Biotechnology for the production of aroma substances from pomace of currants, strawberries, raspberries and wine industry

Abstract

The present invention relates to a biocatalytic process for producing an aroma substance or an aroma substance mixture, comprising the following steps: preparing a transformation medium comprising plant components of Currantaceae, Rosaceae and / or Vitaceae; contacting the transformation medium with at least one fungus from Basidiomycetes, which is capable of forming an aroma substance or an aroma substance mixture on the transformation medium; converting the plant components into the aroma substance or the aroma substance mixture with the help of the fungus; and optionally recovering the aroma substance or the aroma substance mixture, wherein the aroma substance or the aroma substance mixture preferably comprises at least one compound selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool.

Description

Technical Field

[0001] The present invention relates to the field of aroma substances. More specifically, the present invention relates to a method for preparing an aroma substance or an aroma substance mixture as described herein. The present invention also relates to the use of the transformation medium described herein and / or the biocatalyst described herein for producing an aroma substance or an aroma substance mixture, wherein the transformation medium comprises ingredients based on plant parts of the Currantae, Rosaceae or Vitaceae family. In addition, the present invention relates to a composition comprising the compounds described herein and its use in the nutritional, cosmetic, health or edible preparations described herein. Background Art

[0002] Natural aroma substances are usually obtained by extraction from plants. Linalool can be used as an example herein. Linalool is a monohydric alcohol belonging to the terpene family. The compound is colorless, flammable and has a unique aromatic smell. The well-known and abundant sources of linalool are herbs such as basil, mint, coriander, oregano and thyme. Linalool can be used in various applications. For example, due to its aromatizing and / or deodorizing functions, the compound is used as a fragrance in aromatic oils, or as a substitute for bergamot in the spice industry. In addition, it is found that the compound can be a kind of aroma substance in wine.

[0003] In order to meet the strong and steadily increasing demand for linalool, a considerable part of the current market supply is chemically synthesized. Although chemical synthesis offers the possibility of producing a large number of aroma substances, many syntheses are not environmentally compatible. In particular, the reaction rate under normal conditions is almost negligible, which means that acceptable yields can only be achieved by applying harsh synthesis conditions such as high temperature and high pressure. In addition, chemical synthesis often uses heavy metal catalysts, flammable gases and organic solvents, the use of which is full of well-known drawbacks. For this reason, there is an increasing demand for alternative production processes for linalool and for other aroma-active compounds that at least partially overcome the drawbacks of the known methods.

[0004] This can be improved by biotechnological processes based on the use of microorganisms or their enzymes to convert natural precursors into desired aroma substances (Berger, RG, Flavors and Fragrances: Chemistry, Bioprocessing and Sustainability, (2007), Berlin Heidelberg, Springer Verlag). Agricultural byproducts may have great potential in the biotechnological production of linalool and other aroma substances, some of which are rich in dietary fiber and secondary metabolites as potential aroma precursors. Summary of the invention

[0005] Therefore, the main purpose of the present invention is to improve the above-mentioned defects and provide a process for preparing aroma substances such as linalool. In particular, it is of interest to provide a process that is more environmentally friendly than known chemical synthesis.

[0006] WO 2013 / 034613 discloses a method for producing a beverage or a beverage base, wherein a culture medium is fermented in at least one aerobic fermentation process and the culture medium is fermented by hyphae from at least one basidiomycete.

[0007] According to the present invention, this main object is solved by a method for producing an aroma substance or an aroma substance mixture, the method comprising the following steps:

[0008] Preparing a transformation medium (also partly referred to herein as seed medium or substrate) comprising a component based on plant parts of the Currant family (Ricinaceae), the Rosaceae family (family Rosaceae) and / or the Grape family (Vitaceae); contacting the transformation medium with a biocatalyst; transforming the component based on plant parts of the Currant family, the Rosaceae family or the Grape family with the aid of the biocatalyst to form an aroma substance or an aroma substance mixture; and optionally

[0009] The aroma substance or aroma substance mixture is recovered.

[0010] Other objects, aspects and preferred embodiments of the present invention will become apparent from the following description, the additional examples, the drawings and in particular the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : A comparison of the transformation carried out on the culture medium (transformation medium) according to the invention and the transformation carried out on the standard culture medium using the fungus Poria as an example. Photos of the effluent culture (Emerskulturen) of Poria after 11 days on different culture media are shown. A: malt extract agar (MEA), B: MEA + 0.624% monosodium L-aspartate, C: 3% pomace, D: 3% pomace + 0.624% monosodium L-aspartate.

[0012] Figure 2 : A comparison of transformations carried out on a culture medium according to the invention and on a standard culture medium using the fungus Poria cocos as an example. Shown are growth curves of the effluent culture of Poria cocos on four different media: MEA (□); MEA + Asp (); 3TT (x); 3TT + Asp (○). The abbreviations used are: 3TT: 3% Titania variant pomace, Asp: 0.624% monosodium L-aspartate.

[0013] Figure 3: A comparison of the transformation performed on the culture medium according to the invention and the transformation performed on the standard culture medium using the fungus Poria as an example. Shown is the course of the pH value of the medium during the effluent culture of Poria in MEA (□); MEA+Asp(); 3TT (x); 3TT+Asp (○). The abbreviations used are as described above.

[0014] Figure 4 : Using Poria cocos for fermentation on MEA as an example, GC-MS chromatogram (non-divided flow measurement) of transformation on a culture medium not according to the present invention. Shown is a chromatogram of a sample taken after 8 days of cultivation, which shows that the substance is produced.

[0015] Figure 5 : GC-MS chromatogram of the transformation on the culture medium according to the invention using Poria cocos as an example of an aqueous fermentation on 3% red currant pomace, 0.624% monosodium L-aspartate, 3% agar-agar. Shown is the chromatogram of a sample taken after 11 days of cultivation; top: measured with a split ratio of 50:1, bottom without splitting, where the MS detector was turned off between 13.5 minutes and 13.7 minutes (grey background).

[0016] Figure 6 : GC chromatogram of the conversion on the culture medium according to the invention with ODP traces and indicating substance production using Poria cocos on 3% red currant pomace and 0.624% monosodium L-aspartate as an example. Shown is the chromatogram of a sample taken after 10 days of cultivation; between 13.2 and 13.7 minutes, the MS detector stops working (grey background). The black bands with numbers generally indicate the odors expected according to the following overview.

