A method for enhancing the fragrance of in vitro agarwood

Abstract

The present invention relates to the field of biotechnology and discloses a method for enhancing the aroma of an in vitro agarwood block, comprising the following steps: (1) selecting, cleaning, disinfecting, washing, drying, and crushing the in vitro agarwood block to obtain agarwood powder; (2) preparing an aroma-enhancing bacterial liquid: cultivating an aroma-enhancing bacterial strain to obtain an aroma-enhancing bacterial liquid, which is then set aside; (3) adding the agarwood powder to the aroma-enhancing bacterial liquid in a certain proportion, culturing for a certain period of time, filtering out the fermentation liquid, obtaining fermented and aroma-enhanced natural agarwood powder, and drying the powder to obtain the aroma-enhanced in vitro agarwood. The method for enhancing the aroma of an in vitro natural agarwood block is simple, controllable, low-cost, and short-cycle, and can fully utilize and expand the application of agarwood resources.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for enhancing the fragrance of in vitro agarwood. Background Art

[0002] Agarwood is the resinous wood of the Aquilaria plant in the Thymelaeaceae family. It is also a precious medicinal ingredient, known as "one ounce of agarwood is worth one ounce of gold." Over 80 traditional Chinese medicine formulas contain agarwood, and its use is also growing in the tobacco, daily chemical, and food industries. Agarwood essential oil, a rare and premium fragrance known as the "King of Fragrance," is deeply loved by consumers. It possesses the unique top, middle, and base notes of agarwood, boasting a rich, long-lasting aroma, making it a key ingredient in high-end perfumes.

[0003] The formation of agarwood is very unique, and it only forms after being induced to form agarwood after being subjected to external damage such as physical damage, chemical damage, and fungal infection. This is also the main reason for the low agarwood yield of natural agarwood. Increasing the yield of agarwood requires human intervention, and the main methods used include "cutting", "breaking branches", "drilling holes", "chemical reagents for agarwood formation", and "fungal inoculation". In recent years, the use of chemical reagents to form agarwood and fungal inoculation methods have attracted more and more attention. However, these methods all involve artificial intervention in surviving agarwood plants, and the final results cannot be controlled after the intervention. For example, after the agarwood plants are inoculated with fungi, the fungal survival rate and the effect of the fungi are difficult to control. Although agarwood can be formed, the aroma varies greatly. In addition, due to the influence of many factors such as agarwood variety, planting environment and management level, agarwood formation technology, extraction and purification technology, agarwood essential oil samples from various manufacturers vary greatly in flavor, composition and function, making them difficult to standardize and commercialize. This greatly limits the application and promotion of agarwood essential oil, especially agarwood products exported to the Middle East require a strong fragrance. At present, many domestic agarwood essential oil products fail to meet the requirements of the Middle East. Therefore, in addition to promoting agarwood formation, the development of the agarwood industry should find a practical method to enhance the fragrance of agarwood essential oil to obtain agarwood essential oil with a strong fragrance. Summary of the Invention

[0004] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for enhancing the fragrance of agarwood in vitro, which is controllable, low-cost, short-time, not affected by external factors (climate, soil, variety, etc.), and simple to operate.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for enhancing the fragrance of agarwood in vitro, comprising the following steps:

[0007] (1) selecting, cleaning, disinfecting, washing, drying, and crushing the isolated Aquilaria sinensis wood blocks to obtain Aquilaria sinensis wood powder;

[0008] (2) Preparation of flavoring bacterial liquid: Cultivate the flavoring bacterial strain to obtain the flavoring bacterial liquid, which is then set aside;

[0009] (3) Adding agarwood powder to the aroma-enhancing bacterial liquid in a certain proportion, mixing evenly and culturing and fermenting for a certain time, filtering out the fermentation liquid to obtain fermented aroma-enhancing agarwood powder, drying it, and obtaining aroma-enhancing in vitro agarwood powder.

[0010] Preferably, the isolated agarwood in step (1) is one or both of natural agarwood and artificially intervened agarwood.

[0011] Preferably, the particle size of the agarwood powder in step (1) is 60-600 mesh.

