Biotechnological production of aroma substances from currant, strawberry, raspberry and pomace of wine industry
阅读说明：本技术 由醋栗、草莓、覆盆子和葡萄酒工业的果渣生产香气物质的生物技术 (Biotechnological production of aroma substances from currant, strawberry, raspberry and pomace of wine industry ) 是由 霍尔格·佐恩 斯文尼亚·萨默 纳丁·塞拉 克里斯蒂娜·施勒林 马丁·吕尔 马可·弗拉茨 于 2020-05-15 设计创作，主要内容包括：本发明涉及一种生产香气物质或香气物质混合物的生物催化过程,包括以下步骤:准备转化培养基,其包含醋栗科、蔷薇科和/或葡萄科的植物组分；将该转化培养基与来自担子菌纲的至少一种真菌接触,该至少一种真菌能够在该转化培养基上形成香气物质或香气物质混合物；借助于该真菌将该植物组分转化为该香气物质或香气物质混合物；以及可选地回收该香气物质或香气物质混合物,其中该香气物质或香气物质混合物优选地包含至少一种选自由2-辛酮、2-壬酮、2-十一酮、芳樟醇氧化物、苯甲醛、香叶醇、2-辛醇、邻氨基苯甲酸甲酯、2-氨基苯甲醛和芳樟醇组成的组的化合物。(The present invention relates to a biocatalytic process for producing an aroma or an aroma mixture comprising the steps of preparing a transformation medium comprising plant components of the family gooseberry, rosaceous and/or vitidae; contacting the transformation medium with at least one fungus from the class basidiomycetes, the at least one fungus being capable of forming an aroma or a mixture of aromas on the transformation medium; converting the plant component into the aroma or aroma mixture by means of the fungus; and optionally recovering the aroma or mixture of aromas, wherein the aroma or mixture of aromas 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.)
1. A method of producing an aroma or an aroma mixture, comprising the steps of:
preparing a transformation medium comprising plant components of the families gooseberry, rosaceae and/or vitiaceae;
contacting the transformation medium with at least one fungus from the class basidiomycetes, said at least one fungus being capable of forming an aroma or a mixture of aromas on the transformation medium;
converting the plant component into the aroma or aroma mixture by means of the fungus; and optionally
Recovering the aroma or aroma mixture,
wherein the aroma substance or mixture of aroma substances 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.
2. The method of claim 1, wherein
a) The family gooseberry is selected from the group consisting of gooseberry, gooseberry and hybrids thereof, preferably from the group consisting of gooseberry or gooseberry, redcurrant, gooseberry, blackcurrant or blackcurrant and hybrids and varieties thereof,
b) the Rosaceae is selected from strawberry, raspberry and their cultivars and hybrid population, and/or
c) The family Vitaceae is selected from the group of grapevines and their hybrids, and the family Vitaceae is preferably selected from the group of Vitis vinifera and their hybrids and varieties.
3. The method according to any one of the preceding claims, wherein the plant component is selected from the group consisting of leaves, buds, leaf buds, flower buds and berries, preferably from leaves and berries, and further preferably from mature berries, and in each case from their subcomponents, extracts and pomace.
4. The method according to any one of the preceding claims, wherein the at least one fungus from the basidiomycetes is selected from the group consisting of tetrasporium, agrocybe aegerita, armillaria mellea, armillaria tabescens, coprinus comatus, black cornel nest, flammulina velutipes, axillus, sterculia villosa, rabdosia salmoniliformis, corolla gallinarum, lentinus edodes, lepista nuda, lasiosphaera, pilula pilaris, m.
5. The method of claim 4, wherein the fungus is Poria cocos wolf or Trifolium pratense.
6. The method according to any of the preceding claims, wherein the aroma or aroma 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.
7. The process according to any of the preceding claims, wherein the aroma mixture comprises at least 2 and in particular all 3 compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate.
8. The method according to any one of the preceding claims, wherein the transformation medium prepared comprises an aspartic acid source, and the aspartic acid source is preferably present in an amount of 0.1% to 20.0% based on the total weight of the transformation medium.
9. The method according to any one of the preceding claims, wherein the pH range of the transformation medium during the transformation is pH 2 to pH 7, preferably pH 4 to pH 6, and in particular pH 4.2 to pH 5.5.
10. The method according to any of the preceding claims, wherein the transformation is performed in an effluent bacterial species, a fixed bed bacterial species or a submerged bacterial species, preferably in an effluent bacterial species.
11. Use of a transformation medium comprising plant components of the families gooseberry, rosaceae and/or vitidae and/or use of a fungus from the class basidiomycetes for the production of an aroma or an aroma mixture, wherein the aroma or aroma 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.
12. A composition, preferably prepared according to the process of any one of claims 1 to 10, comprising at least 2 and in particular all 3 compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate, preferably the weight of the composition is
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 the benzaldehyde to the methyl anthranilate is between 3:1 and 1: 10.
13. The composition according to claim 12, further comprising at least one additional 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.
14. A nutritional, cosmetic, nutraceutical or edible formulation comprising or consisting of a composition according to any one of claims 12 or 13.
15. Use of the composition according to any one of claims 12 or 13 as or in a nutritional, cosmetic, nutraceutical or edible formulation, preferably as an aroma mixture.
Technical Field
The present invention relates to the field of aroma substances. More specifically, the present invention relates to a process for preparing an aroma or aroma mixture as described herein. The invention also relates to the use of a transformation medium as described herein, comprising ingredients based on plant parts of the family gooseberry, rosaceae or vitidae, and/or of a biocatalyst as described herein for the production of an aroma or a mixture of aromas. Furthermore, the present invention relates to compositions comprising the compounds described herein and their use in the nutritional, cosmetic, nutraceutical or edible formulations described herein.
Background
Natural aroma substances are usually obtained by extraction from plants. Linalool may be taken as an example herein. Linalool is a monohydric alcohol belonging to the terpene family. The compound is colorless, flammable and has a unique aromatic odor. Linalool is a well known and abundant source of herbs such as basil, savory, coriander, oregano and thyme. Linalool can be used in a variety of applications. For example, the compounds are used as fragrances in perfume oils, or as substitutes for bergamots in the perfumery industry, due to their perfuming and/or deodorizing functions. Furthermore, it was found that the compound may be an aroma in wine.
To meet the strong and steadily increasing demand for linalool, a considerable portion of the current market supply is chemically synthesized. Although chemical synthesis offers the possibility of producing large amounts of fragrance material, 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 severe synthesis conditions such as high temperature and high pressure. In addition, chemical synthesis often uses heavy metal catalysts, combustible gases and organic solvents, the use of which is fraught with well-known drawbacks. For this reason, there is an increasing demand for alternative production processes of linalool and for other aroma-active compounds, which at least partially overcome the drawbacks of the known processes.
