Aquilaria sinensis dormancy edgeworthia chrysantha production method

Abstract

The invention provides an aquilaria sinensis dormancy edgeworthia chrysantha producing method. The aquilaria sinensis dormancy edgeworthia chrysantha producing method comprises the following steps: selecting a vigorous growing diameter gt at breast height; the method comprises the following steps: drilling holes in a trunk and thicker lateral branches of aquilaria sinensis with the thickness of 6 cm, forming a certain inclination angle between the drilled holes and the horizontal plane of the trunk or the lateral branches, and injecting agilawood formation liquid which is fermented twice after drilling to induce formation of agilawood. The preparation method of the aquilaria sinensis edgeworthia chrysantha liquid comprises the following steps: mixing and crushing raw materials of the edgeworthia chrysantha liquid to obtain a primary mixed liquid; sealing and fermenting the primary mixed solution to obtain a fermented primary mixed solution; filtering and storing the fermentation primary mixed solution; and performing microbial fermentation on the filtered fermentation primary mixed liquid again, and filtering to obtain the aquilaria sinensis edgeworthia chrysantha liquid. According to the method, the agilawood producing period is shortened, the variety and content of sesquiterpene components in agilawood are increased, and Hp bacteriostasis test results show that the MIC value of the extract is remarkably increased, and a good bacteriostasis effect is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of agarwood formation, and in particular to a method for forming agarwood in a dormant state of white agarwood. Background Art

[0002] Aquilaria sinensis is a tropical and subtropical evergreen tree, also known as native agarwood, tooth incense tree, daughter incense, stack incense, cliff incense, green osmanthus incense, rue, etc. Healthy aquilaria sinensis cannot produce incense, and it is usually stimulated by natural coercion and intervention to produce incense. Under natural conditions, aquilaria sinensis takes a long time to produce incense. There are only a few aquilaria sinensis trees in the mountains that have incense, and agarwood can only be obtained from the wood, so agarwood resources are relatively scarce. At present, the main methods for inducing agarwood formation in Aquilaria sinensis include: spraying saturated salt water through a fire hole, fungal liquid infusion, cutting, half-stem cutting, drilling, burning, and inoculation. Although there has been some progress in the artificial induction of agarwood formation in Aquilaria sinensis, the traditional induction agarwood formation technology has many defects: (1) The traditional artificial incision technology requires a long agarwood formation cycle and low agarwood formation efficiency; (2) The agarwood obtained by artificial incision is of low quality, low volatile oil content, and few varieties. It usually requires older Aquilaria sinensis trees for artificial incision, which cannot meet the sharp market demand for agarwood; (3) The quality of volatile oil is poor, and the addition of chemical additives will cause damage to the Aquilaria sinensis tree and have a negative impact on the environment. (4) Unreasonable artificial incision technology will cause damage to Aquilaria sinensis and affect the later output. Summary of the invention

[0003] In view of this, the present invention develops a dormant agarwood formation method for white agarwood to solve the above problems.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A dormant agarwood formation method for white agarwood comprises the following raw materials of agarwood formation liquid: 2-3 parts of rice, 1-1.4 parts of Eclipta prostrata, 1-2 parts of Polygonum hydropiper, 0.5-1 parts of White Jasmine, and 1-2 parts of Pandan leaves in terms of weight. The agarwood formation method comprises the following steps: selecting a white agarwood plant with vigorous growth, with a breast diameter greater than 6 cm and a side branch greater than 5 cm, drilling a hole obliquely at a height of about 35-45 cm from the ground, and injecting agarwood formation liquid after drilling to induce agarwood formation.

[0006] Furthermore, the preparation method of the fragrance-binding liquid comprises:

[0007] S1, mixing, crushing and stirring the fragrance liquid raw materials;

[0008] S2, sealing the mixture and fermenting it to obtain a fermentation initial mixed liquid, filtering the fermentation initial mixed liquid and storing it;

[0009] S3, taking the filtered fermentation initial mixed liquid for microbial culture again, fermentation, filtering, and obtaining the aroma liquid.

[0010] Furthermore, the crushing has a particle size of 0.1-0.5 mm; S2, the sealed fermentation has a fermentation time of 28-35 days and a fermentation temperature of 25-35°C.