[0017] 1 green 13 flowers, citrus, fragrance

[0018] 2 cheese 14 green

[0019] 3 Solvent 15 slightly sweet, floral

[0020] 4 slightly sweet, green 16 green, fresh, citrus

[0021] 5 fungus, vanilla 17 green, fir

[0022] 6 Green, slightly sweet, floral 18 Fir, floral

[0023] 7 fungus 19 solvent, slightly sweet

[0024] 8 green, floral 20 unclear, smelly, fried

[0025] 9 unclear, green, fir, floral 21 caramel

[0026] 10 Unclear 22 Fruity

[0027] 11 Unclear, green, floral 23 Unclear, green

[0028] 12 marzipan candies, slightly sweetened 24 green

[0029] Figure 7 : Heat map of selected aromas during cultivation of Poria cocos on different media; relative changes during 14 days of cultivation. The EIC peak areas at m / z (benzaldehyde) = 106, m / z (linalool) = 93, m / z (linalool oxides I, II) = 111, m / z (2-undecanone) = 71, m / z (2-nonanone) = 58, m / z (geraniol) = 69, m / z (methylcarbamate) = 119 were used as base peaks. The assignment of the corresponding saturation of the selected fragments was performed based on the relative peak areas of the EIC (0% to 100%) of the selected fragments at various time points (1 to 14 days) according to the scheme shown below.

[0030] Figure 8 : Graph of the evolution of aroma properties of linalool and benzaldehyde during the transformation on a culture medium according to the invention compared to the corresponding transformation on a standard culture medium using a fermentation of Poria cocos as an example. Evolution of linalool on MEA (□); evolution of linalool on 3TT+Asp (○); evolution of benzaldehyde on MEA(); evolution of benzaldehyde on 3TT+Asp (x). For the preparation of the EIC, the fragment at m / z=93 was selected for linalool and the fragment at m / z=106 was selected for benzaldehyde. The abbreviations used are as described above.

[0031] Fig. 9 : Diagram of the progression of aroma characteristics of methyl anthranilate (m / z=119) during transformation on a culture medium according to the invention compared to fermentation using Poria cocos as an example during transformation on a standard culture medium. Progression of methyl anthranilate on MEA (x); Progression of methyl anthranilate on 3TT+Asp (○).

[0032] Fig.10 : Semi-quantitative estimation of the concentration of selected aroma substances (○) after conversion on a culture medium according to the invention using Poria cocos fermented on 3% pomace with 0.624% monosodium L-aspartate as an example. Here, the headspace of the strain was analyzed after 10 days and compared with a calibration series (x) determined by standards. A: linalool; B: benzaldehyde; C: methyl anthranilate. The estimated amounts are indicated in μg / plate.

[0033] Fig.11: Sensory evaluation diagram of the transformation on the culture medium according to the invention using Poria cocos as an example of aqueous fermentation on 3% red currant pomace. The given attributes were evaluated by four trained persons on a scale of 0 to 5. Abbreviations used: JhB: currant pomace, Asp: 0.624% monosodium L-aspartate, d0: uncultured reference medium, d10: cultured with Poria cocos for 10 days.

[0034] Fig.12 : Sensory evaluation diagram of the transformation on the culture medium according to the invention as an example of aqueous fermentation using Poria cocos on 3% grape pomace of the Müller-Thurgau variety. The given attributes were evaluated by four trained persons on a scale of 0 to 5. Abbreviations used: Wine: grape pomace, Asp: 0.624% monosodium L-aspartate, d0: uncultured reference medium, d10: 10 days of culture with Poria cocos.

[0035] Fig.13 : GC-MS chromatogram of transformation on the culture medium according to the invention using Poria cocos fermented on 3% grape pomace, 0.6% monosodium L-aspartate, 1.5% agar-agar of Muscaris var. as an example. The chromatogram of the sample taken after 10 days of cultivation is shown; the split ratio is 3:1 for measurement.

[0036] Fig.14 : GC-MS chromatogram of transformation on the culture medium according to the present invention using Poria cocos for water fermentation on 3% strawberry puree, 0.6% monosodium L-aspartate, 1.5% agar-agar as an example. Shown is the chromatogram of the sample taken after 8 days of cultivation; the split ratio is 3:1 for measurement.

[0037] Fig.15 : GC-MS chromatogram of transformation on the culture medium according to the present invention using Poria cocos for aqueous fermentation on 3% raspberry puree, 0.6% monosodium L-aspartate, 1.5% agar-agar as an example. The chromatogram of the sample taken after 8 days of cultivation is shown; the split ratio is 3:1 for measurement.

[0038] Fig.16: Heat map of selected aromas during cultivation of Poria cocos on different bacterial media and different cultivation times. The EIC peak areas at m / z (benzaldehyde) = 106, m / z (linalool) = 93, m / z (linalool oxides I, II) = 111, m / z (2-undecanone) = 71, m / z (2-nonanone) = 58, m / z (geraniol) = 69, m / z (methylcarbamate) = 119 were used as base peaks. The assignment of the corresponding saturation of the selected fragments was performed based on the relative peak area of ​​the selected fragments in the EIC (0% to 100%) according to the scheme shown below. The effluent bacterial media used: M1: 3% pomace, M2: 3% pomace and 0.4% glucose, M3: 3% pomace and 0.8% glucose. Cultivation time: 0d: uncultured reference medium, 8d: 8 days, 10d: 10 days. Abbreviations used: ER: non-mutant strawberry pomace, HR: non-mutant raspberry pomace, MUT: Muscaris mutant wine grape pomace, Asp: 0.6% monosodium L-aspartate.

[0039] Fig.17 : GC-MS chromatogram of transformation on a culture medium according to the invention using a fixed bed culture of Poria cocos on strawberry puree as an example. Shown is a chromatogram of a sample taken after 28 days of cultivation; measured by SPME-GC-MS in undivided mode. DETAILED DESCRIPTION

[0040] The present invention is essentially based on the discovery that a transformation medium having a composition based on plant parts of the Currant family, Rosaceae or Vitaceae contains aroma precursors of great interest that can be bioconverted into aroma substances. The term aroma substance is used herein to describe a compound that imparts a perceptible taste or odor in an aroma-active amount. In this case, the term "aroma-active" means that the amount of the compound is sufficient to induce a sensory effect on the olfactory and / or taste receptors when a preparation containing the compound is used. This effect can also manifest itself by reducing or masking sensory perceptions based on unpleasant tastes and / or odors.

[0041] The method according to the present invention avoids the need for harsh manufacturing conditions, organic solvents or heavy metal catalysts, which are almost unavoidable in the case of chemical synthesis. Therefore, the present invention provides an environmentally friendly alternative to chemical synthesis, which further meets the growing demand for aroma substances such as linalool.

[0042] Furthermore, the method according to the invention is characterized in that it provides the possibility of using agricultural waste streams such as leaves or pomace of the Currant family, Rosaceae or Vitaceae as a source of high-quality aroma substances and thus being beneficially integrated into the value chain of agricultural production. For this reason, the following process is particularly preferred: the component based on plant parts of the Currant family, Rosaceae or Vitaceae is provided in the form of leaves and / or pomace and / or extracts thereof as described herein. Very particular preference is given to using the pomace of currants, in particular the pomace of black currants as described herein. In the method according to the invention described herein, the proportion of the component based on plant parts of the Currant family, Rosaceae or Vitaceae is generally in the range of 0.2% by weight to 100% by weight, based on the total weight of the transformation medium. If the culture is an aquatic or submerged culture, the proportion of the component based on the plant parts of the family Currantae, Rosaceae or Vitaceae in the method according to the invention described herein, based on the total weight of the conversion medium, is preferably 0.2% to 20% by weight, preferably 0.5% to 10% by weight, further preferably 1% to 5% by weight, and most preferably 2% to 4% by weight. If the culture is carried out in the form of a fixed bed culture, the proportion of the component based on the plant parts of the family Currantae, Rosaceae or Vitaceae in the method according to the invention described herein, based on the total weight of the conversion medium, is preferably 10% to 100% by weight, preferably 30% to 90% by weight, further preferably 50% to 80% by weight, and most preferably 60% to 70% by weight.