This can be improved by biotechnological processes based on the bioconversion of natural precursors into the desired aroma substances using microorganisms or their enzymes (Berger, r.g., Flavors and Fragrances: Chemistry, Bioprocessing and superstatinability, (2007), Berlin Heidelberg, Springer Verlag). Agricultural by-products, some of which are rich in dietary fiber and secondary metabolites as potential aroma precursors, may have great potential in biotechnological production of linalool and other aroma substances.
Disclosure of Invention
It is therefore the main object of the present invention to improve the above mentioned drawbacks and to provide a process for the preparation of aroma substances like linalool. In particular, there is interest in providing a process that is more environmentally friendly than known chemical syntheses.
WO2013/034613 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.
According to the invention, this main object is solved by a method for producing an aroma substance or an aroma substance mixture, comprising the steps of:
preparing a transformation medium (also referred to herein in part as a seed medium or substrate) comprising a composition based on plant parts of the families Ribes (Ribes nigraceae), Rosaceae (Rosaceae) and/or Vitaceae (Vitis amurensis); contacting the transformation medium with a biocatalyst; converting the component based on plant parts of the family gooseberry, rosaceae or vitidae with the aid of the biocatalyst to form an aroma or a mixture of aromas; and optionally
Recovering the aroma or aroma mixture.
Other objects, aspects and preferred embodiments of the present invention will become apparent from the following description, appended examples, drawings and particularly from the appended claims. .
Drawings
FIG. 1 is a graph comparing transformation performed on a seed culture medium (transformation medium) according to the present invention with transformation performed on a standard seed culture medium using fungal Poria as an example. Photographs of the poria cocos outgrowth after 11 days on different bacterial media are shown. A is Malt Extract Agar (MEA), B is MEA + 0.624% L-aspartic acid monosodium salt, C is 3% pomace, D is 3% pomace + 0.624% L-aspartic acid monosodium salt.
FIG. 2 is a graph comparing transformation performed on a seed culture medium according to the present invention with transformation performed on a standard seed culture medium using fungal Poria cocos as an example. The growth curves of Poria cocos effluent species on four different media were shown, MEA (□); MEA + Asp (); 3TT (x); 3TT + Asp (. smallcircle.). The abbreviations used are 3TT: 3% pomace of the Titania variety, Asp: 0.624% monosodium L-aspartate.
FIG. 3 is a graph comparing transformation performed on a seed medium according to the present invention with transformation performed on a standard seed medium using fungal Poria as an example. Shown with the poria in the MEA (□); MEA + Asp (); 3TT (x); 3TT + Asp (. smallcircle.) pH progression of the medium during the hydroponic cultivation. Abbreviations used are as previously described.
FIG. 4 GC-MS chromatogram of the transformation on a seed culture medium not according to the invention (no split measurement) using Poria fermentation on MEA as an example. Shown is a chromatogram of a sample taken after 8 days of incubation, which chromatogram indicates substance production.
FIG. 5 GC-MS chromatogram of transformation on the seed culture medium according to the invention using as an example the outlet water fermentation of Poria cocos on 3% red currant pomace, 0.624% L-monosodium aspartate, 3% agar-agar. Chromatograms of samples taken after 11 days of culture are shown; upper side measured at split ratio 50:1, lower side without split, with MS detector turned off between 13.5 minutes and 13.7 minutes (grey background).
FIG. 6 GC chromatograms of transformations on seed media according to the invention with ODP traces and showing substance production using Poria cocos for the outlet water fermentation on 3% currant pomace and 0.624% L-monosodium aspartate as an example. Shown are chromatograms of samples taken after 10 days of culture; between 13.2 minutes and 13.7 minutes, the MS detector was stopped (grey background). The numbered black bands generally represent the odor expected according to the following summary.
1 green 13 flowers, orange and faint scent
2 cheese 14 Green
3 slightly sweet and flowery flavor of solvent 15
4 slightly sweet, green and fragrant, 16 green and orange
5 fungi, vanilla 17 green, fir
6 green, slightly sweet, 18 fir floral, floral
7 fungal 19 solvent, slightly sweet
8 green, 20 unclear flower fragrance, bad smell and deep-fried
9 unclear, green, fir, flower 21 caramel incense
10 unclear 22 fruity incense
11 unclear, green, floral 23 unclear, green
12 almond protein candy, slightly sweet and 24 green
FIG. 7 is a heat map of the selected aroma during the incubation of Poria cocos wolf on different media; relative change during 14 days of culture. The area of EIC peak at position of m/z (benzaldehyde) 106, m/z (linalool) 93, m/z (linalool oxide I, II) 111, m/z (2-undecanone) 71, m/z (2-nonanone) 58, m/z (geraniol) 69, and m/z (methyl carbamate) 119 was taken as the peak. According to the scheme shown below, an assignment of the respective saturation of the selected fragments is made based on the relative peak areas of the EICs (0% to 100%) of the selected fragments at the various time points (1 to 14 days).
FIG. 8 is a graph showing the development of aroma characteristics during the transformation of linalool and benzaldehyde on the seed culture medium according to the present invention compared to the corresponding transformation on a standard seed culture medium using the fermentation of Poria as an example. Evolution of linalool on MEA (□); evolution of linalool at 3TT + Asp (° o); evolution of benzaldehyde on MEA (); evolution of benzaldehyde on 3TT + asp (x). To prepare EICs, fragments at m/z 93 were chosen for linalool and fragments at m/z 106 were chosen for benzaldehyde. Abbreviations used are as previously described.
FIG. 9 is a graph showing the development of aroma characteristics during the transformation of methyl anthranilate (m/z 119) on a seed culture medium according to the present invention compared to the transformation on a standard seed culture medium using the fermentation of Poria cocos as an example. The evolution of methyl anthranilate on mea (x); the progression of methyl anthranilate at 3TT + Asp (. smallcircle.).
FIG. 10 is a graph of semi-quantitative estimation of the concentration of the selected aroma (. smallcircle.) after transformation on the seed culture medium according to the present invention, using Poria cocos fermented on 3% pomace with 0.624% L-monosodium aspartate as an example. Herein, the headspace of the species was analyzed after 10 days and compared to the calibration series (x) determined by the standard. A is linalool; b is benzaldehyde; and C, methyl anthranilate. The estimated amounts are indicated in μ g/plate.
