METHOD FOR THE PREPARATION OF A WATER-SOLUBLE EXTRACT OF A VEGETABLE BIOMASS

Abstract

The present invention relates to a method for the preparation of a water-soluble extract based on a vegetable biomass of the Melissa officinalis family, which extract is used as a sulphite substitute in the aforesaid winemaking process, which method comprises one or more of the following steps: a) harvesting the herb, b) drying the herb obtained in step a), c) extracting the dried herb obtained in step b), wherein the aforesaid extraction step c) comprises at least two sub steps, a first sub step comprising a first extraction step with water, wherein the aqueous phase thus obtained functions as the starting material for the second sub step comprising an extraction step with an organic solvent, wherein the aqueous phase is acidified before the second extraction step.

Claims

1-37. (canceled)

38. A method for the preparation of a water-soluble extract based on a vegetable biomass of the Melissa officinalis family, which extract is used as a sulphite substitute in a winemaking process, which preparation method comprises one or more of the following steps: a) harvesting the herb, b) drying the herb obtained in step a), c) extracting the dried herb obtained in step b), wherein the aforesaid extraction step c) comprises at least two sub steps, a first sub step comprising a first extraction step with water, wherein the aqueous phase thus obtained functions as the starting material for the second sub step comprising an extraction step with an organic solvent, wherein the aqueous phase is acidified before the second extraction step.

39. A method according to claim 38, characterised in that the organic phase obtained in the second extraction step is subjected to an evaporation step subsequent to the aforesaid extraction step with an organic solvent.

40. A method according to claim 39, characterised in that the fraction obtained after the aforesaid evaporation step is mixed with water to obtain an herbal extract.

41. A method according to claim 40, characterised in that the herbal extract obtained after mixing with water is stored at a temperature in a range of 0-10 C., during a period of at least 5 hours, wherein the cold aqueous mixture of herbal extract is filtered to obtain an herbal filtrate, wherein a solid product obtained during the filtration steps is removed.

42. A method according to claim 41, characterised in that the herbal filtrate obtained after the filtration step is subjected to an additional evaporation step, wherein the herbal filtrate obtained after mixing with water is stored at a temperature in a range of 0-10 C., during a period of at least 5 hours, wherein the cold aqueous mixture of herbal filtrate is filtered to obtain a purified herbal filtrate.

43. A method according to claim 42, characterised in that a diluted herbal extract is obtained by diluting the purified herbal filtrate with water until a FRAP value in a range of 100.000-300.000 micromol Fe.sup.2+/l, as measured according to method as discussed herein, calculated on the total volume of the diluted herbal extract.

44. A method according to claim 43, further comprising the addition of one or more additional components, which components are selected from the group of quinone inhibitors and oxidase inhibitors, the quinone inhibitors being preferably vitamin C and the oxidase inhibitors being preferably kojic acid.

45. A method according to claim 38, characterised in that butanol, n-butyl alcohol and/or iso-butyl alcohol is used as the organic solvent in the aforesaid extraction step with an organic solvent.

46. A method according to claim 38, characterised in that in addition to Melissa officinalis, use is made of one or more components selected from the group of Mentha pperita, Thymus pulegioides, Thymus vulgare and Echinaceae root in the aforesaid step c).

47. A method of preservation an alcoholic beverage, wherein an extract is added to the alcoholic beverage, characterised in that the diluted herbal extract obtained according to claim 43 is added to the alcoholic beverage, in particular wine, in an amount of 0.001-5 wt. %, preferably 0.001-2 wt. %, on basis of the total weight of the alcoholic beverage.

48. A method of preservation an alcoholic beverage, wherein an extract is added to the alcoholic beverage, characterised in that the diluted herbal extract obtained according to claim 44 is added to the alcoholic beverage, in particular wine, in an amount of 0.001-5 wt. %, preferably 0.001-2 wt. %, on basis of the total weight of the alcoholic beverage.

49. A method according to claim 48, wherein the FRAP value of the alcoholic beverage to which the diluted herbal extract has been added, is within a range of 1500-3000 micromol Fe.sup.2+/l, calculated on the total volume of the alcoholic beverage.

50. A method according to claim 48, wherein the amount of oxidase inhibitors of the alcoholic beverage to which the diluted herbal extract has been added, is within a range of 50-250 mg/l, calculated on the total volume of the alcoholic beverage.