[0043] The term "currant family" refers to plants of the family Ribes. Preferred according to the invention are methods using plant parts of species and varieties suitable for soft fruit production, in particular plant parts of currants, gooseberries and hybrids thereof such as blackcurrant trees. Further preferred are methods using plant parts of white or red gooseberries, red currants, white currants, black currants or black currants and hybrids and varieties thereof. Most preferred are methods according to the invention using plant parts of blackcurrants (Ribes nigra) and varieties and cultivars thereof.

[0044] The term "Rosaceae" refers to plants of the family Rosaceae. According to the invention, preference is given to methods which use plant parts of species and varieties suitable for soft fruit production, in particular plant parts of strawberry and raspberry and hybrids and varieties thereof.

[0045] The term "vitaceae" refers to plants of the family Vitaceae. According to the invention, preference is given to methods using plant parts of species and varieties suitable for soft fruit production, in particular plant parts of grapevines and hybrids and varieties thereof. Further preference is given to methods using plant parts of Vitis vinifera (vinifera grapes) and varieties thereof.

[0046] In particular, the plant components include above-ground vegetative or reproductive tissues, preferably leaves, buds, petals and / or flower buds, berries and their subcomponents such as cores, peels and pulps. Preferably, the plant parts are leaves and / or berries and further preferably a whole ripe berry.

[0047] Ingredients based on plant parts are understood in the context of the present invention to be ingredients which are obtained from parts of the plant in question. In this case, the ingredients used according to the method of the invention do not necessarily have to occur in nature in the same way. Instead, the ingredients of the plant in question can also be obtained by further processing of naturally occurring ingredients. Preferred measures for further processing include (partial) drying, (partial) fermentation and / or (partial) pressing. Preferably, the plant is waste from other industries which use the corresponding plant or at least parts thereof as raw material. Particularly preferably, the plant part is a berry (whole) or a residue obtained from juice extraction (so-called pomace).

[0048] Of particular interest to the present invention are pleasant taste and / or odor impressions. The assessment of whether a taste and / or odor impression is considered pleasant or rather unpleasant can be carried out by a trained panel based on a sensory analysis evaluating the sensory impression between negative (pleasant) and positive (unpleasant). Further grades such as strong negative, neutral and strong positive can be set for a more precise classification. The aroma substance to be evaluated occurs in a mixture of other compounds, possibly other aroma substances, and the determination of the aroma of the aroma substance to be evaluated can be carried out, for example, by gas chromatography olfactometry. In the present invention, an aroma substance or an aroma substance mixture is in particular a substance that imparts a pleasant odor impression, and is therefore also referred to as an aromatic substance.

[0049] In the case of preparing aroma substances with a pleasant taste and / or odor impression, a conversion medium containing the following ingredients stands out: the ingredients are based on plant parts of black currants, preferably on plant parts of black currants (black currants), and in particular on plant parts of Cassis Titania var. This ingredient obviously contains aroma precursors, which, through biocatalytic conversion, can produce aroma substances that are extremely pleasant to the senses. Therefore, the conversion medium provided in the method according to the invention preferably contains ingredients based on plant parts of currants, which are preferably black currants and further preferably Cassis Titania var. Thus, the aroma substances that can be produced using suitable biocatalysts have floral, fragrant, fruity or notes reminiscent of wild berries or citrus. When properly converted on a standard bacterial culture medium based on malt extract, these aromas are not easily perceived. On the contrary, the olfactory impression is described herein as a fungal, sour and tropical fruit smell. Other advantages are obtained by the following measures.

[0050] Preferably, the step of recovering the aroma substance or aroma substance mixture comprises at least partially separating the aroma substance produced or at least one aroma substance of the aroma substance mixture from the biocatalyst. Preferably, a substantially complete separation from the biocatalyst is performed. In addition, the step of recovering may comprise enriching, concentrating and / or isolating the aroma substance produced or at least one aroma substance of the aroma substance mixture. As used herein, the term "at least one aroma substance" alternatively refers to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aroma substances.

[0051] For the purposes of the present invention, whole-cell biocatalysts have proven to be particularly effective due to their ability to simultaneously or sequentially regenerate the conversion reactions catalyzed. Thus, the conversion step may include culturing in the sense of "growing" or regenerating the biocatalyst. However, it is generally known to those skilled in the art that conversion using whole cells ultimately relies on one or more catalyzed reactions, and therefore the present invention also includes the following method: the biocatalyst for producing an aroma substance or an aroma substance mixture also includes only the catalytically relevant units of the whole-cell biocatalyst for producing an aroma substance or an aroma substance mixture.

[0052] The biocatalyst is preferably a fungus, a fungal cell thereof or at least one catalytically related unit thereof. The catalytically related unit herein refers to macromolecules such as enzymes or ribozymes, as well as optional cofactors and cosubstrates required for the conversion. Preferably, it is a fungus capable of growing on lignocellulose and / or a fungus selected from the edible fungi group (or its corresponding fungal cell). Therefore, the ability to grow on lignocellulose is advantageous because such fungi are particularly suitable for culturing on the currant-based ingredients described herein. In addition, aroma substances such as benzaldehyde can be obtained directly from the degradation of lignocellulose. When using the produced aroma substances or aroma substance mixtures, edible fungi are advantageous in terms of safety. This preferred requirement profile applies to many fungi of the Basidiomycetes (phylum) (Basidiomycetes). In addition, particularly excellent odor impressions can be obtained using fungi from the Basidiomycetes, especially when the currant is the black currant described herein.

[0053] Of particular note is the finding that the transformation using species belonging to the Basidiomycetes results in an overall pleasant odor. This means that fungal (mushroom), sour and tropical fruity notes, which are known as typical fungal aromas, are not or are difficult to form. Therefore, the following method is preferred: the fungus is selected from the class Basidiomycetes and / or the plant part of the family Currant is selected from the group of plant parts of currants, preferably the group of plant parts of black currants, more preferably the group of plant parts of black currants Titania var.