[0011] Furthermore, the microorganisms are Trichoderma chlororaphis, Fusarium solani and Diplodia cholerae, and the mass ratio of the initial fermentation mixture to Trichoderma chlororaphis, Fusarium solani and Diplodia cholerae is 100:3-5:2-3:4-8.

[0012] Furthermore, the microorganism is cultured at a temperature of 25-35° C. for 5-7 days, at a pH of 5.5-7.5, and under aerobic fermentation conditions.

[0013] Furthermore, the rice is one of indica rice, japonica rice and glutinous rice.

[0014] Furthermore, the horizontal distance between adjacent holes in the drilling is 1.5-2.5 cm, and the vertical distance between two adjacent rows of holes is 6-10 cm.

[0015] Furthermore, the inclined drilling is performed in parallel at an angle of 20-27° downward in the horizontal direction of the main trunk, and the hole diameter is 1-3 mm.

[0016] Furthermore, the fragrance-forming liquid is injected by injecting 0.2-0.5 g of fragrance-forming liquid into each borehole within 60 minutes after drilling.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention uses rice, Eclipta prostrata, Polygonum hydropiper, White orchid and Pandan leaves to ferment and prepare a fragrance liquid, and injects the fragrance liquid into a hole in a tilted manner during the dormant period of Aquilaria sinensis to induce fragrance formation. The fragrance formation method increases the types and contents of sesquiterpenoid components in the Aquilaria sinensis extract, and increases the relative percentage contents of agarospirol, α-copaen-11-ol, (3,8,8-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)methyl acetate and nootkanone. The Aquilaria sinensis extract prepared by the method of the invention also increases the effect of inhibiting inflammation, and the results of the Hp antibacterial test show that the MIC value of the extract is significantly increased, and has a good antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : A picture of the formation of Aquilaria sinensis. In the picture (1), the trunk of Aquilaria sinensis after the formation of agarwood in Example 1; in the picture (2), the trunk of Aquilaria sinensis after the formation of agarwood in Comparative Example 1; in the picture (3), the trunk of Aquilaria sinensis after the formation of agarwood in the blank control. DETAILED DESCRIPTION

[0020] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0022] Trichoderma viride: No. BNCC117922, viable bacteria count greater than 15 billion CFU / g, China Industrial Microbiology Culture Collection Center.

[0023] Fusarium proliferatum: No. BMZ075062, viable bacteria count greater than 12 billion CFU / g, Guangdong Provincial Microbiological Culture Collection Center.

[0024] Lasiodiplodia theobromae: No. BNCC119691, viable bacteria count greater than 10 billion CFU / g, Shanghai Collection Biotechnology Center.

[0025] Preparation Example 1

[0026] A dormant agarwood formation method for white agarwood, wherein the raw materials of the agarwood liquid include: 2 parts of indica rice, 1 part of Eclipta prostrata, 1 part of Polygonum hydropiper, 0.5-1 part of white jasmine, and 1-2 parts of pandan leaves;

[0027] The preparation method of the fragrance liquid comprises:

[0028] Step (1): mixing the raw materials of the fragrance liquid and then crushing them into a powder with a particle size of 0.2 mm, and mixing them to obtain a primary mixed liquid;

[0029] Step (2): sealing and fermenting the initial mixed liquid for 30 days at a temperature of 30° C. to obtain the initial fermentation mixed liquid, filtering the initial fermentation mixed liquid and storing it;

[0030] Step (3): taking the filtered fermentation primary mixed liquid for microbial culture, wherein the microorganisms are Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae, the mass ratio of the fermentation primary mixed liquid to Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae is 100:5:2:4, the culture temperature of the microorganisms is 27°C, the culture time is 5 days, the culture pH value is 6.0, the culture is aerobic, and the fragrance-forming liquid is obtained after filtration.