51. A method according to claim 48, wherein the amount of quinone inhibitors of the alcoholic beverage to which the diluted herbal extract has been added is within a range of 50-150 mg/l, calculated on the total volume of the alcoholic beverage.

52. A method according to claim 48, wherein the amount of sulphite of the alcoholic beverage to which the diluted herbal extract has been added is <10 mg/l, calculated on the total volume of the liquid aqueous medium.

53. A method for the preparation of low-alcoholic beverages, in particular wine, using grapes, which grapes are subjected to a number of process steps, inter alia comprising pre-processing, in particular crushing and pressing and possibly removal of the stems, fermentation, maturing and finally bottling, possibly with interim filtration, characterised in that a herbal extract is added at least once during the aforesaid steps, preferably a diluted herbal extract obtained according to claim 44, which herbal extract comprises Melissa officinalis, said herbal extract further comprises one or more additional components, which components are selected from the group of quinone inhibitor and oxidase inhibitor, wherein vitamin C is used as the quinone inhibitor and kojic acid is used as the oxidase inhibitor, said herbal extract contains Melissa officinalis in an amount of at least 30 wt. %, preferably at least 50 wt. %, in particular at least 80 wt. %, based on the weight of the total amount of herbs used.

54. A method for the preparation of low-alcoholic beverages, in particular wine, using grapes, which grapes are subjected to a number of process steps, inter alia comprising pre-processing, in particular crushing and pressing and possibly removal of the stems, fermentation, maturing and finally bottling, possibly with interim filtration, characterised in that a herbal extract is added at least once during the aforesaid steps, preferably a diluted herbal extract obtained according to claim 45, which herbal extract comprises Melissa officinalis, said herbal extract further comprises one or more additional components, which components are selected from the group of quinone inhibitor and oxidase inhibitor, wherein vitamin C is used as the quinone inhibitor and kojic acid is used as the oxidase inhibitor, said herbal extract contains Melissa officinalis in an amount of at least 30 wt. %, preferably at least 50 wt. %, in particular at least 80 wt. %, based on the weight of the total amount of herbs used.

55. A method according to claim 53, characterised in that said herbal extract comprises one or more natural components selected from the group of Mentha pperita, Thymus pulegioides, Thymus vulgare and Echinaceae root, said herbal extract contains Mentha pperita in an amount of 5-40 wt. % Thymus pulegioides in the amount of 1-20 wt. %, Thymus vulgare in an amount of 1-20 wt. %, Echinaceae root in an amount of 1-20 wt. %, based on the weight of the total amount of herbs used.

56. A method according to claim 54, characterised in that said herbal extract comprises one or more natural components selected from the group of Mentha pperita, Thymus pulegioides, Thymus vulgare and Echinaceae root, said herbal extract contains Mentha pperita in an amount of 5-40 wt. % Thymus pulegioides in the amount of 1-20 wt. %, Thymus vulgare in an amount of 1-20 wt. %, Echinaceae root in an amount of 1-20 wt. %, based on the weight of the total amount of herbs used.

57. A method according to claim 53, characterised in that the addition of said herbal extract is carried out in such a manner during the aforesaid winemaking process that the bacteria count of the wine to be made is at least in accordance with one or more of mesophilic aerobic bacteria count <1 CFU/ml, yeasts<10 CFU/ml, fungi<10 CFU/ml and lactic acid bacteria<1 CFU/ml, in particular in accordance with all of the aforesaid values.

58. A method according to claim 54, characterised in that the addition of said herbal extract is carried out in such a manner during the aforesaid winemaking process that the bacteria count of the wine to be made is at least in accordance with one or more of mesophilic aerobic bacteria count <1 CFU/ml, yeasts<10 CFU/ml, fungi<10 CFU/ml and lactic acid bacteria<1 CFU/ml, in particular in accordance with all of the aforesaid values.

59. Use of a diluted herbal extract based on Melissa officinalis comprising one or more components selected from the group of quinone inhibitor and oxidase inhibitor as a sulphite substitute in alcoholic beverages, in particular wine.

60. Use according to claim 59, characterised in that the herbal extract further comprises one or more components selected from the group of Mentha pperita, Thymus pulegioides, Thymus vulgare and Echinaceae root.

61. Use according to claim 59, characterised that a FRAP value of the diluted herbal extract is in a range of 100.000-300.000 micromol Fe.sup.2+/l, as measured according to method as discussed herein, calculated on the total volume of the diluted herbal extract.