[0012] Preferably, the aroma-enhancing bacterial liquid OD in step (2) 600 =0.6-1.2.

[0013] Preferably, the aroma-enhancing bacteria in step (2) are one or more of Trichoderma sp. and Lasiodiplodia theobromae. More preferably, the aroma-enhancing bacteria are one or both of Trichoderma atroviride MA3-4 and Lasiodiplodia theobromae ASAF12.

[0014] Preferably, the ratio of the agarwood powder to the bacterial solution in step (3) is 1:0.1-10 (g / mL).

[0015] Preferably, the agarwood powder and the bacterial solution in step (3) are evenly mixed and then cultured and fermented for 1-30 days, and the fermentation conditions are constant temperature culture at 26±2°C.

[0016] Preferably, the method further comprises the following steps:

[0017] The scented in vitro agarwood is extracted to obtain agarwood essential oil. The extraction method refers to the document "Gas chromatography-mass spectrometry analysis of agarwood essential oil extracted by supercritical and subcritical methods", and subcritical extraction is used to extract the scented essential oil.

[0018] A kind of agarwood essential oil is obtained by the above method.

[0019] The present invention uses microorganisms to ferment agarwood that has formed fragrance, thereby achieving the purpose of enhancing the fragrance of the agarwood.

[0020] The present invention has the following advantages and beneficial effects compared to the prior art:

[0021] 1) The present invention utilizes microorganisms that have a scent-enhancing effect on agarwood to enhance the scent of in vitro agarwood, and the process is controllable, the time is short, and the scent-enhancing effect is obvious;

[0022] 2) The agarwood with in vitro aroma used in the present invention can be natural agarwood or artificially intervened agarwood, and the degree of aroma can be light agarwood, moderate agarwood, normal agarwood, etc., so as to make full use of agarwood resources and reduce waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the GC-MS spectrum of the changes in agarwood essential oil over time (from top to bottom: 1, 3, 5, 7, 10, 15, 20 days) after the isolated agarwood was treated with the aroma-enhancing fungus (Trichoderma atroviride MA3-4). DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0025] Trichoderma atroviride Horse 3-4: Patent CN 110250211 A has been published.

[0026] Lasiodiplodia theobromae ASAF12: Patent CN 105349430 A has been published.

[0027] The composition of PDA liquid culture medium is as follows: 200 g of potato skin was removed, cut into small pieces, added to distilled water and boiled for 30 min, filtered through 8 layers of gauze, 20 g of glucose was added, the volume was fixed to 1000 mL, chloramphenicol was added and sterilized at 121°C under high pressure for 20 min.

[0028] Example 1

[0029] (1) Treatment of in vitro agarwood blocks: Take artificial intervention agarwood blocks (white agarwood), select and remove the moldy and blackened parts, rinse with sterile water 3 times, wash with 75% ethanol for 1 minute, rinse with sterile water once, rinse with 5% sodium hypochlorite solution for 2 minutes, rinse with sterile water 3 times, dry, crush and set aside.

[0030] (2) Preparation of flavor-enhancing bacterial suspension: Take 3-4 Trichoderma atroviride, add it into PDA liquid culture medium, place it in a constant temperature incubator at 26℃ for activation culture, and adjust the cultured fresh bacterial suspension to OD 600 =0.9 for standby use.

[0031] (3) Take 100 g of the artificial intervention woody wood powder prepared in (1), add it to the bacterial suspension prepared in (2) at a ratio of artificial intervention woody wood powder: bacterial suspension = 1:1 (w / v), and place it in a constant temperature incubator at 26°C for 15 days.

[0032] After 15 days of incubation, the fermented and aromatized agarwood powder was separated from the fermentation broth using a Büchner funnel. The powder was placed in a glass dish, wrapped in plastic wrap, punctured, and dried in a 45°C oven. The essential oil extracted and aromatized by subcritical and subcritical extraction was analyzed by GC-MS, as described in the reference "Gas Chromatography-Mass Spectrometry Analysis of Agarwood Essential Oil." Chromatographic conditions: The column used was an SH-Rxi-5Sil-MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The temperature program was as follows: 50°C, then increased at 5°C / min to 310°C, held for 10 min. The vaporizer temperature was 250°C; the carrier gas was high-purity He (99.999%); the carrier gas flow rate was 1.0 mL / min; the sample was injected without splitting; and the solvent delay time was 4.0 min.

