wine. Comparative Example 4 Preparation and characterization of a nucleotide- free preparation. All nucleotides were removed from MDR 1 in the following way: RNase was added to the product, the pH was adjusted to 5.6, and the solution was heated to 65 °C for 5 V2 hours. The pH was readjusted to 5.6 after 2 hours. The final solution was passed over an ultra-filtration membrane (MW cut-off 2000) to remove all mononucleotides. The retentate was freeze dried and an NMR spectrum was recorded and compared with the NMR spectrum of MDR 1 . The spectra showed that al nucleotides had been removed. Comparative Example 4a Effect of a nucleotide-free preparation on the crystallization of potassium hydrogen tartrate in wine. The freeze dried product was added to unstable wine in the same way and concentrations as described in example 3a . These results show that the nucleotide-free preparation has hardly any stabilizing effect on the wine compared to the nucleotide-containmg preparation. Example 5 Long-term stabilization experiments A nucelotide-containing preparation was prepared as described in Example 3. The sample obtained was labelled MR 1 . Molecular weight distribution of molecules in MR 1 has been determined by size-exclusion chromatography analysis on a TSK G3000SW column. Soluble molecules in MR 1 have molecular weights in the range 5-50 kDa. The dry matter content of the freeze-dried MR1 is 98%. Analyses of the compounds listed below have been performed by standard procedures or as described. Component Percentage (w/w) Total nitrogen 1 0.8 Phosphate 6.3 Sodium 2.0 Potassium 1 .0 Total ashes 3.0 Total carbohydrates 25 Glucose 0.7 Mannose 22.0 Ribose 6.75 Protein 54 Mannoproteins 77 RNA 1 5 Molecular weight distribution 20-30 kDa The RNA content was calculated from the ribose content (6.76%) via the respective molecular weights. The molecular weight of a nucieotide in RNA is 339 Da and of ribose is 1 50 Da. Therefore, the RNA content is 339/1 50 x 6.75 = 1 5 %. The protein content was calculated from the total nitrogen ( 1 0.8%) corrected for the nitrogen present in RNA (2.2%) via ( 1 0.8-2.2) x 6.25 = 54%. Example 5a Effect of MR1 on the crystallization of potassium hydrogen tartrate in a very unstable white wine. Samples of nucieotide preparation MR 1 have been added to wines of different tartaric instabilities (i.e. very unstable and unstable). A 75 g/l solution of MR 1 preparation in water has been prepared. Aliquots of 1 , 2, 3, and 4 ml have been added to 0.5 liter of wine to reach respective final concentrations of 1 50, 300, 450 and 600 mg/l. The results are presented below. They show that the white wine used is very unstable (KHT crystals can be observed already after 1 day of storage at -4°C). After addition of 300 mg/l MR 1 , the time necessary to detect the formation of KHT crystals increased to 43 days. This KHT instability of this white wine is characterized by a DTI of 23%. After addition of 450 and 600 mg/l MR 1 , the DTI value decreased to 1 6 and 7% respectively. The results obtained with the two methods on this very unstable white wine demonstrate that MR 1 prevents both the nucleation of KHT crystals and inhibits their growth. The treated wines can be considered as stable. Example 5b Effect of MR1 on the crystallization of potassium hydrogen tartrate in an unstable white wine The results for the unstable white wine are presented below. They show that this white wine is unstable since KHT crystals can be observed after 1 1 days of storage at - 4°C. After addition of MR 1 at 450 mg/l, no crystals could be detected after 1 20 days of storage at - 4° C. The KHT instability of this white wine is characterized by a DTI of 1 6.1 %. After addition of 450 mg/l of MR 1 , the DTI value decreased to 4.1 %, showing that MR 1 prevents the growth of KHT crystals. The combined use of the two methods on this unstable white wine indicates that MR 1 prevents both the nucleation of KHT crystals and inhibit their growth. Treated wines can be considered as stable.