62. Use according to claim 59, characterised in that the amount of the diluted herbal extract is in a range of 1 ml-20 ml, in particular 5 ml-15 ml, per litre alcoholic beverage.

63. Use according to claim 59, characterised in that the amount of vitamin C as the quinone inhibitor is in a range of 50-150 mg, per litre alcoholic beverage.

64. Use according to claim 59, characterised in that the amount of kojic acid as the oxidase inhibitor is in a range of 50-250 mg, per litre alcoholic beverage.

65. Use of a diluted herbal extract based on Melissa officinalis as an antioxidant in a food medium.

66. Use according to claim 65, characterised in that said food medium to is selected from one or more of the group consisting of potatoes, vegetables, fruit, meat, fish, cheese, milk products, (soft) drinks and food products derived therefrom.

67. Use according to claim 65, characterised in that said diluted herbal extract based on Melissa officinalis is used in the process of the potato processing.

68. Use according to claim 65, characterised in that said diluted herbal extract based on Melissa officinalis is used in the process of the meat processing.

69. Use according to claim 65, wherein said diluted herbal extract further comprise one or more components selected from the group of quinone inhibitor and oxidase inhibitor.

Description

[0075] FIG. 1 is a process flow diagram according to an embodiment of the present invention.

[0076] FIG. 2 is a chromatogram of the aqueous fraction obtained after extraction with an organic solvent.

[0077] FIG. 3 is a chromatogram of the organic fraction obtained after extraction with an organic solvent.

[0078] The present invention will be discussed with reference to the enclosed process flow diagram. This process flow diagram covers an embodiment of the present invention.

[0079] According to FIG. 1, the herb, especially Melissa officinalis, is harvested as indicated by reference number 1. According to a preferred embodiment the herb is sieved and unwanted parts are removed, such as sand, stones etc. The herb thus harvested 50 is dried in unit 2, for example a band dryer. In unit 3 the dried herb 51 is then extracted with water, at room temperature, i.e. a first extraction step with water. The aqueous extract 52 is acidified in unit 4, for example by the addition of phosphoric acid until a pH of about 1.7 and 3.5 to form an acidified plant extract 53. In unit 5 this acidified plant extract 53 is filtrated and the filtrate 54 thus obtained is sent to an extraction unit 6 for carrying out an extraction step with an organic solvent. The solvent used in unit 6 is for example n-butanol. The organic phase 55 originating from this extraction step with an organic solvent is evaporated in unit 7 under vacuum conditions of about 5 mbar and at a temperature of at most 40 C. This evaporation step aims to remove a substantial part of the organic solvent used in unit 6. The concentrate or concentrated fraction 56 obtained after the evaporation step in unit 7 is brought into contact with a liquid phase, i.e. water, in unit 8. In unit 9 there is a cold storage of the aqueous mixture of herbal extract 57 at a temperature of about 4-5 C., during a period of at least 5 hours. The cold aqueous mixture 58 thus stored is filtered in unit 10. The herbal filtrate 59 is sent to a second evaporation step in unit 11. In unit 11 de remainder part of the organic solvent is removed from filtrate 59. The concentrated fraction 60 obtained after the evaporation step in unit 11 is brought into contact with a liquid phase, i.e. water in unit 12. In unit 13 there is a cold storage of the aqueous mixture 61 at a temperature of about 4-5 C., during a period of at least 5 hours and the cold aqueous mixture is filtered in unit 13 as well. The filtrate 62 thus obtained, i.e. the purified herbal filtrate, is sent to unit 14 for standardisation. Unit 14 may receive several filtrates 62 from different batches. The standardisation in unit 14 includes the dilution with water until a specific FRAP value is reached. The thus diluted and standardised stream 63, i.e. the diluted herbal filtrate, is combined with additives in unit 15. Examples of additives comprise one or more additional components, which components are selected from the group of quinone inhibitor and oxidase inhibitor, especially vitamin C is used as the quinone inhibitor and/or kojic acid is used as the oxidase inhibitor. The thus prepared extract 64 is stored in unit 16. From unit 16 several grades of extracts 65 will be delivered to the end suppliers 17.

[0080] Although the process flow diagram has been discussed for only one specific herb Melissa officinalis, the process flow diagram may also include one or more other natural components selected from the group of Mentha piperita, Thymus pulegioides, Thymus vulgare and Echinaceae root. These natural components may be processed simultaneously with Melissa officinalis, but may also be blended with other processed herbs in unit 14.