[0033] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; interface temperature 280°C; emission current 34.6 μA; multiplier voltage 1612 V; mass scan range 40-800 m / z.

[0034] Example 2

[0035] (1) Treatment of in vitro agarwood blocks: Take artificial intervention agarwood blocks (white agarwood), select and remove the moldy and blackened parts, rinse with sterile water 3 times, wash with 75% ethanol for 1 minute, rinse with sterile water once, rinse with 5% sodium hypochlorite solution for 2 minutes, rinse with sterile water 3 times, dry, crush and set aside.

[0036] (2) Preparation of flavor-enhancing bacterial suspension: Take 3-4 Trichoderma atroviride, add it into PDA liquid culture medium, place it in a constant temperature incubator at 26℃ for activation culture, and adjust the cultured fresh bacterial suspension to OD 600 =0.9 for standby use.

[0037] (3) Take 100 g of the artificial intervention woody wood powder prepared in (1), add it to the bacterial suspension prepared in (2) at a ratio of artificial intervention woody wood powder: bacterial suspension = 1:1 (w / v), and place it in a constant temperature incubator at 26°C for 30 days.

[0038] After 30 days of cultivation, the fermented and aromatized agarwood powder was separated from the fermentation broth using a Büchner funnel. The powder was placed in a glass dish, wrapped in plastic wrap, and punctured. The powder was then dried in a 45°C oven. The essential oil extracted and aromatized by subcritical and subcritical extraction was analyzed by GC-MS, as described in the reference "Gas Chromatography-Mass Spectrometry Analysis of Agarwood Essential Oil." Chromatographic conditions: The column used was an SH-Rxi-5Sil-MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The temperature program was as follows: 50°C, then increased at 5°C / min to 310°C, held for 10 min. The vaporizer temperature was 250°C; the carrier gas was high-purity He (99.999%); the carrier gas flow rate was 1.0 mL / min; the sample was injected without splitting; and the solvent delay time was 4.0 min.

[0039] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; interface temperature 280°C; emission current 34.6 μA; multiplier voltage 1612 V; mass scan range 40-800 m / z.

[0040] Example 3

[0041] (1) Treatment of in vitro agarwood blocks: Take artificial intervention agarwood blocks (white agarwood), select and remove the moldy and blackened parts, rinse with sterile water 3 times, wash with 75% ethanol for 1 minute, rinse with sterile water once, rinse with 5% sodium hypochlorite solution for 2 minutes, rinse with sterile water 3 times, dry, crush and set aside.

[0042] (2) Preparation of aroma-enhancing bacterial suspension: Take Lasiodiplodia theobromae ASAF12, add it into PDA liquid culture medium, place it in a constant temperature incubator at 26℃ for activation culture, and adjust the cultured fresh bacterial suspension to OD 600 =0.9 for standby use.

[0043] (3) Take 100 g of the artificial intervention woody wood powder prepared in (1), add it to the bacterial suspension prepared in (2) at a ratio of artificial intervention woody wood powder: bacterial suspension = 1:1 (w / v), and place it in a constant temperature incubator at 26°C for 15 days.

[0044] After 15 days of incubation, the fermented and aromatized agarwood powder was separated from the fermentation broth using a Büchner funnel. The powder was placed in a glass dish, wrapped in plastic wrap, punctured, and dried in a 45°C oven. The essential oil extracted and aromatized by subcritical and subcritical extraction was analyzed by GC-MS, as described in the reference "Gas Chromatography-Mass Spectrometry Analysis of Agarwood Essential Oil." Chromatographic conditions: The column used was an SH-Rxi-5Sil-MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The temperature program was as follows: 50°C, then increased at 5°C / min to 310°C, held for 10 min. The vaporizer temperature was 250°C; the carrier gas was high-purity He (99.999%); the carrier gas flow rate was 1.0 mL / min; the sample was injected without splitting; and the solvent delay time was 4.0 min.

