CN113425641B - Method for producing agilawood by utilizing plant cell culture fermentation and application of agilawood in cosmetics - Google Patents
Method for producing agilawood by utilizing plant cell culture fermentation and application of agilawood in cosmetics Download PDFInfo
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Abstract
The invention relates to the technical field of plant cell culture, and discloses a method for producing agilawood by fermenting plant cell culture technology and application of the agilawood in cosmetics, wherein the method comprises the following steps: the method comprises the steps of preparation of a culture medium, induction culture of aquilaria sinensis embryonic callus, fermentation culture, extraction and identification of a product, and preparation of agilawood balm. The method adopts a plant cell culture technology to realize the induction preparation of embryogenic callus capable of being cultured in a suspension manner, can provide callus cells of aquilaria sinensis trees for large-scale culture, further utilizes a solid fermentation method to prepare the agilawood, is simple to operate, has high-efficiency raw material preparation and less post-treatment pollution, and is close to the agilawood formation process of the aquilaria sinensis trees in the natural environment. The agilawood extract provided by the invention has excellent effects of relieving emotion and improving sleep, and can be used as a compound cosmetic and/or skin care product additive.
Description
Technical Field
The invention relates to the technical field of plant cell culture, in particular to a method for producing agilawood by fermenting embryonic callus of aquilaria sinensis through a plant cell culture technology and application of the aquilaria sinensis in cosmetics.
Background
Eaglewood (gaharu, agarwood, eaglewood) is a traditional and famous spice and medicinal material in China, japan, india, southeast Asia and the middle east, mainly comes from the Aquilaria (Aquilaria) plants in Thymelaeaceae, and 19 species of eaglewood can be recognized to produce fragrance at present. At present, all the agalloch plants are listed in appendix II of International trade convention on endangered species of wild animals and plants (CITES), and the agalloch is also emphatically developed by 'fourth tree' listed after betel nuts, coconuts and rubber in Hainan province in 2020.
Aquilaria sinensis (Aquilaria sinensis) is also named as native Aquilaria sinensis, is a unique tree species capable of producing Aquilaria sinensis in China, and is listed in national key protection wild plant famous book, chinese plant Red book, guangdong rare endangered plant and the like.
The agilawood is derived from the aquilaria sinensis, and is called as the agilawood because the formed agilawood is rich in lipid and sinks when entering water; if the resulting incense is not submerged because of less fat, it is called Huang Shouxiang, lhasiang. The plant of the genus Aquilaria can produce black resin when damaged or stimulated, and the xylem producing the resin is the Aquilaria. The mechanism of its formation is mainly thought to be the result of the cooperative action of the agaric plant and its endophytic fungi.
The agilawood is a rare traditional Chinese medicine and natural spice, is distributed in places such as Guangdong, hainan, guangxi, fujian and the like, is pungent and bitter in taste, and has the effects of promoting qi circulation, relieving pain, warming middle-jiao, preventing vomiting, absorbing qi and relieving asthma. "the heart-spirit deficiency, fire does not drop, water does not rise, palpitation and forgetfulness" can be treated by carrying the agilawood in Ben Cao gang mu, namely the agilawood can be used for treating insomnia; it can also be used for treating wind-damp toxin swelling, removing aversion to qi, treating heart and abdominal pain, cholera, chronic fever due to pathogenic factors, refreshing mind, and tranquilizing mind. At present, the agilawood is widely applied to pharmacy, tasting fragrance, spice and the like.
All species of the aquilaria are endangered species, and compared with the traditional whole plant material, the method replaces the traditional wood chip fermentation production of the aquilaria sinensis by the aquilaria sinensis plant cell culture, and provides a new growth dimension for the utilization and protection of the aquilaria sinensis. The suspension cell has the advantages of high growth speed, strong cell activity, easy control, short experimental period and the like, and is widely used for researches on physiology, cytology, biochemistry, developmental biology, genetics and molecular biology. And the dispersibility and the uniformity of the material are far better than those of the traditional material, so that the experimental repeatability is good, and the result is more convincing. The tissue culture mode can provide high-quality and sufficient agilawood raw materials for the traditional Chinese medicine industry and the big health industry, and effectively promotes the development of the agilawood industry in China.