FIG. 11 is a sensory evaluation chart of transformation on a seed culture medium according to the present invention using as an example the outlet fermentation of Poria cocos on 3% red currant pomace. The given attribute was rated by four trained persons on a scale of 0 to 5. Abbreviations used are JhB currant pomace, Asp 0.624% monosodium L-aspartate, d0 uncultured reference medium, d10 cultured with Poria for 10 days.
FIG. 12 is a graph showing the sensory evaluation of transformation on seed culture media according to the invention using as an example the outlet fermentation of Poria cocos on 3% grape pomace of the Muller-Thurgau variety. The given attribute was rated by four trained persons on a scale of 0 to 5. Abbreviations used were Wine: grape pomace, Asp: 0.624% monosodium L-aspartate, d0: uncultured reference medium, d10: cultured with Poria for 10 days.
FIG. 13 GC-MS chromatogram of transformation on the seed culture medium according to the invention using as an example the outlet water fermentation of Poria cocos on 3% grape pomace of Muscaris variety, 0.6% monosodium L-aspartate, 1.5% agar-agar. Chromatograms of samples taken after 10 days of incubation; the split ratio 3:1 was measured.
FIG. 14 GC-MS chromatogram of transformation on strain medium according to the invention using Poria cocos for outlet water fermentation on 3% strawberry pomace, 0.6% L-monosodium aspartate, 1.5% agar-agar as an example. Chromatograms of samples taken after 8 days of incubation; the split ratio 3:1 was measured.
FIG. 15 GC-MS chromatogram of transformation on a strain medium according to the invention using as an example the outlet water fermentation of Poria cocos on 3% raspberry fruit puree, 0.6% L-monosodium aspartate, 1.5% agar-agar. Shows chromatograms of samples taken after 8 days of culture; the split ratio 3:1 was measured.
FIG. 16 is a heat map of the selected aroma during the incubation of Poria cocos cells on different bacterial species media and at different incubation times. The EIC peak areas at m/z (benzaldehyde) 106, m/z (linalool) 93, m/z (linalool oxide I, II) 111, m/z (2-undecanone) 71, m/z (2-nonanone) 58, m/z (geraniol) 69, and m/z (methyl carbamate) 119 were taken as the base peaks. According to the scheme shown below, the assignment of the respective saturation of the selected fragments is made based on the relative peak area of the selected fragments at EIC (0% to 100%). The used effluent strain media comprise M1, 3 percent of pomace, M2, 3 percent of pomace and 0.4 percent of glucose, and M3, 3 percent of pomace and 0.8 percent of glucose. The culture time is 0d of uncultured reference medium, 8d of uncultured reference medium and 10d of uncultured reference medium. Abbreviations used are ER nonvariant strawberry pomace, HR nonvariant raspberry pomace, MUT Muscaris variant wine grape pomace, Asp 0.6% monosodium L-aspartate.
FIG. 17 GC-MS chromatogram of transformation on strain medium according to the invention using a fixed bed of Poria cocos strain on strawberry pomace as an example. Chromatograms of samples taken after 28 days of incubation; the measurement was performed by SPME-GC-MS in non-split mode.
Detailed Description
The invention is essentially based on the following findings: transformation media having components based on plant parts of the families gooseberry, rosaceous or vitidae contain extremely interesting aroma precursors capable of being bioconverted into aroma substances. The term aroma is used herein to describe compounds that impart a perceptible taste or odor in an aroma-active amount. In this case, the term "aroma activity" means that the amount of the compound is sufficient to elicit an organoleptic effect on the olfactory and/or gustatory receptors when using a formulation containing the compound. This effect can also be manifested by reducing or masking the sensory perception based on unpleasant tastes and/or odours.
The process according to the invention avoids the need for harsh manufacturing conditions, organic solvents or heavy metal catalysts, which is almost unavoidable in the case of chemical synthesis. The present invention thus provides an environmentally friendly alternative to chemical synthesis, which further meets the growing demand for aroma substances such as linalool.
Furthermore, a feature of the method according to the invention is that it provides the possibility of using agricultural waste streams such as leaves or pomace of the families gooseberry, rosaceous or vitiidae as a source of high quality aroma, thereby being beneficially integrated into the value chain of agricultural production. For this reason, the following processes are particularly preferred: ingredients based on plant parts of the family blackcurraceae, rosaceae or vitidae are 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 of blackcurrants as described herein. In the process according to the invention described herein, the proportion of ingredients based on plant parts of the family blackcurraceae, rosaceae or vitidae is generally in the range from 0.2% to 100% by weight, based on the total weight of the transformation medium. If the cultivation is an effluent or submerged cultivation, the proportion of the ingredients based on plant parts of the family blackcurraceae, rosaceae or vitidae in the process according to the invention described herein is preferably from 0.2 to 20% by weight, preferably from 0.5 to 10% by weight, further preferably from 1 to 5% by weight and most preferably from 2 to 4% by weight, based on the total weight of the transformation medium. If the cultivation is carried out as a fixed bed cultivation, the proportion of the constituents based on plant parts of the family gooseberry, rosaceae or vitidae in the process according to the invention described herein is preferably from 10 to 100% by weight, preferably from 30 to 90% by weight, further preferably from 50 to 80% by weight and most preferably from 60 to 70% by weight, based on the total weight of the transformation medium.
The term "blackcurraceae" refers to plants of the family blackcurrant. Preferred according to the invention are the following methods: the method uses plant parts of species and varieties suitable for soft fruit production, in particular of gooseberry, gooseberry and hybrids thereof such as blackcurrant tree. Further preferred is a method using plant parts of gooseberry or gooseberry, redcurrant, gooseberry, blackcurrant or blackcurrant and hybrids and varieties thereof. Most preferred is a method according to the invention using blackcurrants (blackcurrants) and variants and cultivars of plant parts thereof.
The term "rosaceae" refers to plants of the family rosaceae. According to the invention, the following method is preferred: the method uses plant parts of species and varieties suitable for soft fruit production, in particular of strawberries and raspberries and hybrids and varieties thereof.
The term "Vitaceae" refers to plants of the family Vitaceae. According to the invention, the following method is preferred: the method uses plant parts of species and varieties suitable for soft fruit production, in particular of grapevine and its hybrids. Further preferred is a method using plant parts of the noble grape (vitis vinifera) and its varieties.
In particular, the plant component comprises above ground vegetative or reproductive tissue, preferably leaves, buds, petals and/or flower buds, berries and their pits, peels and pulp etc. Preferably, the plant part is a leaf and/or a berry and further preferably an entire mature berry.
