JP2005087084A - Plant aroma enhancing method and aroma enhancing agent to be used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an aroma enhancing method for continuously enhancing aroma of plants, especially cut flowers by the action of an enzyme exemplified by diglycosidase or β-glucosidase and decomposing aroma precursors which are produced by microorganisms, and to obtain an aroma enhancing agent to be used for the method. <P>SOLUTION: The plant aroma enhancing method comprises an enzyme treatment process of making the enzyme decomposing the aroma precursors which are produced by the microorganisms act on plants. The enzyme decomposing the aroma precursors comprises a sugar-decomposing enzyme. The sugar-decomposing enzyme is the diglycosidase or the β-glucosidase. The aroma enhancing agent containing as the active ingredient the enzyme decomposing the aroma precursors is used for the plant aroma enhancing method. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention enhances the fragrance of a plant by absorbing the enzyme solution of the enzyme produced by the microorganism from the conduit of the plant or spraying the enzyme on the plant to cause the fragrance component to be released from the plant by an enzymatic reaction. The present invention relates to a method and an aroma enhancer used therefor.

In recent years, plants for cut flowers are often cultivated in a greenhouse, but the fragrance of cut flowers cultivated in the greenhouse is generally said to be weaker than that of alley cultivation, and has a fragrance comparable to alley cultivation. A cut flower having been desired. Therefore, conventionally, a method for enhancing the aroma of such a plant has been proposed. For example, by adding a fragrance enhancer for cut flowers containing a water-soluble fragrance or a water-dispersible fragrance as an active ingredient to the water in which cut flowers are placed, the fragrance of the fragrances is continuously emitted from the flowers or leaves of the cut flowers. There is a proposal of a method (refer patent document 1). Further, as a method for enhancing the aroma of plants, there is a proposal of a method of absorbing a fragrance from the root (see Patent Document 2). However, in these methods, since the fragrance is hardly absorbed from the stem and root due to the relationship between the polarity and the molecular weight, the effect of enhancing the fragrance could not be sufficiently expected. Moreover, although the method of spraying a fragrance | flavor directly on a plant body is also proposed (refer patent document 3), since the persistence of a fragrance is low in this method, since it harms a plant body, especially a flower, discoloration and browning, It could not be a useful method.
Japanese Patent No.2922177 Japanese Unexamined Patent Publication No. 4-108317 JP 59-220125 A

  None of the above conventional methods for enhancing the fragrance of plants was an invention of the concept of adding a fragrance synthesized exogenously to the original fragrance of the plant, so that sufficiently satisfactory results could not be obtained. By the way, at present, as aroma components of plants, monoterpene alcohols such as geraniol and aromatic alcohols such as phenylethyl alcohol are known. These aroma components are contained in fruits, flowers and leaves derived from plants, and are also contained in wines, teas, foods, etc. of these processed products in a free state, but it is also apparent that they are contained as glycosides. It is becoming. And it has been reported that endogenous β-primeverosidase plays an important role in the process of producing these aromatic components (IEAckermann et.al., J. Plant Physiol., 134, 567-572). (1989), Sakata Kanzo et al., Applied Glycoscience, Vol. 45, No. 2, 123-129 (1998)). That is, the aroma of the plant body is caused by a mechanism in which the β-glycoside of the glycoside, which is an aroma precursor, is decomposed by a kind of β-primeverosidase of diglycosidase and the aroma component is released.

  Therefore, if the action of the enzyme that degrades the fragrance precursor exemplified by β-primeverosidase is enhanced, the degradation of β-glycoside of the fragrance precursor is promoted, and more fragrance components in the plant body are generated. It is thought that you can. Or it is thought that the same effect can be anticipated by making the enzyme which decomposes | disassembles an aromatic precursor act from the outside of a plant body.

  An object of the present invention is to provide an aroma enhancing method and an aroma enhancing agent capable of continuously enhancing the aroma of plants, particularly cut flowers, by the action of an enzyme that degrades aroma precursors produced by microorganisms. And Another object of the present invention is to provide a plant in which aroma components are enhanced by the action of an enzyme that decomposes aroma precursors.