The mechanism of agilawood formation is mainly considered as a result of the combined action of agilawood plants and endophytic fungi of the agilawood plants, and artificial induction experiments show that the inoculation of the endophytic fungi separated from agilawood plant tissues can accelerate the generation of agilawood resin, and artificial agilawood formation is used as another way for forming agilawood, so that the time for forming agilawood can be greatly shortened, and the requirements of consumers are met.
How to research a mode which has an industrialization prospect, is simple in production process, is environment-friendly and can produce agilawood in a large scale based on the existing plant cell culture technology is a problem to be solved urgently.
Disclosure of Invention
The invention aims to develop a new method for producing agilawood by using a plant cell culture technology and apply the method to cosmetics, solve the problems of long growth period, shortage of raw material resources, great environmental influence and the like of the traditional plants, and provide a new idea for applying agilawood to cosmetics.
The technical scheme of the invention comprises the following steps:
a method for producing lignum Aquilariae Resinatum by fermenting with plant cell culture technology and its application in cosmetics comprises the following steps:
(1) Preparation of white incense wood callus cell induction culture medium
The induction culture medium comprises B5 basic salt culture medium, sucrose 20-30g/L, agar 6-8g/L, inositol 80-120mg/L, 6-benzylaminopurine (6-BA) 2-3.5mg/L, alpha-naphthylacetic acid (NAA)
0.2-0.6mg/L, 0.4-1.2mg/L indole-3-acetic acid (IAA), 1-1.8g/L choline and water.
(2) White incense wood callus cell induction preparation method
Taking a stem section newly germinated by a aquilaria sinensis plant, performing surface sterilization treatment on the stem section, then inoculating the stem section into the induction culture medium prepared in the step (1), culturing to obtain an aquilaria sinensis embryonic callus, transferring the dispersed embryonic callus particles into a tissue culture bottle for subculture, then transferring the tissue culture bottle into a liquid shake culture medium, performing shake culture at 26 ℃, at a rotating speed of 115r/min for 15 days, removing 50% of the supernatant of a culture solution, and then supplementing a fresh culture medium for subculture; when the dry thallus concentration in the culture solution reaches above 25g/L, the supernatant can be removed by filtration to obtain the Aquilaria sinensis callus
Drying the cells at 85 ℃ for later use;
the tissue culture bottle culture medium comprises the following components: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.0002g/L of 6-benzylaminopurine (6-BA), 0.004g/L of alpha-naphthylacetic acid (NAA) and poly (I-N-acetyl-L-alanine)
2g/L of vinyl pyrrolidone (PVP), 8-10g/L of agar powder and 5.8-6.0 of pH;
the liquid shake flask culture medium comprises the following components: 3.21g/L of B5 culture medium, 30g/L of cane sugar, 2g/L of polyvinylpyrrolidone (PVP) and 5.8-6.0 of pH.
(3) Strain activation for preparing microorganism from agilawood
Selecting Fusarium (Fusarium sp. A2) strain stored in low temperature refrigerator (isolated from 5-age radix Aristolochiae Mollissimae, purchased from Microbiol research institute of Guangdong province), inoculating a small amount of mycelium onto the slant of sterilized potato glucose agar culture (PDA) medium, and culturing at 28 deg.C for 7 days to activate strain;
the potato glucose agar culture liquid (PDA) culture medium comprises the following components: 200g/L of potato, 20g/L of glucose and 1000ml of distilled water, wherein the pH is natural, and the potato is sterilized at the temperature of 115 ℃ for 25min in advance for later use;
(4) Microbial fermentation culture of white incense wood callus cells
Sucking the aquilaria sinensis suspension cells by using a big-head straw under the aseptic condition, and putting the aquilaria sinensis suspension cells into an aquilaria sinensis cell liquid culture medium. Washing Fusarium (Fusarium sp.A2) slant strain with 5mL sterile water, sucking bacterial liquid with big head straw, inoculating into seed shake flask, and performing seed culture; the cultured fusarium sp.a2 seed solution was inoculated to a Potato Dextrose Agar (PDA) medium in an inoculum size of 10%, and placed in the dark at 27 ℃ for 20 days.
The aquilaria sinensis cell liquid culture medium comprises the following components: 20g/L of balsamiferous tree callus cells, 20g/L of glucose and 1g/L of corn steep liquor, wherein the pH value is natural, and the balsamiferous tree callus cells are sterilized at the temperature of 115 ℃ for 25min in advance;
(5) Extraction and identification of products
Crushing the fermentation liquor by a high-pressure homogenizer, adding 95% ethanol into the whole fermentation liquor after cell crushing, soaking overnight, centrifuging to remove precipitates, evaporating the extracting solution to dryness by a rotary evaporator, dissolving by 1.5mL of chloroform, passing through a 0.22-micron microporous filter membrane, and detecting and identifying components.