[0031] Preparation Example 2

[0032] A dormant agarwood formation method for white agarwood, wherein the raw materials of the agarwood liquid include: 2.5 parts of polished rice, 1.2 parts of Eclipta prostrata, 1.5 parts of Polygonum hydropiper, 0.7 parts of white jasmine, and 1.5 parts of pandan leaves;

[0033] The preparation method of the fragrance liquid comprises:

[0034] Step (1): mixing the raw materials of the fragrance liquid and then crushing them into a powder with a particle size of 0.3 mm, and mixing them to obtain a primary mixed liquid;

[0035] Step (2): sealing the initial mixed liquid and fermenting it for 31 days at a temperature of 32° C. to obtain the initial fermentation mixed liquid, filtering the initial fermentation mixed liquid and storing it;

[0036] Step (3): taking the filtered fermentation initial mixed liquid for microbial culture, wherein the microorganisms are Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae, the mass ratio of the fermentation initial mixed liquid to Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae is 100:3:2:5, the microbial culture temperature is 26°C, the time is 6 days, the pH value is 6.2, the culture is carried out aerobically, and the fragrance-forming liquid is obtained after filtration.

[0037] Preparation Example 3

[0038] A dormant agarwood method for white agarwood, wherein the raw materials of the agarwood liquid include: 3 parts of glutinous rice, 1.4 parts of Eclipta prostrata, 2 parts of Polygonum hydropiper, 1 part of white jasmine, and 2 parts of pandan leaves;

[0039] The preparation method of the fragrance-forming liquid comprises:

[0040] Step (1): mixing the raw materials of the fragrance liquid and crushing them into a powder with a particle size of 0.4 mm, and mixing them to obtain a primary mixed liquid;

[0041] Step (2): sealing the initial mixed liquid and fermenting it for 32 days at a temperature of 35° C. to obtain the initial fermentation mixed liquid, filtering the initial fermentation mixed liquid, and storing it;

[0042] Step (3): taking the filtered fermentation initial mixed liquid for microbial culture again, the microorganisms are Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae, the mass ratio of the fermentation initial mixed liquid to Trichoderma chlororaphis, Fusarium spp. and Diplodia cholerae is 100:4:3:7, the microbial culture temperature is 26°C, the time is 7 days, the pH value is 7.0, aerobic fermentation is carried out, and the aroma-forming liquid is obtained after filtration.

[0043] Example 1

[0044] The dormant agarwood formation of white agarwood comprises the following steps: in the dormant period of white agarwood, a diameter at breast height of 7 cm and a side branch of 6 cm are selected, holes are drilled at a height of 35 cm from the ground, the horizontal distance between adjacent holes is 1.7 cm, the vertical distance between two adjacent rows of holes is 6 cm, inclined drilling is adopted, holes are drilled side by side at a 20° downward angle on the main trunk, and the hole diameter is 1.8 mm. At 30 minutes of drilling, 0.2 g of the agarwood formation liquid prepared in Preparation Example 1 is injected into each borehole at one time to induce agarwood formation.

[0045] Example 2

[0046] The dormant agarwood formation of white agarwood comprises the following steps: in the dormant period of white agarwood, a diameter at breast height of 8 cm and a side branch of 7 cm are selected, holes are drilled at a height of 43 cm from the ground, the horizontal distance between adjacent holes is 2.5 cm, the vertical distance between two adjacent rows of holes is 10 cm, and holes are drilled side by side at a 27° downward angle on the main trunk, with a hole diameter of 2.5 mm. After drilling, the agarwood formation liquid prepared in Preparation Example 2 is injected, and 0.5 g of the agarwood formation liquid is injected into each borehole at once at 35 minutes of drilling to induce agarwood formation.

[0047] Example 3

[0048] The dormant agarwood formation of white agarwood comprises the following steps: in the dormant period of white agarwood, a diameter at breast height of 8 cm and a side branch of 6 cm are selected, holes are drilled at a height of 42 cm from the ground, the horizontal distance between adjacent holes is 2.5 cm, and the vertical distance between two adjacent rows of holes is 8 cm. When drilling, inclined drilling is adopted, and holes are drilled side by side at an angle of 25° downward on the main trunk, and the hole diameter is 2 mm. After drilling, the agarwood formation liquid prepared in Preparation Example 3 is injected, and 0.4 g of agarwood formation liquid is injected into each borehole at 50 minutes of drilling to induce agarwood formation.