The present inventors added an enzyme produced by a microorganism to water in which cut flowers are alive, so that the enzyme is absorbed into the plant body through the conduit at the same time as landing, and then efficiently sent to the flower or leaf. It has been found that the fragrance precursors inherent in the leaves are decomposed by an enzymatic reaction due to the activity of the enzyme and the fragrance components are emitted, and a stronger fragrance is produced from the cut flowers, and the present invention has been completed.
That is, the gist of the present invention is a plant body aroma enhancement method comprising an enzyme treatment step in which an enzyme that degrades aroma precursors produced by microorganisms acts on a plant body. In the present invention, the enzyme that degrades the aromatic precursor may be a saccharide-degrading enzyme, and the saccharide-degrading enzyme may be diglycosidase or β-glucosidase. Diglycosidase refers to an enzyme that cleaves sugar into disaccharide units. By this enzyme, β-glycosides of glycosides that are fragrance precursors are decomposed to release fragrance components. -Primeverosidase is one of them. Β-Glucosidase is an exo-type saccharide-degrading enzyme that refers to an enzyme that cleaves glucose in monosaccharide units. By acting on this enzyme, the β-glycoside of the fragrance precursor is decomposed to release the fragrance component. It became clear. In the present invention, the plant body may be a petal, the plant body may be a rose family plant or the iris family plant, or the plant body may be a cut flower. In the present invention, the plant body may be immersed in a solution to which an enzyme for decomposing the fragrance precursor is added, or a solution to which an enzyme for decomposing the fragrance precursor is added to the plant body may be sprayed.

  Further, the gist of the present invention is an aroma enhancer for use in a method for enhancing the aroma of a plant comprising an enzyme that degrades the aroma precursor described in the above method as an active ingredient.

  The gist of the plant is enhanced in aroma by the method described above.

  According to the present invention, an aroma enhancer containing, as an active ingredient, an enzyme that degrades an aroma precursor produced by a microorganism exemplified by diglycosidase or β-glucosidase is added to water in which a plant body can live, or to leaves and petals. Direct spraying is extremely useful because it allows the fragrance of the fragrances to be continuously emitted from the flowers and leaves of the plant to impart sufficient fragrance to the plant.

  The fragrance enhancer used in the plant fragrance enhancing method of the present invention is an enzyme that degrades a fragrance precursor produced by a microorganism, preferably a saccharide-degrading enzyme, more preferably a glycolytic enzyme diglycosidase or β- Glucosidase is an active ingredient. Microorganisms can be of any origin as long as they produce diglycosidase, but the genus Aspergillus, Penicillium, Rhizopus, Rhizomucor, Talaromyces, Mortierella ( Microorganisms of the genus Mortierella, the genus Cryptococcus, the genus Microbacterium, the genus Corynebacterium or the genus Actinoplanes are preferred. The microorganism may be of any origin as long as it produces β-glucosidase, but microorganisms of the genus Aspergillus, Penicillium, Rhizopus or Trichoderma are preferred. Among these, Aspergillus niger IFO4407, Aspergillus niger IAM2020, Aspergillus niger AMA9328, Aspergillus fumigatus IAM2046, penicillium multi-color 715 More preferred is the enzyme produced by Trichoderma viride AMA9061.

  The fragrance enhancer used in the present invention is added with an appropriate excipient, for example, dextrin, starch, modified starch, etc., to the enzyme that decomposes the fragrance precursor as an active ingredient, and is dried by means such as spray drying or vacuum drying. Can be powdered. In addition, the fragrance enhancer can be used by adding a fragrance glycoside, a surfactant, and ethanol as necessary. Furthermore, the aroma enhancer includes conventionally used nutrient sources such as nitrogen, phosphate, potassium, sucrose and vitamin C, trace nutrients such as iron, zinc, manganese, copper and boron, B-nine, Plant growth regulators such as benzyladenine and brassinolide can also be mixed.

  A pretreatment agent such as Coat Fresh (registered trademark) can be added to the aroma enhancer for the purpose of improving the flower life.