[0054] In particular, the method described herein is preferred, wherein the fungi of the class Basidiomycetes are selected from the group consisting of Agaricus bisporus, Agrocybe oleraceus, Armillaria mellea, Cigarette pipe mushroom, Coprinus comatus, Rough-margin black egg nest, Flammulina velutipes, Clover, Clustered yellow toughness umbrella, Salmon shell rake tooth mushroom, Corydalis, Lentinula edodes, Lilac mushroom, Puffball, Grouped small leather umbrella membrane cover, M. pseudocorticola, Garlic small leather umbrella, Late-growing fan mushroom, Yellow spore original hairy leather mushroom, Fan-shaped pleurotus, Purple spore pleurotus, Hydrangea, Hairy leather mushroom, Inospora odorifera, Thin-skinned cheese mushroom and Poria cocos. These species are clearly superior to a large number of other fungi or fungal transformation medium combinations that have been tested and not specified herein in terms of their ability to form aroma substances with a pleasant odor impression.

[0055] Further preferred is the method described herein, wherein the fungus is selected from the group consisting of Agaricus truncatus, Agrocybe chinensis, Coprinus comatus, Clover, Agaricus fasciatus, Agaricus salmoides, Agaricus robur, Lentinula edodes, Puffball, Agaricus gregatus, M. pseudocorticola, Agaricus scaly, Pseudocorticola tarda, Pseudocorticola tarda, Pseudocorticola lutea, Pseudocorticola chrysosporus, Pleurotus eryngii, Pleurotus eryngii, Hydrangea, Agaricus odoratus, Agaricus thunbergii, Poria cocos and Poria cocos. The selection results in a particularly pleasant aroma in at least one possible culture form.

[0056] Further preferred is the method described herein, wherein the fungus is selected from the group consisting of Agrocybe edulis, Coprinus comatus, Clover, Acanthocephala fasciata, Cockscomb, M. pseudocorticola, Acanthocephala scabra, Pleurotus eryngii, Pleurotus purpurogenus, Hydrangea spp., T. suaveolens and Poria cocos. The above selection results in a particularly pleasant aroma in at least two possible cultivation forms.

[0057] Further preferred is the method described herein, wherein the fungus is selected from the group consisting of Poria cocos and Clover. The fungi Clover and Poria cocos show very strong odor impressions in all tested culture forms on all tested substrates. Clover produces a citrus aroma in this context, and the aroma of Poria cocos is floral, fruity and reminiscent of wild strawberry. Thus, this selection results in a particularly pleasant aroma, regardless of the possible culture form.

[0058] As mentioned above, preferred biocatalysts include single cells or the above preferred catalytically active units and further preferred are fungi.

[0059] In addition, according to the present invention, the following method is preferred: the conversion medium provided comprises an aspartic acid source, preferably an L-aspartic acid source, further preferably sodium L-aspartate, most preferably monosodium L-aspartate. Therefore, in the course of conducting experiments, it was found that the addition of monosodium L-aspartate offsets the pH drop otherwise observed and ultimately obtains a particularly strong wild strawberry smell. After about 10 days of cultivation, the smell is enhanced to a floral and strong fruity aroma reminiscent of wild strawberries. When cultivated on the conversion medium supplemented with aspartic acid described herein, the signal intensity of the aroma is significantly higher than when cultivated on a standard strain medium based on malt extract. The intensity of linalool is particularly high, which is very related to the floral odor of the strain. Some aroma active substances, such as benzaldehyde, linalool, linalool oxide I, II and 2-undecanone, are particularly well formed on the conversion medium containing aspartic acid, which is preferably provided in the form of pomace as described herein. The content of the aspartic acid source is based on the total weight of the conversion medium, typically 0.1% to 20.0%. If the culture is in the form of aquaculture or submerged culture, in the method according to the invention described herein, the content of the aspartic acid source is preferably 0.1 to 5% by weight, more preferably 0.2 to 3% by weight, further preferably 0.3 to 2% by weight, and most preferably 0.4 to 1% by weight, based on the total weight of the conversion medium. If the culture is in the form of fixed bed culture, in the method according to the invention described herein, the content of the aspartic acid source is preferably 0.1 to 20% by weight, more preferably 2.5 to 17.5%, further preferably 5 to 15% by weight, and most preferably 7.5 to 12.5% ​​by weight, based on the total weight of the conversion medium.

[0060] Alternatively, the pH drop can be counteracted by other buffer substances. Preferably, the pH range of the conversion medium during the conversion is pH 2 to pH 7, preferably pH 4 to pH 6, and in particular pH 4.2 to pH 5.5. In the case where the conversion lasts for several days, this means that this pH range is at least substantially maintained during the entire period.

[0061] Additional nutrient sources can be added to the conversion medium to optimize aroma properties and intensity. In addition to the aspartic acid source described herein, these additional nutrient sources particularly include glucose; trace elements and other amino acids selected from the group consisting of Mg2+, Zn2+, Fe3+, Mn2+, Cu2+, K+, Cl-, SO42- and PO43-.

[0062] In the context of the present invention, all forms of cultivation are basically contemplated, even though the specific form may influence the aroma substances formed. For example, particularly good results were obtained with transformations using effluent strains, especially when Poria cocos and / or pomace were used as the corresponding biocatalyst and the ingredients based on the family Currantae described herein. Submerged and fixed bed strains also gave very pleasant aromas, depending on the fungal species used as described herein and the specific form of the ingredients based on the family Currantae, Rosaceae or Vitaceae described herein.

[0063] It has been found that it is particularly advantageous for the aroma substances that can be produced that the transformation medium containing the plant parts of the gooseberry described herein and the aspartic acid source described herein is added is transformed by means of the basidiomycetes described herein. Instead of the aspartic acid source, a pH buffer component can also be added to the transformation medium as described herein above so that the pH is maintained within the pH range described herein during the conversion. In particular, culturing Poria cocos on the gooseberry residue of Titania variants produces a floral aroma and a fruity smell reminiscent of wild strawberries. Poria cocos is also an edible fungus (belonging to the phylum Basidiomycetes) used in traditional Chinese medicine. In addition to a variety of minor components, linalool, benzaldehyde and methyl anthranilate also form key aroma components.

[0064] The method according to the present invention is particularly suitable for producing an aroma substance or an aroma substance mixture, which comprises at least one, preferably two or three, more preferably four or five and most preferably six, seven, eight, nine or ten compounds selected from 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, linalool and 2-aminobenzaldehyde. Particularly preferred is a method involving the production of at least one compound, preferably two compounds and further preferably all compounds of linalool, benzaldehyde and methyl anthranilate. These compounds are unique features of the aroma substance mixture that can be produced by the transformation culture medium described herein. In particular, methyl anthranilate shows a strong fruity odor of wild strawberry.

[0065] The exact composition of the type and intensity of the aroma substances (also referred to herein as aroma compositions) produced can therefore be influenced by changing the time of recovery of the aroma substances or aroma substance mixtures. In this case, it has been proven to be effective according to the collection time in the method of the present invention, wherein the extraction of the aroma substances or aroma substance mixtures is carried out 3 to 14 days, preferably 5 to 14 days and in particular 9 to 13 days after the start of contact. In addition to this, it is also envisaged that the extraction time of the aroma substances or aroma substance mixtures is determined as a function of a predetermined aroma composition. Among other aspects, this enables collection at a time when there is a particularly pleasant aroma substance mixture and / or a particularly pleasant aroma substance that is clearly superior to other aroma substances that may be formed.