By plant part based ingredients is meant in the present invention ingredients obtained from the parts of the plant in question. In this case, the components used in the process according to the invention do not necessarily have to occur in the same way in nature. Conversely, the constituents of the plants involved can also be obtained by further processing of naturally occurring constituents. Preferred measures for further processing include (partial) drying, (partial) fermentation and/or (partial) pressing. Preferably, the plant is waste from other industries that use the corresponding plant, or at least a part thereof, as a feedstock. Particularly preferably, the plant part is a berry (whole) or a residue obtained from the extraction of a fruit juice (so-called pomace).
Of particular interest for the present invention are pleasant taste and/or odor impressions. Assessing whether a taste and/or odor impression is considered pleasant or rather unpleasant can be performed by a trained panel conducting a sensory analysis based on assessing the sensory impression between negative (pleasant) and positive (unpleasant). Further levels of strong negatives, neutrality, and strong positives may be set for more accurate classification. The aroma to be evaluated is present in other compounds, possibly mixtures of other aromas, and the determination of the note of the aroma to be evaluated can be carried out, for example, by gas chromatography olfactometry. In the context of the present invention, an aroma substance or an aroma substance mixture is in particular a substance which imparts a pleasant odor impression and is therefore also referred to as fragrance substance.
In the case of the preparation of aroma substances with a pleasant taste and/or odor impression, transformation media containing the following components stand out: the ingredient is based on plant parts of blackcurrants, preferably blackcurrants (blackcurrants), and in particular blackcurrants of the variety Titania. This ingredient obviously contains aroma precursors which, by biocatalytic conversion, are able to produce an extremely pleasant aroma. Thus, the transformation medium provided in the method according to the invention preferably contains ingredients based on plant parts of blackcurrants, preferably blackcurrants and further preferably blackcurrant variety Titania. Thus, the aroma that can be produced using a suitable biocatalyst has a floral, faint scent, fruity or reminiscent of wild berry or citrus notes. These aromas are not readily noticeable when properly transformed on standard seed culture media based on malt extract. Instead, olfactory impressions are described herein as fungal, sour, and tropical fruit odors. Other advantages result from the following measures.
Preferably, the step of recovering the aroma or aroma mixture comprises at least partially separating at least one aroma of the produced aroma or aroma mixture from the biocatalyst. Preferably, substantially complete separation from the biocatalyst is performed. Furthermore, the step of recovering may comprise enriching, concentrating and/or sequestering the at least one aroma of the produced aroma or aroma mixture. As used herein, the term "at least one aroma" optionally refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aromas.
For the purposes of the present invention, whole-cell biocatalysts have proven to be particularly effective because of their ability to perform the catalytic conversion reaction by simultaneous or sequential regeneration. Thus, the conversion step may comprise culturing in the sense of "growing" or regenerating the biocatalyst. However, it is generally known to those skilled in the art that transformation with whole cells ultimately relies on one or more catalyzed reactions, and therefore the present invention also includes the following methods: the biocatalyst used for the production of the aroma or the aroma mixture also comprises only the catalytically relevant units of the whole-cell biocatalyst used for the production of the aroma or the aroma mixture.
The biocatalyst is preferably a fungus, a fungal cell thereof or at least one catalytically relevant unit thereof. Catalytically relevant units herein refer to macromolecules required for the conversion, such as enzymes or ribozymes, and optionally cofactors and co-substrates. Preferably, it is a fungus capable of growing on lignocellulose and/or a fungus (or its corresponding fungal cell) selected from the group of edible fungi. The ability to grow on lignocellulose is therefore advantageous, since this fungus is particularly suitable for cultivation on currant-based ingredients as described herein. In addition, aromatic substances such as benzaldehyde can be directly obtained by degradation of lignocellulose. When the produced aroma or aroma mixture is used, the edible fungi are advantageous in terms of safety. This preferred demand profile applies to many fungi of the class (phylum) of basidiomycetes (basidiomycota). Furthermore, a particularly good odor impression can be obtained with fungi from the basidiomycetes class, in particular when the currants are blackcurrants as described herein.
Of particular note are the following findings: transformation with a variety belonging to basidiomycetes gives a smell that is pleasant in the overall impression. This means that the notes of the fungus (mushroom), sour taste and tropical fruit smell, which are called typical fungus aromas, are not or hardly formed. Therefore, the following method is preferred: the fungi is selected from plant parts of Basidiomycetes and/or Ribes nigrum family, preferably from plant parts of blackcurrant, more preferably from plant parts of blackcurrant variety Titania.
In particular, the method described herein is preferred, wherein the fungus of the basidiomycetes is selected from the group consisting of tetraspora mushroom, agrocybe aegerita, armillaria mellea, schizophyllum, coprinus comatus, crinis nigra, flammulina velutipes, axanthus axyridis, sterculia villosa, corolla gallinarum, lentinus edodes, lepista nuda, puffball pear, pileus, m. These species are clearly superior to a large number of other fungi or combinations of fungal transformation media that have been tested and are not specified herein in terms of their ability to form aroma with a pleasant odor impression.
Further preferred is a method as described herein, wherein the fungus is selected from the group consisting of tetraspora mushroom, agrocybe aegerita, coprinus comatus, axillus, sterculia fulvescens, rabdosia salmonis, corolla gallinarum, shiitake mushroom, puffball, epididymal pileus, m. This selection results in a particularly pleasant aroma in at least one possible culture format.
Further preferred is a method as described herein, wherein the fungus is selected from the group consisting of agrocybe cylindracea, coprinus comatus, axillus pulchrus, sterculia fulva, cockscomb, m. The above selection results in a particularly pleasant aroma in at least two possible culture formats.
Further preferred is the method described herein, wherein the fungus is selected from the group consisting of poria cocos wolf and clover. The fungi Trifolium pratense and Poria cocos showed a very strong odor impression in all tested culture forms on all tested substrates. Axletoe herein produces a citrus aroma, and the aroma of poria is floral, fruity and reminiscent of strawberries. This selection therefore results in a particularly pleasant aroma, regardless of the possible culture format.
As mentioned above, preferred biocatalysts comprise single cells or the above preferred catalytically active units and further preferred fungi.