  Diglycosidase or β-glucosidase, for example, is a commercially available crude enzyme agent such as galactosidase, cellulase, lactase, or other enzyme products or preparations, and diglycosidase or β-glucosidase contained as an accessory component in these acts. It is also possible to make it.

  The concentration in the case of using an aroma enhancer as an aqueous solution varies depending on various conditions such as the type of target cut flower and cannot be specified unconditionally, but for 100 mL of water in the container holding the cut flower, The range is 0.001 to 4 g, preferably 0.004 to 0.4 g, more preferably 0.01 to 0.1 g. However, the bulk powder usually has about 2000 AU of diglycosidase or β-glucosidase activity per gram, but if the activity value is low, the input amount may be increased.

  The aroma enhancement method of the present invention can be widely used for plants, but is particularly preferably used for cut flowers. Plants used for cut flowers include, for example, hidden flower plants that are houseplants cultivated in alleys, houses, flower pots, etc., or flowering plants that appreciate flowers.

  Examples of flowering plants include gypsophila, chrysanthemums, roses, tulips, lilies, carnations, orchids, stocks, sweet peas, freesia, and staticis, with roses and irises being particularly preferred.

  The fragrance enhancing method of the present invention can be achieved by immersing the cut end of a cut flower in an enzyme solution of a fragrance enhancing agent and absorbing it through a cut flower conduit, or by directly adding the fragrance enhancing agent to a vase containing a plant body, a basin or the like. They can be absorbed through the conduit, and any of them does not require any special treatment and can be carried out easily and simply. More specific examples include a method for enhancing the aroma while immersed in an aqueous solution of an aroma enhancer, or a method for enhancing the subsequent aroma by dipping for a predetermined time. As the former, for example, when selling cut flowers in a container such as a bucket in a store, or when keeping cut flowers in a vase at home or at an exhibition, the latter, for example, by a fragrance enhancer only in the process of transporting cut flowers This is the case when the cuts are processed and the fragrance is already sufficiently enhanced. Thus, the fragrance enhancing method of the present invention can be used in various modes.

  Moreover, although the aroma enhancer used by this invention is easy to absorb through the conduit | pipe of a plant body as mentioned above, the method of spraying a plant body can enhance a fragrance more efficiently. Although it varies depending on various conditions such as the type of cut flower to be targeted and cannot be defined unconditionally, preferably 1 mL of diglycosidase or β-glucosidase with a concentration of 0.001 to 20 (AU) per plant, More preferably, it is desirable to spray about 1 mL of 0.01 to 1 (AU) concentration. It is also possible to enhance the fragrance by appropriately combining spraying on the plant body and absorption through the conduit of the plant body.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1] Preparation of aroma enhancer (1-1) Preparation of diglycosidase using Penicillium multicolor IAM7153 strain (hereinafter referred to as producing bacterium)
A growth medium (pH 5.6) containing 2.0% defatted soybeans, 3.0% glucose, 0.5% potassium dihydrogen phosphate, 0.4% ammonium sulfate, and 0.3% dry yeast was sterilized at 121 ° C. for 20 minutes. 100 mL of the sterilized medium was inoculated with 1 ase of the producing bacteria, and precultured at a shaking speed of 27 ° C. and 140 min ⁻¹ . After 5 days, 20 L of this pH 4.9 medium containing 1.0% Sunfiber®, 2.0% potassium dihydrogen phosphate, 1.0% ammonium sulfate, 3.13% Mist P1G in a 30 L jar fermenter, 150 min ⁻¹ The mixture was sterilized at 121 ° C. for 20 minutes with stirring. The main culture medium is inoculated with the above-mentioned preculture medium at 1.5% (v / v), the stirring rate is 250 min ⁻¹ , the air flow rate is 0.75 vvm (15 L / min), and the internal pressure is 0.5 Kg / cm ² (48 kPa). And cultured at 27 ± 1 ° C. for 8 days.