[0066] Furthermore, the present invention relates to the use of a conversion medium comprising a component based on a plant part of the Currantaceae family as described herein and / or a biocatalyst as described herein for producing an aroma substance or an aroma substance mixture as described herein. In particular, the component based on a plant part of the Currantaceae family is a component based on a plant part such as leaves or pomace of blackcurrant as described herein. In particular, the biocatalyst is a fungus from the class of Basidiomycetes as described herein.

[0067] In addition, the present invention relates to a composition, in particular a composition prepared (preparable) according to the method described herein, comprising 2 or more particularly all compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate. Preferred is the following composition according to the present invention: the volume weight ratio of linalool to benzaldehyde is between 100:1 and 4:1 and / or the volume weight ratio of linalool to methyl anthranilate is between 50:1 and 2:1 and / or the volume weight ratio of benzaldehyde to methyl anthranilate is between 3:1 and 1:10. The composition according to the present invention further preferably comprises at least one, preferably 2 or 3, more preferably 4 or 5 and most preferably 6, 7, 8, 9 or 10 compounds selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool.

[0068] In addition, the present invention relates to the use of the composition according to the invention described herein as a nutritional, cosmetic, health or edible preparation or in a nutritional, cosmetic, health or edible preparation, in particular as a mixture of aroma substances (preferably for the purposes described herein). Therefore, the present invention also relates to a nutritional, cosmetic, health or edible preparation comprising or consisting of the composition according to the invention described herein.

[0069] The present invention is explained in more detail below with reference to the following examples.

[0070] Example

[0071] 1. Experimental part

[0072] 1.1 Materials and Chemicals

[0073] The side stream used by the gooseberry industry and the wine industry was provided by the University of Geisenheim, Germany. Raspberry or strawberry pomace was prepared by pureeing and sieving fresh, commercially available, non-mutant berries. The remaining pomace was used as the medium component. The sources of the Basidiomycota fungi used are shown in Table 1.

[0074] Table 1: Overview of the tested fungi, their sources and culture media; DSMZ: Leibniz Institute DSMZ-German Collection of Microorganisms, Braunschweig, Germany; CBS: CBS-KNAW Collection, Westerdijk Fungal Biodiversity Institute, Utrecht University, The Netherlands; S: Institute of Food Chemistry and Food Biotechnology, University of Giessen, Giessen; MEA: malt extract agar; SNL: standard nutrient solution agar.

[0075]

[0076]

[0077] 1.2 Strain maintenance

[0078] The fungi to be studied (Table 1) were cultured on malt extract agar plates (MEA) or standard nutrient solution (SNL) agar plates for strain maintenance. All media were steam sterilized at 121°C for 20 minutes. The media composition was as follows:

[0079] MEA: 20g·L -1 Malt extract (ME), 15g·L -1 Agar - Agar in deionized water.

[0080] SNL: 30g·L -1 Glucose monohydrate, 15 g·L -1 Agar-agar, 4.5 g·L -1 Asparagine monohydrate, 3 g·L -1 Yeast extract, 1.5 g·L-1 potassium dihydrogen phosphate, 0.5 g·L -1 Magnesium sulfate hydrate, 400 μg·L -1 EDTA, 90 μg·L -1 Zinc sulfate (II) heptahydrate, 80 μg·L -1 Iron chloride hexahydrate, 30 μg·L -1 Manganese(II) sulfate monohydrate, 5 μg·L -1 Copper (II) sulfate pentahydrate in deionized water and adjusted to pH 6.0.

[0081] For cultivation, approximately 0.5 cm from an 80% overgrown agar plate was 2 The mycelial fragments were placed on new agar plates and The plate was sealed and incubated at 24°C protected from light.

[0082] 1.3 Screening of the blackcurrant residue stream

[0083] The cultivation of fungi of the phylum Basidiomycota on leaves and pomace of blackcurrant residue streams was carried out in submerged, effluent and fixed bed cultures. Initially, the residue stream was used as the sole nutrient source for the 26 tested fungi.

[0084] In order to cultivate fungi in the effluent strains, agar plates with different concentrations of substrate were prepared. Depending on the substrate, different concentrations of agar were required. Keeping similar to the strains, they were inoculated with 26 selected fungi (Table 1). After 4 to 7 days, the sensory properties of the plates were checked for the first time, depending on the growth of the fungi. The effluent strains of Poria cocos were cultivated on gooseberry pomace, wine grape pomace, strawberry puree pomace or raspberry puree pomace for further experiments, with 3% pomace added to 30% deionized water and 70% deionized water with 1.5% or 3% agar autoclaved and mixed before pouring. Each plate contained approximately 16 mL of culture medium.

[0085] To grow the fungus in submerged culture, first prepare a pre-culture stock (100 mL) in 2% ME medium. This is done by placing approximately 1 cm of hyphae overgrown from a strain holding plate. 2 The agar fragments were transferred to a conical flask containing ME medium, where the fragments were homogenized using a disperser (Ultraturrax, 10000rpm, 30s). The pre-made strains were incubated at 150rpm in the dark at 24°C for eight days. For inoculation of the main strain, the pre-made strains were homogenized (Ultraturrax, 10000rpm, 30s), centrifuged (3000g, 10 minutes) and the supernatant was poured. The mycelium was washed twice with sterile water and then suspended in 100ml of sterile water. 4mL of this suspension was added to 40mL of the main strain medium. The main strain medium consisted of 15g·L -1 or 30 g·L -1 The medium consisted of deionized water. The strains were grown at 150 rpm at 24 °C in the dark. Sensory analysis was performed by sniffing directly on the flasks starting from the third day of cultivation. For comparison, ME medium and the main strain on a medium not inoculated with the main strain served as blanks.

[0086] To cultivate fungi in fixed bed culture, the pre-prepared culture was cultured and homogenized in the manner described above. 2 mL of the suspension was added to the fixed bed culture medium. To prepare the fixed bed culture medium, 20 g of pomace, 10 g of water and 2 g of monosodium L-aspartate (Asp) were homogenized in a flask and autoclaved. The inoculated fixed bed culture medium was statically cultured at 150 rpm at 24 ° C in the dark. For comparison, uncultured fixed bed medium was used as a blank.

[0087] 1.4 Optimization of the culture on the blackcurrant residue stream

[0088] To optimize aroma characteristics and intensity, other sources of nutrients are added to the substrate. In addition to pH adjustment, glucose, trace elements (Mg2+, Zn2+, Fe3+, Mn2+, Cu2+, K+, Cl-, SO42-, PO43-), monosodium L-aspartate and other amino acids are also supplemented. In addition, the substrate concentration of the currant side stream is varied.

[0089] 1.5 Sensory analysis

[0090] The sensory analysis was performed by a trained panel (n=4) to preselect the fungus-substrate combinations. For this purpose, the species were examined in a "simple descriptive test" and the overall impression over several incubation days was evaluated. The scale for evaluating the overall impression included six levels from strongly negative (0) to strongly positive (5). The aroma was identified by GC-MS-O.