Furthermore, according to the present invention, the following method is preferred: the transformation medium provided comprises a source of aspartic acid, preferably a source of L-aspartic acid, further preferably sodium L-aspartate, most preferably monosodium L-aspartate. Thus, during the course of the experiments, it was found that the addition of monosodium L-aspartate counteracts the otherwise observed pH drop and finally gives a particularly strong strawberry odour. After approximately 10 days of cultivation, the odor was enhanced to floral and reminiscent of the strong fruit aroma of wild strawberries. When cultured on the aspartate-supplemented transformation medium described herein, the signal intensity of the aroma is significantly higher than when cultured on standard seed culture media based on malt extract. Linalool is particularly strong and is very relevant to the floral odor of the species. Some aroma actives, such as benzaldehyde, linalool oxide I, II, and 2-undecanone, form particularly well on the transformation medium containing aspartic acid, which is preferably provided in the form of pomace as described herein. The level of aspartate source is typically from 0.1% to 20.0% based on the total weight of the transformation medium. If the form of the culture is an effluent or submerged culture, the aspartic acid source is preferably present in an amount of from 0.1 to 5 wt.%, more preferably from 0.2 to 3 wt.%, even more preferably from 0.3 to 2 wt.%, and most preferably from 0.4 to 1 wt.%, based on the total weight of the transformation medium, in the process according to the invention described herein. If the form of cultivation is a fixed bed cultivation, the content of the source of aspartic acid in the process according to the invention described herein is preferably from 0.1% to 20% by weight, more preferably from 2.5% to 17.5%, further preferably from 5% to 15% by weight, and most preferably from 7.5% to 12.5% by weight, based on the total weight of the transformation medium.
Alternatively, the pH drop may be counteracted with other buffer substances. Preferably, the pH of the transformation medium during said transformation ranges from pH 2 to pH 7, preferably from pH 4 to pH 6, and in particular from pH 4.2 to pH 5.5. In case the transition lasts several days, this means that the pH range is at least substantially maintained during the whole period.
Additional nutrient sources may be added to the transformation medium to optimize aroma characteristics and intensity. In addition to the aspartic acid sources described herein, these additional nutrient sources include, inter alia, glucose; trace elements selected from the group consisting of Mg2+, Zn2+, Fe3+, Mn2+, Cu2+, K +, Cl-, SO42-, and PO43-, and other amino acids.
In the context of the present invention, essentially all forms of cultivation are envisaged, even though a particular form may influence the formed aroma. For example, transformation with an aquatic species gives particularly good results, especially when poria cocos and/or pomace are used as the respective biocatalyst and the herein described gooseberry based ingredient. Submerged and fixed bed species also give a very pleasant aroma according to the fungal species used as described herein and the specific form of the herein described constituents based on the family gooseberry, rosaceous or vitidae.
It has been found to be particularly advantageous for the producible aroma substance to transform a transformation medium comprising plant parts of currants as described herein and added with an aspartate source as described herein, with the aid of basidiomycetes as described herein. Instead of an aspartic acid source, a pH buffering component can also be added to the transformation medium as described herein above to maintain the pH within the pH range described herein during this transition. In particular, the cultivation of Poria on currant pomace of the Titania variety produces a floral aroma and a fruity aroma that lets associate with Fragaria maackii. Poria is also an edible fungus used in traditional Chinese medicine. In addition to a variety of minor components, linalool, benzaldehyde and methyl anthranilate also form key aroma components.
The process according to the invention is particularly suitable for producing fragrances or fragrance mixtures comprising at least 1, 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, linalool and 2-aminobenzaldehyde. Particularly preferred is a process involving the production of at least 1 compound, preferably 2 compounds and further preferably all compounds of linalool, benzaldehyde and methyl anthranilate. These compounds are unique features of the aroma mixture that can be produced by the transformation media described herein. In particular, methyl anthranilate showed a strong fruity odor of wild strawberry.
The exact composition of the type and intensity of the aroma (also referred to herein as aroma composition) produced can therefore be influenced by varying the time over which the aroma or aroma mixture is recovered. In this case, it has proven effective according to the collection time in the process of the invention, wherein the extraction of the aroma or aroma mixture takes place 3 to 14 days, preferably 5 to 14 days and in particular 9 to 13 days after the start of the contact. In addition to this, it is also envisaged to determine the extraction time of the aroma or aroma mixture as a function of the predetermined aroma composition. This enables, among other things, acquisition at the following times: the presence of a particularly pleasant aroma mixture at this time and/or a particularly pleasant aroma is clearly superior to other aromas that may be formed.
Furthermore, the present invention relates to the use of a transformation medium comprising the herein described constituents based on plant parts of the family gooseberry and/or the herein described biocatalyst for the production of the herein described aroma or aroma mixture. In particular, the component based on plant parts of the family blackcurrant is a component based on plant parts described herein, such as the leaves or pomace of blackcurrant. In particular, the biocatalyst is a fungus from the basidiomycetes class described herein.
Furthermore, the present invention relates to a composition, in particular a composition prepared (preparable) according to the process described herein, comprising 2 or more particularly all compounds selected from the group consisting of linalool, benzaldehyde and methyl anthranilate. Preferred are the following compositions according to the 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 1, 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.
Furthermore, the present invention relates to the use of the composition according to the invention described herein as or in a nutritional, cosmetic, nutraceutical or edible formulation, in particular as a mixture of aroma substances (preferably for the purposes described herein). Thus, the present invention also relates to a nutritional, cosmetic, nutraceutical or edible formulation comprising or consisting of the composition according to the invention as described herein.
The present invention is explained in more detail below with reference to the following examples.
Examples
1. Experimental part
1.1 materials and chemicals
The side stream used by the gooseberry industry and the wine industry is provided by the university of gessom, germany. Raspberry or strawberry pomace is prepared by mashing and sieving fresh, commercially available, non-variant berries. The remaining puree residue is used as a vehicle component. The sources of the basidiomycota fungi used are shown in table 1.
Table 1: the profile of the tested fungi, their origin and the culture medium; DSMZ: the research institute of Lei Bunitz DSMZ-German culture Collection of microorganisms, Blorex Germany; and (3) CBS: CBS-KNAW culture Collection, Westdek institute of fungus biodiversity, university of Udele, Netherlands; s: university of giseng, institute of food chemistry and food biotechnology, university of giseng; MEA: malt extract agar; SNL: standard nutrient solution agar.
1.2 Strain maintenance
The fungi to be investigated (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 ℃ for 20 minutes. The medium consists of:
MEA：20g·L-1malt Extract (ME), 15 g.L-1Agar-deionized water of agar.
SNL：30g·L-1Glucose monohydrate, 15 g.L-1Agar-agar, 4.5 g.L-1Asparagine monohydrate, 3 g.L-1Yeast extract, 1.5 g.L-1 potassium dihydrogen phosphate, 0.5 g.L-1Magnesium sulfate hydrate, 400. mu.g.L-1EDTA、90μg·L-1Zinc sulfate (II) heptahydrate, 80. mu.g.L-1Iron chloride hexahydrate, 30. mu.g.L-1Manganese sulfate (II) monohydrate, 5. mu.g.L-1Copper (II) sulfate pentahydrate in deionized water and adjusted to pH 6.0.
For the culture, about 0.5cm from an 80% overgrown agar plate was used2The hypha fragments of (A) were placed on new agar plates and usedThe plate was sealed and incubated at 24 ℃ protected from light.