(1-2) Partial purification of diglycosidase
To the culture broth, Zemlite Super 56M and Fine Flow A were added to the culture broth as 2% of the total liquid volume, respectively, and diatomaceous earth filtration was performed. The membrane was concentrated 20 times with an ultrafiltration membrane UF AIP-2020 (MW 6,000) and replaced with 20 mM acetate buffer at pH 4.7. This was aseptically filtered and lyophilized to obtain crude diglycosidase (hereinafter referred to as GLY).

(1-3) β-glucosidase contained as an accessory component in enzyme products A certain percentage of enzyme preparations sold as α-D-Galactosidase “AMANO” (hereinafter ADG), a galactosidase manufactured by Amano Enzyme, Inc. Has been found to contain β-glucosidase. Therefore, this preparation was also subjected to the following evaluation as an aroma enhancer.

(1-4) Method for measuring diglycosidase and β-glucosidase activity Using an automated chemical analyzer (TBA-30R, manufactured by Toshiba Corporation), 30 μL of GLY sample was mixed with acetic acid so that paranitrophenyl (pNP) primeveroside was 2 mM. Mix with 200 μL of buffer solution (pH 5.5) and react for 9.75 minutes at 40 ° C with a cycle time of 22.5 seconds. Add 250 μL of sodium carbonate and measure the absorbance at 412 nm. The blank from the sample is measured in the same manner using 20 mM acetate buffer (pH 5.5) instead of the substrate solution. In addition, 30 μL of ADG sample is mixed with 200 μL of paranitrophenyl (pNP) glucoside dissolved in acetic acid buffer (pH 5.5) to 2 mM, and the absorbance is measured under the same conditions. The amount of enzyme that increases the absorbance by 1 under these conditions is defined as 1 unit (AU).
The above-mentioned pNP-primeveroside is prepared by reacting pNP-glucoside (Merck) and xylooligosaccharide (Wako Pure Chemical Industries) using the enzyme xylosidase (Sigma) and binding xylose to pNP-glucoside by β-1,6 It was synthesized by transferring one residue at

[Example 2] Evaluation of aroma enhancement effect on rose (2-1) Sensory evaluation by panelists 0.01% (w of GLY (2527 AU / g) and ADG (13.3 AU / g) of the aroma enhancer obtained above / v) Each aqueous solution was prepared. 20 ml of these aqueous solutions (GLY contains about 5 AU, ADG contains about 0.03 AU) and 3 Rosette varieties rose and treated in a closed container for 24 hours. Evaluation of the fragrance was performed. On the other hand, as a control without enzyme treatment, three roses of three roterosa varieties were placed in a container containing only water, and sensory evaluation was performed by five panelists on the aroma emitted from both cut flowers.

  The sensory test is evaluated in five levels (score 5: strong, score 4: slightly strong, score 3: normal, score 2: slightly weak, score 1: weak). The higher the value, the higher the evaluation of the item. To do. Regarding the items, two kinds of senses of “sweet scent” and “fruity” were evaluated. Moreover, the average which totaled the evaluation value of all five panelists was displayed as a reference numerical value. This sensory test evaluation method is the same in the following examples. The results are shown in Table 1.

  As is clear from Table 1, all five panelists from A to E responded that the roses treated with GLY had a “sweet scent” compared to untreated, and 4 out of 5 panelists other than panel A were more He responded that it was “fruity”. On the other hand, roses treated with ADG responded that 4 out of 5 people other than Paneler A had a “sweet scent” and 4 out of 5 people other than Paneler C responded that they were “fruity”. It was. In both GLY treatment and ADG treatment, the average score of “sweet scent” and “fruity” was at least 0.8 points higher than untreated (1.2 points or more for GLY). From these results, the effect of enhancing aroma by enzyme treatment was clearly recognized.

  Rose aroma precursors are present in leaves, stems and petals. In particular, a glycoside of phenylethyl alcohol, which is one of the main aroma components of roses, is synthesized in petals. Monoterpene alcohol, on the other hand, is synthesized in the leaves and moves to the stem as the buds develop and flower. Therefore, it is considered that the method of directly acting the enzyme on the leaves, stems and petals, that is, the action site, that is, the spraying method is more effective than the absorption of the enzyme by water, and the same five panelists as above. Sensory evaluation was performed.