[0091] 1.6 Determination of hyphal growth

[0092] During the incubation period, fungal growth was recorded daily by drawing the overgrowth area on the back of the agar plates and determining the diameter. From this, the hyphal area of ​​overgrowth was calculated.

[0093] 1.7 Determine pH

[0094] To determine the course of the pH of submerged strains, a sample (2 mL) was taken and the pH was determined using a pH meter. For effluent strains, the pH of the strain was determined as follows: one plate of agar was homogenized after adding 15 mL of deionized water using an Ultratrurrax (10000 rpm, 10 s), then centrifuged (10 min, 4000 g) and the pH of the supernatant was measured using a pH meter.

[0095] 1.8 GC-MS-O analysis

[0096] The aroma of the bacterial strains in the water of currant pomace was extracted by stir bar sorptive extraction (SBSE) at 24 ° C for 60 minutes. For this purpose, a 10 mm magnetic stirring bar with a 0.5 mm PDMS (polydimethylsiloxane) coating (Twister, GERSTEL, Mühlheim an der Ruhr, Germany) was fixed to the head space of the culture vessel with a magnet. After incubation, the magnetic stirring bar was removed with a magnetic bar, rinsed with deionized water, dried with a lint-free cloth, and placed on a conditioned thermal desorption unit (TDU) pad (GERSTEL). TDU desorption was performed without splitting. The analytes were cold collected using a glass evaporator tube with a silanized glass filler (GERSTEL) in a cold injection system 4 (CIS) (GERSTEL). Gas chromatography was performed using a GC-MS-O consisting of an A7890BGC (Agilent Technologies, Waldbronn, Germany) coupled to a 5977B MSD (Agilent Technologies) and an olfactory detection port (ODP) (GERSTEL). An Agilent J&W VF-WAXms (30 m×0.25 mm ID×0.25 μm) was used as a polar column, and an Agilent J&W DB-5ms (30 m×0.25 mm ID×0.25 μm) was used as a nonpolar separation phase. The column was concentrated at 1.56 mL min -1 Helium was used as carrier gas at a constant flow rate of 1.5 mmol / l. The gas flow was directed to the MS and ODP with a split ratio of 1:1. Further settings are listed as follows: Septum purge flow 3 mL min -1 , scan mode total ion current (TIC), scan range m / z 33-300, ionization energy 70 eV, EI source temperature 230 °C, quadrupole temperature 150 °C, MS transfer line temperature 250 °C, helium quench gas 2.25 mL·min -1 , N2 collision gas 1.5 mL min -1 , ODP3 transmission line temperature 250°C, ODP mixing chamber temperature 150°C, ODP supplementary gas N2.

[0097] To investigate the changes in aroma relative intensity and identification using VF-WAXms, the -1 The oven temperature was heated from 40°C to 150°C for 3 minutes, and then -1 The temperature was raised to 240°C and held at this temperature for 7 minutes. The TDU temperature program was heated from 40°C (0.5 minutes) to 250°C (10 minutes) at 360°C·min-1. After a holding time of 0.5 minutes in CIS, the temperature was raised at 12°C·s-1. -1 The temperature was raised from -20°C to 250°C and held for 5 minutes. The CIS split ratio was varied between splitless and 50:1.

[0098] For semi-quantitative estimation and determination of retention index using DB-5ms, the oven was set at 5 °C min -1 The temperature was raised from 40°C to 320°C in 3 minutes and maintained at this temperature for 7 minutes. -1 The TDU was heated to 250° C. Cold collection was performed differentially by CAS at 100° C. The split ratio at the CIS was varied between splitless and 50:1.

[0099] The aroma of the effluent strain of Poria cocos on wine grape pomace, strawberry pomace or raspberry pomace was extracted using headspace SBSE in the manner described above. Gas chromatography analysis was then performed using a GC-MSFID consisting of an 8890GC (Agilent Technologies) coupled to a 5977B MSD (Agilent Technologies) and a flame ionization detector. The column used was an Agilent VF-WAXms (30 m×0.25 mm ID×0.25 μm). The oven temperature was set at 3°C ​​min -1 From 40°C to 230°C and hold for 30 minutes. TDU temperature program from 30°C to 150°C at 60°C·min-1 and hold for 10 minutes. After 0.1 minute hold time, CIS at 12°C·s-1 -1 The temperature was raised from -20°C to 250°C and maintained for 10 min. The CIS split ratio used was 3:1. The flow rate was 2.4 mL min -1 Helium was used as carrier gas at a constant flow rate of 1.3 Å. This gas flow was directed to the MS and FID with a split ratio of 4:1. Different from the previous system, the other settings are listed as follows: scan range m / z 25-370, MS transfer line temperature 280 °C, FID H2 gas flow 35 mL·min -1 , FID supplementary gas N2.

[0100] In order to analyze the fixed bed strains, 3g of the fixed bed strain culture medium of mycelial growth was weighed into a 20mL headspace vial and analyzed by solid phase microextraction (SPME) in the headspace. For this purpose, the sample was incubated at 45°C at 250rpm for 20 minutes and extracted with polydimethylsiloxane / divinylbenzene fiber for 5 minutes. Desorption was performed in CIS 4 at 230°C without splitting for 60 minutes. Gas chromatography analysis was performed using GC-MS, which consisted of a 7890B GC coupled to a 5977B MSD. Agilent DB-FFAP (60m×0.25mm ID×0.25μm) was used as a column. After 5 minutes, the oven temperature was increased to 10°C·min -1 The temperature was raised from 40°C to 230°C and maintained for 10 minutes. -1Helium was used as carrier gas at a constant flow rate of 100 ℃. This gas flow was directed to the MS and FID with a split ratio of 10:1. Other deviating settings are listed as follows: scan range m / z 25-550, MS transfer line temperature 250 °C, FID H2 gas flow 30 mL·min -1 , FID supplementary gas N2.

[0101] 1.9 Identification of aroma substances

[0102] For identification of the substance, 10-30 mg of the reference substance was mixed with 50-1000 μL DMSO (Carl Roth, Karlsruhe, Germany) depending on the solubility and topped up to 1 mL with deionized water if necessary. The following aroma substances were used for analysis: 2-aminobenzaldehyde ≥98% (Sigma Aldrich), 2-nonanone ≥99% (Accos Organics, Hull, Belgium), linalool 97% (Accos Organics), methyl anthranilate 99% (Acros Organics), (+)-2-octanol 98% (Alfa Aesar, Haverhill, USA), geraniol 97% (Alfa Aesar), 2-octanone 98% (Sigma Aldrich, St. Louis, USA), linalool oxide isomer mixture (Sigma Aldrich), 2-undecanone pure (Honeywell Fluka, Bucharest, Romania), benzaldehyde pure (Applichem, Darmstadt, Germany). From this, 1:200 and 1:1000 dilutions were prepared with water and 0.5 mL of the solution was plated on agar (1.5% agar in deionized water). Adherence to the headspace and analysis by GC-MS using the method described previously in 1.8. Retention indices were calculated according to van den Dool and Kratz (van Den Dool, H.; Dec. Kratz, P., Journal of Chromatography A, (1963) 11, 463).