[0045] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; interface temperature 280°C; emission current 34.6 μA; multiplier voltage 1612 V; mass scan range 40-800 m / z.

[0046] Example 4

[0047] (1) Treatment of in vitro agarwood blocks: Take artificial intervention agarwood blocks (white agarwood), select and remove the moldy and blackened parts, rinse with sterile water 3 times, wash with 75% ethanol for 1 minute, rinse with sterile water once, rinse with 5% sodium hypochlorite solution for 2 minutes, rinse with sterile water 3 times, dry, crush and set aside.

[0048] (2) Preparation of flavor-enhancing bacterial suspension: Take Trichoderma atroviride Ma 3-4 and Lasiodiplodia theobromae ASAF12, add them into PDA liquid culture medium, place them in a constant temperature incubator at 26℃ for activation culture, and adjust the fresh bacterial suspension of the two cultures to OD 600 =0.9, mix and set aside.

[0049] (3) Take 100 g of the artificial intervention woody wood powder prepared in (1), add it to the bacterial suspension prepared in (2) at a ratio of artificial intervention woody wood powder: bacterial suspension = 1:1 (w / v), and place it in a constant temperature incubator at 26°C for 15 days.

[0050] After 15 days of incubation, the fermented and aromatized agarwood powder was separated from the fermentation broth using a Büchner funnel. The powder was placed in a glass dish, wrapped in plastic wrap, punctured, and dried in a 45°C oven. The essential oil extracted and aromatized by subcritical and subcritical extraction was analyzed by GC-MS, as described in the reference "Gas Chromatography-Mass Spectrometry Analysis of Agarwood Essential Oil." Chromatographic conditions: The column used was an SH-Rxi-5Sil-MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The temperature program was as follows: 50°C, then increased at 5°C / min to 310°C, held for 10 min. The vaporizer temperature was 250°C; the carrier gas was high-purity He (99.999%); the carrier gas flow rate was 1.0 mL / min; the sample was injected without splitting; and the solvent delay time was 4.0 min.

[0051] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; interface temperature 280°C; emission current 34.6 μA; multiplier voltage 1612 V; mass scan range 40-800 m / z.

[0052] Example 5

[0053] (1) Treatment of in vitro agarwood blocks: Take artificial intervention agarwood blocks (Agarwood), select and remove the moldy and blackened parts, rinse with sterile water 3 times, wash with 75% ethanol for 1 minute, rinse with sterile water once, rinse with 5% sodium hypochlorite solution for 2 minutes, rinse with sterile water 3 times, dry, crush and set aside.

[0054] (2) Preparation of flavor-enhancing bacterial suspension: Trichoderma atroviride 3-4 and Lasiodiplodia theobromae ASAF12 were placed in PDA liquid culture medium and activated in a 26°C constant temperature incubator. The fresh bacterial suspensions of the two cultures were adjusted to OD 600 =0.9, mix and set aside.

[0055] (3) Take 100 g of the artificial intervention woody wood powder prepared in (1), add it to the bacterial suspension prepared in (2) at a ratio of artificial intervention woody wood powder: bacterial suspension = 1:1 (w / v), and place it in a constant temperature incubator at 26°C for 15 days.

[0056] After 15 days of incubation, the fermented and aromatized agarwood powder was separated from the fermentation broth using a Büchner funnel. The powder was placed in a glass dish, wrapped in plastic wrap, punctured, and dried in a 45°C oven. The essential oil extracted and aromatized by subcritical and subcritical extraction was analyzed by GC-MS, as described in the reference "Gas Chromatography-Mass Spectrometry Analysis of Agarwood Essential Oil." Chromatographic conditions: The column used was an SH-Rxi-5Sil-MS (30 m × 0.25 mm × 0.25 μm) elastic quartz capillary column. The temperature program was as follows: 50°C, then increased at 5°C / min to 310°C, held for 10 min. The vaporizer temperature was 250°C; the carrier gas was high-purity He (99.999%); the carrier gas flow rate was 1.0 mL / min; the sample was injected without splitting; and the solvent delay time was 4.0 min.