  A 0.01% (w / v) aqueous solution of GLY and ADG was prepared. Three roterosa varieties were sprayed onto each leaf, stem and petal with 5 mL of each enzyme solution (activity of about 1.3 AU for GLY and about 0.007 AU for ADG). The fragrance produced per day was evaluated by treating the sprayed roses in a closed container for 24 hours. On the other hand, as an untreated control, the same amount of water was sprayed for sensory evaluation. The results are shown in Table 2.

  As is clear from Table 2, all the five panelists A to E responded that the rose treated with GLY had a “sweet scent” and more “fruity” than untreated. On the other hand, all five panelists from A to E also responded that the treatment had a “sweeter scent” and that four out of five other panelists were “fruity”. In both GLY treatment and ADG treatment, the average score of “sweet scent” and “fruity” was at least 1.4 points higher than untreated (1.8 points or more for GLY). Since there was no petal damage due to the spray of the enzyme solution, it was found that spraying directly on the leaves, petals, and stems would be more efficient as a means to enhance the aroma than to absorb the enzyme from the water. .

(2-2) Analysis of aroma components by gas mass analyzer (GC / MS)
In addition to sensory evaluation, a quantitative test using a mass spectrometer was conducted to evaluate the increase and decrease of the substance level with and without enzyme treatment. Triangular Kolben was charged with 40 g of rose buds and 80 mL of purified water, heated in a microwave oven (Sharp, RE-B3, 1380 W) and boiled until stopped and mixed with liquid. This process was repeated several times and heat-treated for a total time of 6 minutes. The filtrate obtained by removing the residue with filter paper was used as an extract for enzyme treatment. GLY was added at a concentration of 0.001, 0.01, 0.1% (w / v) to 5 mL of the extract. ADG was added at respective concentrations of 0.004 and 0.04% (w / v). After adding these GLY or ADG to each at 50 ° C. for 2 hours, 3 mL was used as a sample for GC / MS analysis. A headspace (HP7694 Headspace sampler, manufactured by Agilent technoligies) -GC / MS (HP5973GC / HP6890MSD, manufactured by Agilent technologies) system was used for analysis of aroma components. The column was HP-WAX (60 x 0.25 mm x 0.25 µm), the moving bed was helium, and the flow rate was 1.0 mL / min. A 3 mL analytical sample was placed in a vial and heated at 85 ° C. for 20 minutes before the gas was sampled and injected into the GC / MS at a split ratio of 25: 1. The initial oven was held at 40 ° C. for 5 minutes, heated to 100 ° C. at a rate of 5 ° C./min, further heated to 230 ° C. at a rate of 10 ° C./min, and held for 10 minutes. The results are shown in Table 3.

  As is clear from Table 3, phenylethyl alcohol, one of the main fragrance components of roses, increases more than about 2 times compared to untreated when the GLY concentration is 0.001% (0.025 AU / mL). ing. Furthermore, when the enzyme concentration is 10 times 0.01% (0.25 AU / mL), the enzyme concentration is increased to about 15 times or more compared to the untreated. Furthermore, when the enzyme concentration is 10%, 0.1% (2.5 AU / mL), it increases by about 45 times or more compared to untreated. The 0.004% ADG treatment (0.005 AU / mL) also increased about 45 times more than the untreated. Similarly, linalool, one of the major aroma components of roses, is increased about 5 times or more with 0.1% GLY, and increased about 8 times or more with 0.04% ADG treatment (0.05 AU / mL). Dihydro β-ionol is known as an aroma component of cinnamon rather than rose, but it is about 170 times more than 0.1% GLY and about 180 times more than 0.04% ADG treatment (0.05 AU / mL). It has increased. Similarly, the results for theaspiran A and theaspiran B increased by about 300 times by the enzyme treatment. As described above, even at the substance level, the significant enhancement effect of the main aroma component of rose was confirmed by GLY treatment. On the other hand, although it was not expected that it was not the main ingredient of rose, it turned out that it produces in large quantities by enzyme treatment. Benzaldehyde, a refreshing scent that is considered a green note, was slightly increased by enzyme treatment.