[0103] 1.10 Semi-quantitative estimation of the concentrations of methyl anthranilate, linalool and benzaldehyde

[0104] For semi-quantitative estimation, linalool, methyl anthranilate, and benzaldehyde were dissolved in deionized water. For benzaldehyde, 3 mg·L -1 , 4mg·L -1 , 8mg·L -1 , 12mg·L -1 and 16 mg·L -1For methyl anthranilate, the concentration was 9 mg·L -1 、14mg·L -1 , 19mg·L -1 , 41mg·L -1 and 80mg·L -1 For linalool, the concentration was 120 mg·L -1 、241mg·L -1 、361mg·L -1 and 722 mg·L -1 Specifications. Among them, on agar plates ( Linalool, methyl anthranilate and benzaldehyde were precipitated 0.5 mL each on 16 mL of 1.5% agar (deionized water). Attached to the headspace and analyzed similarly to the samples. The calculated peak areas are determined by the extracted ion chromatogram (EIC) of the specific substance fragments, and the intensity detected in the samples is compared with that of the standard substances to assign a concentration range to the samples.

[0105] 2. Results

[0106] In order to investigate the bioconversion of blackcurrant sidestreams to synthesize natural aromas, a total of 26 fungi from the phylum Basidiomycetes were tested. Table 2 summarizes the evaluation of the odor impression of the tested fungi-substrate combinations in both effluent and submerged species.

[0107] Table 2: Sensory evaluation of the overall impression of the fungus-matrix combinations between 0 (strong negative) and 5 (strong positive). -: No cultivation was performed.

[0108]

[0109]

[0110] Based on the sensory analysis, several fungus-substrate combinations of interest were identified. The fungi Trifolium and Poria cocos showed very strong odor impressions on all tested substrates of all tested culture types. Trifolium here produced a citrus-like aroma, and the aroma of Poria cocos was floral, fruity and reminiscent of wild strawberry.

[0111] In order to analyze the growth characteristics of Poria cocos in more detail, the fungus was cultivated on different media. In addition to pomace, malt extract agar was chosen, which is very suitable as a complete medium for fungal growth. When cultivated on pomace, the addition of monosodium L-aspartate gave a particularly strong aroma. In addition to the smell, the cultures differed visually and in growth (see Figure 1 ).

[0112] After addition of aspartic acid, mycelium and nutrient medium turned brown. In addition, the growth was reduced compared to the reference medium (see Figure 2 ). Formation of less biomass and very flat hyphae.

[0113] Adding 0.624% monosodium L-aspartate reduced the pH less than in the strain without aspartate (see Figure 3 ). In the fermentation of pomace and MEA medium, the pH value decreased to about 2.5 during the culture process. In contrast, no pH decrease was observed in the culture medium of pomace supplemented with aspartic acid. The smell of wild strawberry was particularly strong in these strains.

[0114] For the analysis of aroma composition, SBSE analysis was performed in combination with GC-MS-O. Poria fermentation on blackcurrant pomace supplemented with 0.624% monosodium L-aspartate was identified as being of particular interest. The aromas detected were clearly different from those detected in fermentations performed on MEA or on currant pomace or currant leaves without the addition of aspartic acid.

[0115] In particular, C8 aromas such as 3-octanone, 1-octen-3-ol, 1-octanol, and (E)2-octen-1-ol were detected in MEA medium (see Figure 4 ). They are described as typical fungal aromas (Hofrichter, M., ed., Industrial Applications, (2011), Berlin, Heidelberg, Springer Berlin Heidelberg). This is closely related to the olfactory impression, which is described as fungal, sour and tropical fruity.

[0116] In contrast, when Poria cocos was cultivated on currant pomace with aspartic acid, the aroma was significantly different. After only a few days, the strain began to have a floral and fresh aroma. In addition, no aroma reminiscent of fungi was detected. After about 10 days of cultivation, the smell was stronger, with floral and strong fruity aromas reminiscent of wild strawberries.

[0117] When cultured on blackcurrant pomace supplemented with aspartic acid, the aroma signal intensity was significantly higher than when cultured on MEA (see Figure 5 ). Linalool is particularly strong and is closely associated with the floral aroma of this species.

[0118] In addition to linalool, further compounds were identified by GC-MS-O investigations that were characterized by an aroma of a fungus-matrix combination. In particular, methyl anthranilate showed a strong fruity odor of wild strawberry.

[0119] Compounds identified by olfaction also highlight the complexity of the aroma composition. For this purpose, the effluent strains of Poria cocos were examined by GC-MS-O at defined culture days in order to detect particularly strong aromas (see Figure 6 ).

[0120] In the olfactory evaluation of the strain thus carried out, the substances linalool and methyl anthranilate were very strongly detected. Benzaldehyde, which has a characteristic odor of bitter almonds, was also detected by means of ODP. In addition to these key aroma substances, a large number of other odors can be detected, the classification of which remains to be determined.

[0121] Table 3 shows a suggestion for selected substances contained in the samples and their retention index (RI) according to van den Dool and Kratz and their odors compared with the retention index of standard substances and odors described in the literature. By using a sample from the effluent inoculum of Poria cocos on 3% pomace + 0.624% monosodium L-aspartate, the formation of 2-octanone, 2-nonanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, 2-aminobenzaldehyde, methyl anthranilate and linalool was confirmed by correlation mass spectra (not shown) compared with the corresponding reference spectra obtained from the NIST database.

[0122] Table 3: Retention index (RI) according to van den Dool and Kratz and odor of the substances detected in the samples compared with standard substances (STD) and odors described in the literature. VF-WAXms and DB-5ms indicate the respective separation phases used (see Experimental part, 1.8).

[0123]

[0124]

[0125]

[13] Wood, William F.; Brownson, Mary; Smudde, R. Allen; Largent, David L.

[0126] (1992): 2-Aminobenzaldehydes. The Source of the “Sweet Odor” of Hebelomasacchari-olens. In: Mycologia84(6), p.935. DOI: 10.2307 / 3760296.

[0127] The retention indices of samples and standards as well as mass spectra and odors were very consistent.

[0128] The kinetics of the formation of the main aromas were recorded at 24-h intervals for 14 days using headspace SBSE (see Figure 7 ).

[0129] Some aroma active compounds such as benzaldehyde, linalool, linalool oxides I, II and 2-undecanone were formed almost exclusively on the medium with pomace and aspartic acid (3TT+Asp). Other compounds such as 2-nonanone, geraniol and methyl anthranilate were formed on the medium with pomace and aspartic acid and on the medium with MEA and aspartic acid (MEA+Asp).

[0130] Aroma analysis data (see Figure 8 and 9 ) correlated well with the aroma analysis data from the sensory analysis, where the characteristic odor was described as being strongest on the 10th day of incubation.