(6) Preparation of agilawood balsam
And preparing the agilawood paste from the agilawood extract subjected to component identification.
The agilawood balsam comprises the following raw materials in parts by weight: 75-85 parts of denatured ethanol; 2-4 parts of diethylene glycol; 2-4 parts of glycerol; 4-6 parts of stearic acid; 1-3 parts of agilawood extract; 0-0.5 parts of preservative; the balance of water, and the pH value is adjusted to 4.6-6.8.
The preservative is one or more of potassium sorbate, sodium benzoate, ethylhexyl glycerin and chlorphenesin. The water is deionized water.
The preparation steps of the agilawood balm in the step (6) are as follows: under the condition of continuously stirring, in a closed stainless steel kettle, mixing and heating diethylene glycol, glycerol and stearic acid to 70 ℃; a) Slowly adding water; b) Sampling, titrating and determining excessive alkalinity or acidity; c) Adding denatured ethanol and lignum Aquilariae Resinatum extract; d) Poured into a mold at 55 ℃.
The specific steps of inoculating the stem segments subjected to surface sterilization treatment in the step (2) into the induction culture medium in the step (1) are as follows: washing young and tender stem segments of picked aquilaria sinensis with clear water for about 2h, shearing the stem segments into about 1cm by using sterilized scissors on a sterile super clean bench, soaking the stem segments in 70% ethanol for 30s, soaking the stem segments in 4% sodium hypochlorite solution for 15-20min, and then inoculating the stem segments into a tissue culture bottle containing hormones (6-benzylaminopurine (6-BA) and alpha-naphthylacetic acid (NAA)) to induce callus cells. And (3) standing and culturing in dark at the temperature of 25 ℃. During the culture process, the stem section is horizontally placed, so that the wounds at two ends of the stem section are fully contacted with the culture medium until embryogenic callus grows out of the culture medium. Standing and culturing in dark at 25 ℃, inoculating 5 stem segments into each bottle of culture medium, and repeating each treatment;
transferring the dispersed embryogenic callus particles into a tissue culture bottle for subculture in the step (2) specifically comprises the following steps: selecting the callus particles with loose texture by using tweezers on an aseptic super clean bench, dividing the callus particles into small pieces, and transferring the small pieces to a tissue culture bottle for subculture. In the process of subculture, browned cells are removed, and callus with good color and state is selected for continuous culture. If the callus surface contains a lot of non-embryogenic white floc, the white floc can be scraped off with a scalpel and then cut into pieces for culturing. Subcultured every 10 days.
Evaporating the extracting solution in the step (5) to dryness by using a rotary evaporator to obtain an agilawood extract, and detecting and identifying components of the obtained agilawood extract by using a gas chromatography-mass spectrometer, wherein the component detection and identification instrument is an Shimadzu gas chromatography-mass spectrometer (gas chromatography end GC-2010PLUS [ equipped with AOC-5000PLUS automatic sample injector; mass spectrometer end GC-MS TQ 8030).
Instrument parameters injection port temperature: 250 ℃; the interface temperature is 280 ℃; detector temperature: 230 ℃; the split ratio is 2:1; flow rate of carrier gas: 22.9cm/s; purge flow rate: 3mL/min.
And (4) carrying out qualitative determination by adopting mass spectrum database retrieval of an NIST14 mass spectrum database, and carrying out relative quantification by adopting a chromatographic peak area normalization method.
The agilawood paste prepared by the method is applied to the external surface of skin in cosmetics.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. because the traditional whole plant material is not adopted, the aquilaria sinensis plant cell culture is used for replacing the aquilaria sinensis production, the resource can be saved, and the method has important significance for protecting wild resources, maintaining ecological balance and promoting industrial development.
2. The formed suspension cells have the advantages of high growth speed, strong cell activity, easy control, short experimental period, simple operation, high efficiency of raw material preparation, less pollution of post treatment, close to the agilawood formation process of the aquilaria sinensis wood in the natural environment, better dispersibility and uniformity than the traditional materials and good experimental repeatability.
3. The agilawood production method by adopting the plant cell culture mode can provide a high-quality and sufficient agilawood raw material for the traditional Chinese medicine industry and the big health industry, and effectively promote the development of the agilawood industry in China.