1.3 screening of the Ribes nigrum residue stream
The culture of Basidiomycota fungi on black currant residue leaves and fruit residues is carried out on submerged strains, effluent strains and fixed bed strains. Initially, the residue stream was used as the sole source of nutrition for the 26 fungi tested.
To culture the fungi in the effluent species, agar plates with different concentrations of the substrate were prepared. Agar is required in different concentrations depending on the substrate. They were inoculated with 26 selected fungi (table 1), keeping the strain similar. After 4 to 7 days, the plates were first examined for organoleptic properties, depending on the growth of the fungus. For further experiments, poria cocos strain cultivation was performed on currant pomace, wine grape pomace, strawberry pomace or raspberry pomace, 3% pomace was added to 30% deionized water and 70% deionized water of 1.5% or 3% agar was autoclaved and mixed before pouring. Each plate contained approximately 16mL of medium.
To culture the fungus in submerged species, a pre-strain (100mL) was first prepared in 2% ME medium. This was achieved by overgrowing hyphae approximately 1cm from the strain holding plates2The agar fragment of (a) was transferred to a conical flask containing ME medium to completion, wherein the fragment was homogenized using a disperser (Ultraturrax, 10000rpm, 30 s). The pre-strain was incubated at 150rpm for eight days at 24 ℃ in the absence of light. For inoculation of the main species, the pre-strain was homogenized (Ultraturrax, 10000rpm, 30s), centrifuged (3000g, 10 min) and the supernatant poured in. The mycelia were washed twice with sterile water and then suspended in 100ml of sterile water. 4mL of the suspension was addedAdd to 40mL of main seed medium. The culture medium of main strain contains 15 g.L-1Or 30 g.L-1Deionized water composition of the substrate. These strains were grown at 150rpm in the dark at 24 ℃. Sensory analysis was performed by sniffing directly on the flask starting from day 3 of culture. For comparison, the blank values were used for ME medium and the host strain on the media without inoculation of the host strain.
For the cultivation of fungi in a fixed bed of strains, the pre-strains were cultivated and homogenized in the manner described previously. To a fixed bed of seed culture medium was added 2mL of the suspension. To prepare a fixed bed seed culture medium, 20g of pomace, 10g of water and 2g L-monosodium aspartate (Asp) were homogenized in flasks and autoclaved. The inoculated fixed bed strain media was statically cultured at 150rpm at 24 ℃ in the dark. For comparison, the uncultured fixed bed media was used as a blank.
1.4 optimization of the culture on the Ribes nigrum residue stream
To optimize aroma characteristics and intensity, other sources of nutrition are added to the matrix. In addition to adjusting pH, glucose, trace elements (Mg2+, Zn2+, Fe3+, Mn2+, Cu2+, K +, Cl-, SO42-, PO43-), L-aspartic acid monosodium and other amino acids are also supplemented. Additionally, the substrate concentration of the currant side stream was varied.
1.5 sensory analysis
Sensory analysis was performed by a trained panel (n-4) to pre-select the fungus-matrix combination. For this purpose, the bacterial species were examined in a "simple descriptive test" and the overall impression was evaluated over several days of cultivation. The rating for overall impression was evaluated to include six levels from strong negatives (0) to strong positives (5). The aroma was identified by GC-MS-O.
1.6 determination of hyphal growth
During the incubation period, fungal growth was recorded daily by drawing overgrowth areas on the back of the agar plates and determining the diameter. From this, the overgrowth hyphal regions were calculated.
1.7 determination of the pH value
To determine the progress of the pH of the submerged species, an aliquot (2mL) was taken and the pH was determined using a pH meter. For the effluent bacterial species, the pH of the species was determined as follows: one plate of agar was homogenized after adding 15mL of deionized water using an ultraturrax (10000rpm, 10s), then centrifuged (10 minutes, 4000g) and the pH of the supernatant was measured using a pH meter.
1.8GC-MS-O analysis
The aroma of the strain yielding water from the currant pomace is extracted by a stirring rod adsorption extraction method (SBSE) for 60 minutes at 24 ℃. For this purpose, a 10mm magnetic stirring bar with a 0.5mm PDMS (polydimethylsiloxane) coating (Twister, GERSTEL, Muhlheim an der Ruhr, Germany) was fixed with a magnet in the headspace of the culture vessel. After incubation, the magnetic stir bar was removed with a magnetic bar, rinsed with deionized water, dried with lint free cloth, and placed in a conditioned Thermal Desorption Unit (TDU) pad (GERSTEL). The TDU desorption was carried out without splitting. The analytes were cold-concentrated using a glass evaporator tube with silanized glass filler (GERSTEL) in a cold injection system 4(CIS) (GERSTEL). Gas chromatography was performed using a GC-MS-O consisting of a7890B GC (agilent technologies, waldbolon, germany) coupled to a 5977B MSD (agilent technologies) and Olfactory Detection Port (ODP) (GERSTEL). Using Agilent J&WVF-WAXms (30 m.times.0.25 mm ID. times.0.25 μm) as a polar column, and Agilent J was used&W DB-5ms (30m × 0.25mm ID × 0.25 μm) as the nonpolar separation phase. At a rate of 1.56 mL/min-1Helium was used as the carrier gas. The gas stream was directed to the MS and ODP with a split ratio of 1: 1. Further settings are listed below: the purging flow of the isolation pad is 3 mL/min-1Total Ion Current (TIC) in scanning mode, scanning range m/z 33-300, ionization energy 70eV, EI source temperature 230 ℃, quadrupole temperature 150 ℃, MS transmission line temperature 250 ℃, helium quenching gas 2.25 mL/min-1、N21.5 mL/min of collision gas-1ODP3 line temperature 250 ℃, ODP mixing chamber temperature 150 ℃, ODP make-up gas N2。
To investigate the change in relative intensity of fragrance and to identify using VF-WAXms, 10 ℃ min-1Heating the oven for 3 min to 40-150 deg.C, and heating at 20 deg.C/min-1The temperature was raised to 240 ℃ and maintained at this temperature for 7 minutes. TDU temperature program at 360 ℃ min-1 from 40 DEG C(0.5 min) to 250 deg.C (10 min). In CIS, after a holding time of 0.5 min, the temperature is 12 ℃ s-1The temperature was raised from-20 ℃ to 250 ℃ and held for 5 minutes. The CIS split ratio varies between no split and 50: 1.