[0049] Comparative Example 1

[0050] The main difference between Comparative Example 1 and Example 3 is that the fragrance-forming liquid prepared in Preparation Example 3 is different, and the white jasmine and hot polygonum in the raw material components of Preparation Example 3 are removed, and the rest of the preparation method is the same as Example 3.

[0051] Comparative Example 2

[0052] The main difference between Comparative Example 2 and Example 3 is that the fragrance-forming liquid prepared in Preparation Example 3 is different, the microbial fermentation step of Preparation Example 3 is cancelled, and the rest of the preparation method is the same as Example 3.

[0053] Comparative Example 3

[0054] The main difference between Comparative Example 3 and Example 3 is that the horizontal distance between adjacent holes is increased to 5 cm, and the vertical distance is increased to 15 cm. The rest of the preparation method is the same as Example 3.

[0055] Comparative Example 4

[0056] The main difference between Comparative Example 4 and Example 3 is that the inclined drilling is replaced by vertical drilling, and the amount of fragrance-forming liquid is replaced by 0.1 g from 0.4 g, and the rest of the preparation method is the same as Example 3.

[0057] Blank control

[0058] White agarwood without any intervention in the formation of fragrance.

[0059] test

[0060] The xylem sample of Aquilaria sinensis was collected from the experimental field of the Haikou R&D Center of the Hainan Branch of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences, and was identified as Aquilaria sinensis (Lour.) Gilg by the Hainan Branch of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences. The voucher specimens are preserved in the Hainan Provincial Chinese Medicinal Materials Herbarium of the Hainan Branch of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences. The volatile oil was extracted from the extracts of the embodiment, comparative example and blank control group by steam distillation, the mass volume ratio of the extract to water was 1g: 5-8mL, the extraction time was 30-50min, the temperature was 20-30°C, and the volatile oil obtained by distillation was dried with anhydrous sodium sulfate and weighed.

[0061] The volatile oil extracted above was subjected to GC-MS determination and analysis. The GC-MS system consisted of a two-dimensional gas chromatograph 3300GC×GC connected in series with a TOF-MS detector. The one-dimensional gas chromatograph column was AgilentDB-5MS (0.25mm×30m, 0.25μm), and the two-dimensional gas chromatograph column was AgilentDB-17HT (0.25mm×2.5m, 0.15μm). The injection port temperature was 300°C, with a 30:1 split; the carrier gas was high-purity helium, the column flow rate was N2, and the carrier gas flow rate was 1mL·min -1 Heating program (starting temperature 70℃, hold for 1min, start from 70℃ and increase at 6℃·min -1 The TOF-MS source was heated to 270°C, the ionization voltage was 70 eV, the collection voltage was 1800 V, and the mass scanning range was m / z 50-500.

[0062] Table 1 GC-MS analysis of sesquiterpenoid components and relative percentages (%)

[0063]

[0064]

[0065] The extracts of Example 1-3, Comparative Example 1-4 and blank control were subjected to Hp (Helicobacter pylori, HP) antibacterial test in Test 1. The culture and identification of Hp refer to the document "In-vitro anti-Helicobacter pyloriactivity and preliminary mechanism of action of Canarium album raeusch". The specific operation is: Hp is revived and inoculated into a plate culture medium. During the culture, the oxygen content is 5%, the carbon dioxide content is 10%, and the nitrogen content is 85%; the culture temperature is 37°C and the culture time is 6 days. Hp is identified by urease reaction method. Scrape the cultured Hp colonies and dilute them with BHI to adjust the bacterial solution concentration to 5×108 CFU / mL. The compound was dissolved in DMSO, and the initial screening drug concentration was 25 μmol / L. The anti-Hp activity was detected, and 3 replicates were set for each experimental group. The absorbance was measured at 600nm using an ELISA reader, and the turbidity in the well was combined to determine the concentration MIC at which Hp was inhibited without obvious growth. The test results show that the white wood fragrance solution obtained according to the method of Examples 1-3 has a higher antibacterial effect.

[0066] Table 2 MIC value content table

[0067]

[0068] Note: * indicates significant difference at P<0.05.