Claims: CLAIMS 1 . A process for stabilizing wine, which process comprises the addition of an oligonucleotide or polynucleotide containing preparation to the grape must or to the wine. 2. A process according to claim 1 , wherein the oligonucleotide or polynucleotide containing preparation also comprises yeast cell wall components or parts thereof. 3. A process according to claim 2, wherein the yeast cell wall component is a mannoprotein. 4. A process according to claim 1 -3, wherein the oligonucleotide or polynucleotide containing preparation is derived from bacteria, fungi or yeasts. 5. A process according to claims 1 -4, wherein the oligonucleotide or polynucleotide containing preparation is derived from the yeasts Saccharomyces or Kluyveromyces. 6. A process according to claim 1 -5 wherein the oligonucleotide or polynucleotide containing preparation is derived from Saccharomyces cerevisiae. 7. A process according to claims 1 -6 wherein the oligonucleotide or polynucleotide containing preparation is added to give final nucleic acid concentrations between 1 and 400 mg per liter of must or wine. 8. A process according to claim 1 -7 wherein the oligonucleotide or polynucleotide is DNA, RNA or a combination of both. 9. A process according to claim 1 -8 wherein the oligonucleotide or polynucleotide has a molecular weight between 0.4 and 500 kDa as determined by size-exclusion chromatography using a column that is calibrated with protein molecular weight standards. 1 0. A process for preparing an oligonucleotide or polynucleotide containing preparation according to claims 1 -9, which process comprises: autolysis of yeast cells at pH 5.4, 52 ° C for 40 hours - followed by separation of the cell wall containing fraction by centrifugation in such a way that a dry matter content of 1 2- 1 5 % of the cell wall containing fraction is reached followed by the addition of buffer to obtain a solution of pH 7 followed by a heatshock at 1 20 ° C for 2 hours - followed by centrifugation in which most of the insolubles are removed. followed by filtration to clarify the liquid/broth followed by ultra-filtration of the clarified liquid to obtain a fraction with molecular weight between 3 and 1 00 kDa and concentration factor of about 1 0. 1 1 . Biologically stabilized wine characterized by a T crys staDlllzed w,n 7T crvs unstable w,ne of > 2, preferably > 5, more preferably > 1 0 and most preferably > 40 and by a DTI sta ,,,zed wιn DTI unstable w,ne of < 0.8, preferably < 0.5 and more preferably < 0.30. 1 2. Use of an oligonucleotide or polynucleotide containing preparation to stabilize wine. 1 3. Use according to claim 1 2, wherein the oligonucleotide or polynucleotide containing preparation is used to prevent or retard the crystallisation of salts of tartaric acid. 14. Use according to claim 1 2 or 1 3, wherein the oligonucleotide or polynucleotide containing preparation is derived from bacteria, fungi or yeasts. 1 5. Use according to claim 1 4, wherein the yeast is Brettanomyces, Saccharomyces or Kluyveromyces. 1 6 Use according to claims 1 5, wherein the yeast is Saccharomyces cerevisiae. 1 7 Use according to claims 1 2- 1 6, wherein the oligonucleotide or polynucleotide containing preparation contains between 0. 1 and 1 00% nucleic acid. 1 8. Use according to claims 1 2- 1 7, wherein the nucleic acid is DNA, RNA or a combination of both. 1 9. Use according to claims 1 2- 1 8, wherein the oligonucleotide or polynucleotide has a molecular weight between 0.4 and 500 kDa as determined by size-exclusion chromatography using a column that is calibrated with protein molecular weight standards.

Inventors: LANKHORST PETER PHILIP, PELLERIN PATRICE JACQUES MARIE, LANKHORST, PETER, PHILIP, PELLERIN, PATRICE, JACQUES, MARIE
Applicants: DSM NV, LANKHORST PETER PHILIP, PELLERIN PATRICE JACQUES MARIE, DSM N.V., LANKHORST, PETER, PHILIP, PELLERIN, PATRICE, JACQUES, MARIE
Application year: 2000
Main classification: C12H1
IPC codes: C12G1, C12H1, C12P19