For semi-quantitative estimation and determination of retention index using DB-5ms, the oven was run at 5 ℃ min-1The heating was carried out for 3 minutes from 40 ℃ to 320 ℃ and the temperature was maintained for 7 minutes. Except that the temperature is 120 ℃ min-1The TDU was heated to 250 ℃. The cold set was carried out differentially by CAS at 100 ℃. The split ratio at the CIS varies between no split and 50: 1.
The aroma of the outlet 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 was then performed using a GC-MSFID consisting of 8890GC (agilent technologies) coupled to 5977B MSD (agilent technologies) and a flame ionization detector. The column used was Agilent VF-WAXms (30 m. times.0.25 mm ID. times.0.25 μm). The temperature of the oven is 3 ℃ min-1From 40 ℃ to 230 ℃ and held for 30 minutes. The TDU temperature program was heated from 30 ℃ to 150 ℃ at 60 ℃ min-1 and held for 10 minutes. After a holding time of 0.1 min, the CIS had a temperature of 12. degreesS-1The temperature was raised from-20 ℃ to 250 ℃ and held for 10 minutes. The CIS split ratio used was 3: 1. At a rate of 2.4 mL. min-1Helium was used as the carrier gas. The gas stream was directed to the MS and FID at a 4:1 split ratio. Other arrangements, unlike the previous system, are listed below: the scanning range m/z is 25-370, the MS transmission line temperature is 280 ℃, and the FID H2 airflow is 35 mL/min-1FID make-up gas N2.
To analyze fixed bed strains, 3g of fixed bed strain medium for hyphal growth was weighed into a 20mL headspace vial and analyzed by Solid Phase Microextraction (SPME) in the headspace. For this purpose, the samples were incubated at 45 ℃ for 20 minutes at 250rpm and extracted with polydimethylsiloxane/divinylbenzene fibers for 5 minutes. Desorption was carried out in CIS 4 at 230 ℃ for 60 minutes without splitting. Gas chromatography analysis was performed using GC-MS consisting of 7890B GC coupled to 5977B MSD. Agilent DB-FFAP (60 m.times.0.25 mm ID. times.0.25) was usedμ m) as a column. After 5 minutes the oven temperature was 10 ℃ min-1From 40 ℃ to 230 ℃ and hold the temperature for 10 minutes. At a rate of 1.5 mL/min-1Helium was used as the carrier gas. The gas stream was directed to the MS and FID at a split ratio of 10: 1. Other offset settings are listed below: scanning range m/z25-550, MS transmission line temperature 250 ℃, FID H2 airflow 30mL min-1FID make-up gas N2.
1.9 identification of aroma substances
To identify the substance, 10-30mg of reference substance were mixed according to solubility with 50-1000. mu.L DMSO (Carl Roth, Germany, Carlsreue) and, if necessary, topped up to 1mL with deionized water. The following aromas were used for the analysis: 2-aminobenzaldehyde > 98% (Sigma Aldrich), 2-nonanone > 99% (Accos Organics, Hell, Belgium), linalool 97% (Accos Organics), methyl anthranilate 99% (Acros Organics), (+) -2-octanol 98% (Alfa Aesar, Black Flory, USA), geraniol 97% (Alfa Aesar), 2-octanone 98% (Sigma Aldrich, St Louis, USA), linalool oxide isomer mixture (Sigma Aldrich), 2-undecanone pure (Honeywell Fluka, Bugarrett, Romania), benzaldehyde pure (Applichem, Damschott, Germany). Thus, 1:200 and 1:1000 dilutions were made with water and 0.5mL of solution was precipitated onto agar (1.5% agar in deionized water). After 1 hour, the mixture is stirred with a magnetAttached to the headspace and analyzed by GC-MS using the method described in 1.8, supra. 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).
1.10 semi-quantitative estimation of the concentrations of methyl anthranilate, linalool, and benzaldehyde
For semi-quantitative estimation, linalool, methyl anthranilate, and benzaldehyde were dissolved in deionized water. For benzaldehyde, the concentration is 3 mg.L-1、4mg·L-1、8mg·L-1、12mg·L-1And 16 mg. L-1The specification of (a); for methyl anthranilate, a concentration of 9 mg. L was prepared separately-1、14mg·L-1、19mg·L-1、41mg·L-1And 80 mg. L-1The specification of (a); for linalool, the concentration is 120 mg.L-1、241mg·L-1、361mg·L-1And 722 mg. L-1The specification of (1). Wherein, on agar plates (16mL of 1.5% agar deionized water) 0.5mL of each of linalool, methyl anthranilate, and benzaldehyde were precipitated. After one hour, use the magnet toAttached in the headspace and analyzed similarly to the sample. The calculated peak area is determined by Extracted Ion Chromatography (EIC) of the specific species fragment and the intensity detected in the sample is compared to the intensity of the standard species to assign a range of concentrations to the sample.
2. Results
To investigate the biotransformation of the blackcurrant side stream to synthesize a natural aroma, a total of 26 fungi of the basidiomycota were tested. Table 2 summarizes the evaluation of odor impressions of tested fungus-substrate combinations in the effluent and submerged species.
Table 2: sensory evaluation of the overall impression of the fungus-matrix combination between 0 (strong negative) and 5 (strong positive). -: no culture was performed.
Based on sensory analysis, several fungal-matrix combinations of interest were identified. The fungi clover and poria show a very strong odor impression on all tested substrates of all tested culture types. The axletree grass herein produces a citrus-like aroma, and the aroma of poria is floral, fruity and reminiscent of strawberries.
To analyze the growth characteristics of poria in more detail, fungi were cultured on different media. In addition to the pomace, malt extract agar was chosen, which is very suitable as a complete medium for fungal growth. During the cultivation on pomace, the addition of monosodium L-aspartate results in a particularly strong aroma. The cultures differed visually and in growth except for odor (see FIG. 1).
After addition of aspartic acid, the hyphae and nutrient medium turned brown. Furthermore, the growth was reduced compared to the reference medium (see fig. 2). Less biomass and very flat hyphae were formed.
The pH was reduced less than in the case of the aspartic acid-free species by the addition of 0.624% monosodium L-aspartate (see FIG. 3). In the fermentation of pomace and MEA medium, the pH is lowered to about 2.5 during the course of the culture. In contrast, no pH decrease was observed in the medium of the pomace added with aspartic acid. Among these species, the odor of wild strawberry is particularly strong.
For analysis of the aroma composition, SBSE analysis was performed in combination with GC-MS-O. The fermentation of poria cocos on blackcurrant pomace with addition of 0.624% monosodium L-aspartate is considered to be of particular interest. The aromas detected were significantly different from those detected in the fermentations carried out on MEA or currant pomace or currant leaves without added aspartic acid.