[0131] Linalool, methyl anthranilate and benzaldehyde are considered to be characteristic features of the aroma. For this reason, a semi-quantitative estimate of aroma concentration was made when Poria cocos was cultured on pomace with aspartic acid for 10 days (see Fig.10 ).

[0132] Based on the calibration series, it was possible to estimate 95–135 μg of linalool, approximately 5 μg of benzaldehyde, and 5–20 μg of methyl anthranilate per plate. Considering the plate volume of approximately 16 mL, this corresponds to approximately 7000 ± 1500 μg L -1 Linalool, 350±50μg·L -1 Benzaldehyde, 700±350μg·L -1 Corresponding to methyl anthranilate.

[0133] A trained panel conducted a sensory evaluation of Poria cocos cultures on a transformation medium based on blackcurrant pomace with and without aspartic acid addition. The effluent culture supplemented with aspartic acid was rated as significantly more intense in terms of the attributes "fruity", "floral" and "wild berry" (see Fig.11 ).

[0134] Interesting aroma properties can also be produced by cultivating Poria cocos in transformation media with components based on plant parts of the Vitaceae or Rosaceae families. Thus, cultivation of Poria cocos on media consisting of wine grape pomace of Müller-Thurgau, Gewürztraminer or Muscaris resulted in aroma properties with fruity and floral aromas reminiscent of wild strawberries, depending on the duration of cultivation (see Table 4).

[0135] Table 4: Sensory evaluation of transformations on the culture medium according to the invention using Poria cocos fermented with 3% wine grape pomace of different varieties MT: Müller-Thurgau, GT: Gewürztraminer, MU: Muscaris as an example. Summarized aroma attributes for different incubation durations.

[0136]

[0137] Sensory evaluation of the Poria cocos effluent culture on a transformation medium based on grape pomace of the Müller-Thurgau variety supplemented with aspartic acid showed similarities to the effluent culture based on red currant pomace (see Fig.12 ).

[0138] By means of gas chromatography, aroma-active compounds 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, methyl anthranilate and linalool were detected in the culture of Poria cocos on media containing components of Rosaceae or Vitaceae. Fig.13 , 14 15 and 16 show example chromatograms of bacterial species in the effluent of Poria cocos on Muscaris wine grape pomace, strawberry pomace or raspberry pomace, all of which were supplemented with 0.6% monosodium L-aspartate.

[0139] Fig.16 Comparison of the amounts of aroma-active compounds formed when Poria cocos is grown in water on media containing components of the Rosaceae or Vitaceae family and additionally varying the glucose content. The aroma-active compounds indicated could not be detected or were detected only in very low amounts in the uncultured reference medium (M1 (0d)) without added glucose. Cultivation on media based on strawberry and raspberry puree, additionally containing 0.4% and 0.8% glucose (M2 and M3, respectively), resulted in the formation of smaller amounts of the presented aroma-active compounds compared to the medium M1 (see Table 1). Fig.16 ). Thus, the formation of the present aroma-active compounds is mainly achieved by biotechnological transformation of the plant components present in the culture medium.

[0140] The culture method used has a significant effect on the quantitative composition of the aroma of the strain. Therefore, the aroma active compounds, which are the aroma characteristics of the water-derived strain of Poria cocos, are also detected in deviated amounts by submerged culture or fixed bed culture of the fungus. Fig.17 An example chromatogram showing fixed bed culture of Poria cocos on medium with strawberry pomace and aspartic acid.

Claims

1. A method for producing an aroma substance or an aroma substance mixture, comprising the following steps: preparing a transformation medium comprising plant components of the Currant family; contacting the transformation medium with at least one fungus from the class Basidiomycetes, the at least one fungus being capable of forming an aroma substance or a mixture of aroma substances on the transformation medium; converting the plant components into the aroma substance or aroma substance mixture with the aid of the fungus; and recovering the aroma substance or aroma substance mixture, wherein the aroma substance or aroma substance mixture comprises at least one compound selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool, wherein the plant component is selected from berries and sub-components, extracts and pomace thereof, wherein the fungus is Poria cocos, and The prepared transformation medium contains an aspartic acid source, and the content of the aspartic acid source is 0.1% to 20.0% based on the total weight of the transformation medium.

2. The method according to claim 1, wherein The Currantaceae family is selected from the group consisting of red currant, gooseberry and hybrids thereof.

3. The method according to claim 2, wherein the Currantaceae is selected from the group consisting of white or red gooseberry, red currant, white currant, black currant or dark black currant and their hybrids and variants.

4. The method according to claim 1, wherein the plant component is selected from ripe berries and selected from sub-components, extracts and pomace thereof.

5. The method according to claim 1 or 2, wherein the aroma substance or aroma substance mixture comprises at least one compound selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool.

6. The method according to claim 1 or 2, wherein the aroma substance mixture comprises at least 2 compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate.

7. The method according to claim 6, wherein the aroma substance mixture comprises all three compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate.

8. The method according to claim 1 or 2, wherein the pH of the transformation medium during the transformation is in the range of pH 2 to pH 7.

9. The method of claim 8, wherein the pH of the transformation medium during the transformation is in the range of pH 4 to pH 6.

10. The method of claim 8, wherein the pH of the transformation medium during the transformation is in the range of pH 4.2 to pH 5.

5.

11. The method according to claim 1 or 2, wherein the conversion is carried out in an effluent culture (Emerskultur), a fixed bed culture (Festbettkultur) or a submerged culture (Submerskultur).

12. The method of claim 11, wherein the transformation is carried out in an effluent culture.

13. Use of a transformation medium comprising plant components of the Currantaceae family and a fungus from the Basidiomycetes for producing an aroma substance mixture, wherein the aroma substance mixture comprises at least one compound selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool, wherein the plant components are selected from berries and are selected from subcomponents, extracts and pomace thereof, wherein the fungus is Poria cocos, and wherein the prepared transformation medium comprises an aspartic acid source, and the content of the aspartic acid source is 0.1% to 20.0% based on the total weight of the transformation medium.

14. A composition prepared according to the method of any one of claims 1 to 7, comprising 3 compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate, wherein the weight of the composition is The volume to weight ratio of linalool to benzaldehyde is between 100:1 and 4:1, and / or The volume-to-weight ratio of linalool to methyl anthranilate is between 50:1 and 2:1, and / or The volume weight ratio of benzaldehyde to methyl anthranilate is between 3:1 and 1:

10.

15. The composition according to claim 14, further comprising at least one other compound selected from the group consisting of 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, methyl anthranilate, 2-aminobenzaldehyde and linalool.

16. A nutritional, cosmetic, health or edible preparation comprising or consisting of the composition according to claim 14 or 15.

17. Use of the composition according to claim 14 or 15 as a nutritional, cosmetic, health care or edible preparation or use in a nutritional, cosmetic, health care or edible preparation.

18. Use according to claim 17, wherein the composition is used as an aroma mixture.

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