[0057] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; interface temperature 280°C; emission current 34.6 μA; multiplier voltage 1612 V; mass scan range 40-800 m / z.

[0058] Example 6

[0059] According to the method of Example 2, the essential oil after fragrance enhancement was extracted after culturing for 1, 3, 5, 7, 10, 15, and 20 days, and the content (%) of the characteristic components of the agarwood essential oil was detected by GC-MS. Figure 1 and Table 2.

[0060] Comparative Example 1

[0061] The difference from Examples 1, 2, 3 and 4 is that the fermentation was not performed using aroma-enhancing bacteria.

[0062] Comparative Example 2

[0063] The difference from Example 5 is that the fermentation process with flavor-enhancing bacteria was not performed.

[0064] Table 1 Contents of characteristic components of agarwood essential oil

[0065]

[0066] Note: “-” means not detected

[0067] According to the results in Table 1, the method provided by the present invention can effectively increase the characteristic components (linalool, γ-eudesmol, guaiacol, atractylol, (+)-agarwood and benzyl acetone) in agarwood essential oil, achieving the purpose of fragrance enhancement. Compared with the comparative example, the characteristic components of agarwood essential oil increased after 15 days of fermentation with aroma-enhancing bacteria, and a new characteristic component, atractylol, was also produced. The agarwood essential oil after fermentation and fragrance enhancement has a rich aroma, which can meet the needs of the Middle East market.

[0068] Depend on Figure 1 As shown in Table 2, during the fermentation process, the characteristic components in agarwood essential oil gradually increased with the extension of fermentation time, and then the increase gradually slowed down. Among them, the content of agarwood spirol increased by about 3 times after 20 days of fermentation, and the contents of γ-eudesmol, guaiacol and benzyl acetone increased by more than 2 times.

[0069] In Examples 1-5, the test results of the characteristic component content of the agarwood essential oil of each sample after fermentation and fragrance enhancement are similar to the test results at the corresponding time in Table 2, indicating that the technology provided by this patent is stable and controllable, and the fermentation and fragrance enhancement time can be adjusted according to actual needs to obtain stable agarwood essential oil.

[0070] Table 2 Changes of main characteristic components of agarwood essential oil over time after treatment with aroma-enhancing bacteria in vitro

[0071]

[0072] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for enhancing the fragrance of agarwood in vitro, characterized in that: The following steps are involved: (1) selecting, cleaning, disinfecting, washing, drying, and crushing the isolated Aquilaria sinensis wood blocks to obtain Aquilaria sinensis wood powder; (2) Preparation of flavoring bacterial liquid: Cultivate the flavoring bacterial strain to obtain the flavoring bacterial liquid, which is then set aside; (3) adding agarwood powder to the aroma-enhancing bacterial solution in a certain proportion, mixing evenly and then culturing and fermenting for a certain time, filtering out the fermentation solution to obtain fermented aroma-enhancing agarwood powder, drying to obtain aroma-enhancing isolated agarwood, and extracting the aroma-enhancing isolated agarwood to obtain agarwood essential oil; The isolated Aquilaria sinensis wood in step (1) is one or both of natural Aquilaria sinensis wood and artificially intervened Aquilaria sinensis wood; The aroma-enhancing fungus in step (2) is Trichoderma atroviride MA3-4 and / or Lasiodiplodia theobromae ASAF12; The OD value of the aroma-enhancing bacterial solution in step (2) 600 =0.6-1.2; The ratio of the agarwood powder to the bacterial solution in step (3) is 1:1 (g / mL); The agarwood powder and the bacterial solution are mixed evenly in step (3), and then cultured and fermented for 10-30 days at a constant temperature of 26±2°C; The method can effectively increase the characteristic components of agarwood essential oil, such as linalool, γ-eudesmol, guaiacol, atractylodesyl alcohol, (+)-bergamotene and benzyl acetone.

2. The method for enhancing the fragrance of agarwood according to claim 1, wherein: The particle size of the agarwood powder in step (1) is 60-600 mesh.

3. An agarwood essential oil, characterized in that: Obtained by the method of claim 1.

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