[0081] In addition, in some embodiments the addition of additives, such as one or more additional components, in unit 15 may also take place before the standardisation in unit 14. This means that the dilution with water in unit 14 may take place after the addition of additives. In another embodiment of the present invention the concentrate 56 may undergo only one time the steps of mixing with water, cold storage and filtration. In such an embodiment the herbal filtrate 59 is sent directly to unit 14 for standardisation. As mentioned above, unit 14 may receive several filtrates, for example filtrates that have undergone both the first evaporation in unit 7 and the second evaporation in unit 11 and filtrates that have only undergone a single evaporation step in in unit 7.

[0082] As mentioned in this application, the extraction step with an organic solvent is preferably carried out with butanol, n-butyl alcohol and/or iso-butyl alcohol. The present inventors found that by using these preferred organic solvents a broad spectrum of anti-oxidant components is extracted. This can be seen with the chromatographic fingerprints of the discarded water fraction after the organic extraction, as shown in FIG. 2. The amount of rosmarinic acid in this water fraction is high. Application tests with white wine showed that the extract with high rosmarinic acid purity had a detrimental effect on the taste of the wine and did not stop brown discoloration during storage of the wine.

[0083] The chromatogram of the organic phase obtained after the organic extraction step as shown in FIG. 3 shows much more peaks besides the rosmarinic acid peak. The present inventors assume that the presence of these other components provides the unexpected anti-oxidative properties of the herbal extract obtained according to the present invention.

[0084] An example of the method for measurement Ferric reducing abilityantioxidant power (FRAP) will now be discussed.

[0085] Preparing reagents:

[0086] 1. Stock solution of 300 mM acetate buffer, pH 3.6 (store at 4.c for maximum 1 month) For 100 ml:

[0087] weigh 0.31 g sodium acetate (C.sub.2H.sub.3NaO.sub.2.3H.sub.2O), dissolve in 50 ml distilled water

[0088] add 1.6 ml acetic acid (C.sub.2H.sub.4O.sub.2)

[0089] add distilled water up to 75 ml

[0090] measure the pH, if necessary adjust to 3.6

[0091] add distilled water up to 100 ml.

[0092] 2. Stock solution of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) (store at 4 C. for maximum 1 month) For 10 ml:

[0093] weigh 31 mg TPTZ, dissolve in 5 ml of distilled water

[0094] add 34 l HCl (36%, d=1.18 kg/l)

[0095] add distilled water up to 10 ml.

[0096] 3. Stock solution of 20 mM FeC.sub.3.6H.sub.2O (store at 4 C. for maximum 1 month)

For 10 ml:

[0097] weigh 54.1 mg FeCl.sub.3.6H.sub.2O, dissolve in 5 ml of distilled water

[0098] add distilled water up to 10 ml.

[0099] 4. Work solution: FRAP reagent

[0100] Mix 25 ml acetate buffer (solution 1), 2.5 ml TPTZ-reagent (solution 2) and 2.5 ml FeCl.sub.3.6H.sub.2O (solution 3).

[0101] Place the solution at 37 C. 30 minutes before starting the analysis.

Calibration:

[0102] Weigh on the analytical balance 27.8 mg FeSO.sub.4.7H.sub.2O in a measuring flask of 100 ml, note the weight, dissolve in a little distilled water, and add distilled water up to 100 ml

[0103] Make dilutions 1/10, 2/10, 4/10, 6/10, 8/10 and 10/10

[0104] Add 225 l of FRAP reagent in all needed wells of the 96 well plates

[0105] Add to the appropriate well 25 l of the standard dilutions, 25 l of distilled water for the blank, mix by carefully pipetting up and down

[0106] Measure the optical density at 593 nm at 0, 5, 10, 15 and 20 minutes, incubate the plate in the spectrophotometer, place the incubator of the spectrophotometer at 37 C.

Samples:

[0107] If possible dilute the samples within the range of the calibration curve (100-1000 pmol/l Fe.sup.2+)

[0108] Add for each sample 225 l of FRAP reagent (37 C.) in a well of the 96 well plates

[0109] Add 25 l of the sample to the appropriate well, 25 l of distilled water for the blank, mix by carefully pipetting up and down

[0110] Measure the optical density at 593 nm at 0, 5, 10, 15 and 20 minutes, incubate the plate in the spectrophotometer, place the incubator of the spectrophotometer at 37 C.

[0111] Determine the FRAP value by recalculation with the use of the calibration curve/