In particular, C8 aromas such as 3-octanone, 1-octen-3-ol, 1-octanol, and (E) 2-octen-1-ol were detected in the MEA medium (see FIG. 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 a fungal, sour and tropical fruit smell.
In contrast, when poria cocos was cultured on currant pomace having aspartic acid, there was a significant difference in the aroma. Only after a few days, the strain started to be floral and fragrant. Furthermore, no aroma was perceived that was reminiscent of fungi. After about 10 days of culture, the odor was more intense, floral and reminiscent of the strong fruity aroma of strawberries.
When cultured on blackcurrant pomace supplemented with aspartic acid, the intensity of the aroma signal was significantly higher than when cultured on MEA (see figure 5). Linalool is particularly intense, being strongly linked to the floral odor of the species.
In addition to linalool, additional compounds characterized by an aroma of the fungus-substrate combination were identified by GC-MS-O investigation. In particular, methyl anthranilate showed a strong fruity odor of wild strawberry.
Compounds identified by olfaction also highlight the complexity of the fragrance composition. For this purpose, in order to detect particularly strong aromas, the outlet species of Poria cocos were examined by GC-MS-O on a defined number of cultivation days (see FIG. 6).
In the olfactory evaluation of the strains thus carried out, the substances linalool and methyl anthranilate were very strongly perceived. Benzaldehyde, which is also perceived by means of ODP and has the characteristic odour of bitter almond flavour. In addition to these key aroma substances, a large number of other odors are perceived, and the substance classification is to be determined.
Table 3 shows the recommendations for the selected substances contained in the samples and their Retention Indices (RI) according to van den dol and Kratz and the odor compared to the retention index of the standard substances and the odor described in the literature. Correlation mass spectrometry (not shown) confirmed the formation of 2-octanone, 2-nonanone, linalool oxide, benzaldehyde, geraniol, 2-octanol, 2-aminobenzaldehyde, methyl anthranilate and linalool by comparison with corresponding control spectra obtained from the NIST database using samples of the aqueous seed species from poria on 3% pomace + 0.624% L-monosodium aspartate.
Table 3: the Retention Index (RI) according to van den dol and Kratz and the odor of the substances detected in the samples were compared to the standard Substances (STD) and the odors described in the literature. VF-WAXms and DB-5ms represent the respective separate phases used (see experimental part, 1.8).
[13]Wood,WilliamF.；Brownson,Mary；Smudde,R.Allen；Largent,David L.
(1992):2-Aminobenzaldehydes.The Source of the“Sweet Odor”of Hebeloma sacchari-olens.In:Mycologia84(6),p.935.DOI:10.2307/3760296。
The retention indices of the samples and standards were very consistent with the mass spectrum and odor.
The kinetics of formation of the primary aroma was recorded at 24 hour intervals for 14 days using headspace SBSE (see figure 7).
Some aroma-active compounds like benzaldehyde, linalool oxide I, II and 2-undecanone are formed almost exclusively on pomace and aspartic acid (3TT + Asp) medium. Other compounds such as 2-nonanone, geraniol and methyl anthranilate were formed on pomace and aspartic acid media and on MEA and aspartic acid (MEA + Asp) media.
The aroma analysis data (see fig. 8 and 9) is closely related to that of sensory analysis, where characteristic odor is described as most intense at day 10 of culture.
Linalool, methyl anthranilate, and benzaldehyde are considered to be characteristic features of the aroma. For this reason, semi-quantitative estimation of aroma concentration was performed after culturing poria cocos on pomace having aspartic acid for 10 days (see fig. 10).
Based on the calibration series, it can be estimated that each plate contains 95-135. mu.g of linalool, about 5. mu.g of benzaldehyde, and 5-20. mu.g of methyl anthranilate. This correlates with a plate volume of about 7000 + -1500. mu.g.L, considering a plate volume of about 16mL-1Linalool, 350 + -50 mug.L-1Benzaldehyde, 700 +/-350 mu g.L-1Methyl anthranilate corresponds.
The sensory evaluation of the poria cocos cultures on transformation media based on blackcurrant pomace with and without aspartic acid addition was performed by trained groups. The aspartic acid added yielding water species rated significantly more strongly in terms of "fruity", "floral" and "wildberry" attributes (see figure 11).
By culturing Poria cocos in a transformation medium with a component based on a plant part of the family Vitaceae or Rosaceae, it is also possible to produce a very interesting aroma profile. Thus, depending on the duration of the cultivation, the cultivation of Poria cocos on a medium consisting of the pomace of Vitis vinifera from Muller-Thurgau, Gew ü rztraminer or Muscaris gives aroma characteristics with fruity and floral aroma that are reminiscent of wild strawberries (see Table 4).
Table 4: poria cocos was used in different varieties MT: Muller-Thurgau, GT: gew ü rztraminer, MU: effluent fermentation on 3% vitis vinifera pomace of Muscaris as an example, sensory evaluation of transformation was performed on the seed culture medium according to the invention. Odor profile summarized by different incubation durations.
Sensory evaluation of the effluent bacterial species of Poria cocos on transformation media based on grape pomace of the Muller-Thurgau variety with addition of aspartic acid showed similarities to the effluent bacterial species based on red currant pomace (see FIG. 12).
By means of gas chromatographic analysis, the species of Poria cocos on a medium containing Rosaceae or Vitaceae components detected the aroma active compounds 2-octanone, 2-nonanone, 2-undecanone, linalool oxide, benzaldehyde, geraniol, methyl anthranilate and linalool. Examples of chromatograms of Poria cocos over Muscaris s.cerevisiae pomace, strawberry pomace or raspberry pomace all supplemented with 0.6% monosodium L-aspartate are shown in FIGS. 13, 14 and 15.
Fig. 16 shows a comparison of the amount of aroma active compounds formed when poria cocos was grown over water on media containing rosaceous or vitiidae components with an additional change in glucose content. The aroma-active compounds shown were not detectable or only very low amounts were detectable in the uncultured reference medium (M1(0d)) without added glucose. In comparison with the culture medium M1, the culture was carried out on media based on strawberry and raspberry fruit puree additionally containing 0.4% and 0.8% glucose (M2 and M3, respectively), with formation of a small amount of the aroma-active compound present (see fig. 16). The formation of the aroma-active compounds thus present is mainly achieved by biotechnological transformation of the plant constituents present in the culture medium.
The culture method used had a significant impact on the quantitative composition of the aroma of the strain. Therefore, the aroma-active compound, which is an aroma characteristic of the outlet strain of poria cocos, is also detected by the amount of deviation in submerged culture or fixed bed culture of the fungus. FIG. 17 shows an example chromatogram of a fixed bed culture of Poria cocos wolf on a medium with strawberry pomace and aspartic acid.