[Example 3] Evaluation of aroma enhancement effect on freesia (3-1) Sensory evaluation by panelists
A 0.01% (w / v) aqueous solution of GLY and ADG was prepared. 10 mL of freesia was sprayed onto leaves, stems, and petals with 5 mL of each enzyme solution (activity of about 1.3 AU for GLY and about 0.007 AU for ADG). The fragrance produced per day was evaluated by treating the sprayed freesia for 24 hours in a closed container. On the other hand, as an untreated control, the same amount of water was sprayed for sensory evaluation. The results are shown in Table 4.

  As is clear from Table 4, all five panelists A to E responded that the freesia treated with GLY had a “sweet scent” and more “fruity” than untreated. On the other hand, 4 out of 5 people except panel B responded that the ADG treatment was more “sweet” and that all 5 panelists A to E were “fruity”. . In both GLY treatment and ADG treatment, the average score of “sweet scent” and “fruity” was at least 1.0 points higher (1.4 points for GLY) than untreated.

Claims (17)

  A method for enhancing an aroma of a plant comprising an enzyme treatment step in which an enzyme that decomposes an aroma precursor produced by a microorganism is allowed to act on the plant.   The method for enhancing an aroma of a plant according to claim 1, wherein the enzyme that decomposes the aroma precursor is a saccharide-degrading enzyme.   The method for enhancing aroma of a plant according to claim 2, wherein the saccharide-degrading enzyme is diglycosidase or β-glucosidase.   Diglycosidase is genus Aspergillus, Penicillium, Rhizopus, Rhizomucor, Talaromyces, Mortierella, Cryptococcus Micro, Bacteria 4. The method for enhancing aromas of a plant according to claim 3, which is produced by any microorganism of the genus Corynebacterium or Actinoplanes.   The method for enhancing aroma of a plant according to claim 3, wherein β-glucosidase is produced by any microorganism of the genus Aspergillus, Penicillium, Rhizopus or Trichoderma. .   Aspergillus microorganism is Aspergillus niger or Aspergillus fumigatus, Penicillium genus is Penicillium multicolor, Trichoderma microorganism is Trichoderma The method for enhancing aroma of a plant according to claim 4 or 5, wherein the method is Trichoderma viride.   Aspergillus niger I Aspergillus niger IFO4407, Aspergillus niger IAM2020 or Aspergillus niger AMA9328, Aspergillus fumigatus The plant aroma enhancement according to claim 6, wherein IAM2046, Penicillium multicolor is Penicillum multicolor IAM7153, Trichoderma viride is Trichoderma viride AMA9061 Law.   The method for enhancing aroma of a plant according to claim 7, wherein the diglycosidase produced by Penicillium multicolor IAM7153 is an extracellular enzyme.   The plant body fragrance enhancing method according to any one of claims 1 to 8, wherein the plant body is a petal.   The plant body fragrance enhancing method according to any one of claims 1 to 9, wherein the plant body is a Rosaceae plant or Iridaceae plant. The method for enhancing aroma of a plant according to claim 10, wherein the rose family plant is a rose genus plant and the iris family plant is a freesia genus plant. The method for enhancing aroma of a plant according to claim 11, wherein the rose genus plant is a rose and the freesia genus plant is freesia. The plant body fragrance enhancing method according to any one of claims 1 to 12, wherein the plant body is a cut flower. The method for enhancing an aroma of a plant according to any one of claims 1 to 13, wherein the plant is immersed in a solution to which an enzyme that decomposes an aroma precursor is added. The method for enhancing an aroma of a plant according to any one of claims 1 to 13, wherein the plant is sprayed with a solution to which an enzyme that decomposes an aroma precursor is added. The fragrance enhancer used for the fragrance enhancement method of the plant body which contains the enzyme which decomposes | disassembles the fragrance precursor in any one of Claims 1-8 as an active ingredient. A plant whose aroma is enhanced by the method according to any one of claims 1 to 15.