[0069] Test results: The present invention uses rice, Eclipta prostrata, Polygonum hydropiper, White jasmine and Pandan leaves in combination to prepare aroma liquid through secondary fermentation, performs inclined drilling during the dormant period of Aucklandia odorifera, injects aroma liquid after drilling to induce aroma formation, and increases the types and contents of sesquiterpenoid components in the obtained aroma extract, wherein the sesquiterpenoid components in the extract mainly contain agarospirol, α-copaen-11-ol, (3,8,8-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)methyl acetate, longifolenal dehyde and nootkanone, etc. In the embodiment, the relative percentage of agarospirol is 11.1%-11.9%, and the relative percentage of α-copaen-11-ol is 7.6%-8.8%, both of which are higher than the comparative example and the control. At the same time, the effect of the extract in inhibiting inflammation is also increased. The results of the Hp antibacterial test show that the MIC value of the extract obtained by the method of Examples 1-3 is in the range of 7.3-7.6umol / L, which has a good antibacterial effect. After the preparation method of the agarwood liquid and the dormant agarwood method of white agarwood were changed in Comparative Examples 1-4, the types and contents of the agarwood extract were reduced, and the effect of the extract in inhibiting inflammation was reduced.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for dormant agarwood formation in white agarwood, characterized in that: The method for forming incense comprises the following steps: selecting a white agarwood plant with a breast diameter greater than 6 cm and a side branch greater than 5 cm, drilling an inclined hole at a position 35-45 cm above the ground on the trunk, and injecting an incense-forming liquid after drilling to induce incense formation; Calculated by weight, the raw materials of the fragrance-forming liquid include: 2-3 parts of rice, 1-1.4 parts of Eclipta prostrata, 1-2 parts of Polygonum hydropiper, 0.5-1 parts of White Jasmine and 1-2 parts of Pandan leaves.

2. A method for dormant agarwood formation in claim 1, characterized in that: The preparation method of the fragrance-forming liquid comprises: S1: Mix, crush and evenly stir the raw materials of the fragrance liquid; S2: sealing and fermenting the mixture to obtain a fermentation initial mixed liquid, filtering the fermentation initial mixed liquid and storing it; S3: taking the filtered fermentation initial mixed liquid for microbial culture, fermentation, and filtering to obtain the aroma liquid.

3. A method for forming dormant agarwood in Aquilaria sinensis according to claim 2, characterized in that: Step S1, the crushing, the crushing particle size is 0.1-0.5mm; Step S2, the sealed fermentation, the fermentation time is 28-35 days, and the fermentation temperature is 25-35°C.

4. A method for dormant agarwood formation in Aquilaria sinensis according to claim 2, characterized in that: Step S3, the microorganisms include Trichoderma chlororaphis, Fusarium solani and Diplodia theobromin, and the mass ratio of the initial fermentation mixture to Trichoderma chlororaphis, Fusarium solani and Diplodia theobromin is 100:3-5:2-3:4-8.

5. A method for dormant agarwood formation in Aquilaria sinensis according to claim 2, characterized in that: Step S3, the microorganisms are cultured at a temperature of 25-35° C. for 5-7 days, a pH value of 5.5-7.5, and aerobic fermentation conditions.

6. A method for dormant agarwood formation in Aquilaria sinensis according to claim 1, characterized in that: The rice is at least one of indica rice, japonica rice or glutinous rice.

7. A method for forming dormant agarwood in Aquilaria sinensis according to claim 1, characterized in that: The inclined drilling is performed side by side at an angle of 20-27 degrees downward in the horizontal direction of the main trunk, and the hole diameter is 1-3 mm.

8. A method for dormant agarwood formation in Aquilaria sinensis according to claim 7, characterized in that: The horizontal distance between adjacent holes in the drilled holes is 1.5-2.5 cm, and the vertical distance between two adjacent rows of holes is 6-10 cm.

9. A method for dormant agarwood formation in Aquilaria sinensis according to claim 1, characterized in that: The injection of the fragrance-forming liquid is performed by injecting 0.2-0.5 g of the fragrance-forming liquid into each borehole within 60 minutes after drilling.

10. The method for forming agarwood during dormancy of Aquilaria sinensis according to claim 1, characterized in that: The method for forming fragrance starts to form fragrance continuously after 1-1.5 months after the fragrance liquid is injected into the white agarwood.

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