4. By utilizing the advantages of fusarium (fusarium sp.a2) which is an endophytic fungus in the aquilaria sinensis edgeworthia chrysantha, the generation of agilawood resin can be accelerated, the incense forming time is greatly shortened, and the market demand is met.
5. The agilawood balm prepared by the invention widens the application of agilawood in cosmetics, has the effects of improving sleep, relieving emotion and the like, and brings comfortable fragrance and emotion improvement experience to people.
Drawings
FIG. 1 is a total ion flow diagram of GC-MS analysis of fermentation liquor: and (4) carrying out qualitative determination by adopting mass spectrum database retrieval of an NIST14 mass spectrum database, and carrying out relative quantification by adopting a chromatographic peak area normalization method.
Detailed Description
The present invention will be described in detail with reference to specific examples.
The various organic reagents used in the examples of the present application are analytical grade reagents.
Example one
A method for producing lignum Aquilariae Resinatum by plant cell suspension culture technology comprises the following steps:
(1) Preparation of white incense wood callus cell induction culture medium
The induction culture medium is prepared from a B5 basic salt culture medium, 20-30g/L of sucrose, 6-8g/L of agar, 80-120mg/L of inositol, 2-3.5mg/L of 6-benzylaminopurine (6-BA), 0.2-0.6mg/L of alpha-naphthylacetic acid (NAA), 0.4-1.2mg/L of indole-3-acetic acid (IAA), 1-1.8g/L of choline and water;
(2) White incense wood callus cell induction preparation method
Taking a newly germinated stem section of a aquilaria sinensis plant, washing the picked tender stem section of the aquilaria sinensis plant with clear water for about 2 hours, shearing the stem section into about 1cm by using sterilized scissors on a sterile super clean bench, soaking the stem section in 70% ethanol for 30s, soaking the stem section in 4% sodium hypochlorite solution for 15-20min, and then inoculating the stem section into a tissue culture bottle containing hormone (6-benzylaminopurine (6-BA) and alpha-Naphthalene Acetic Acid (NAA)) to induce callus cells. And (3) standing and culturing in dark at the temperature of 25 ℃. During the culture process, the stem section is horizontally placed, so that the wounds at two ends of the stem section are fully contacted with the culture medium until embryogenic callus grows out of the culture medium. Standing and culturing in dark at 25 ℃, and inoculating 5 stem segments in each bottle of culture medium. And (3) inoculating the stem sections subjected to surface sterilization treatment into an induction culture medium, culturing to obtain aquilaria sinensis embryonic callus, and transferring the dispersed embryonic callus particles into a tissue culture bottle for subculture. Selecting loose callus particles by using forceps on an aseptic super clean bench, dividing the loose callus particles into small pieces, and transferring the small pieces into a tissue culture bottle for subculture. During subculture, browned cells are removed, and callus with good color and state is selected for continuous culture. If the callus surface contains a lot of non-embryogenic white floc, the white floc can be scraped off with a scalpel and then cultured in pieces. Subcultured every 10 days. Transferring the culture medium into a liquid shake flask culture medium after subculture, carrying out shake culture at 26 ℃ and a rotating speed of 115r/min for 15 days, removing 50% of culture solution supernatant, and supplementing a fresh culture medium for subculture; when the concentration of the dry thallus in the culture solution reaches more than 25g/L, filtering to remove supernatant to obtain the white incense wood callus cells, and drying at 85 ℃ for later use;
(3) Activation of strains for preparing microorganisms from agilawood
Selecting a small amount of mycelia of Fusarium sp.A2 (isolated from 5-year-old radix Aristolochiae Mollissimae and purchased from Microbiol research institute in Guangdong province) preserved in a low-temperature refrigerator, inoculating onto a slant of a sterilized potato glucose agar (PDA) culture medium, and culturing at 28 deg.C for 7 days to activate the strain;
the potato glucose agar culture liquid (PDA) culture medium comprises the following components: 200g/L of potato, 20g/L of glucose and 1000ml of distilled water, wherein the pH is natural, and the potato is sterilized at the temperature of 115 ℃ for 25min in advance for later use;
(4) Fermentation culture
Sucking the aquilaria sinensis suspension cells by using a big-head straw under the aseptic condition, and putting the aquilaria sinensis suspension cells into an aquilaria sinensis cell liquid culture medium. Washing Fusarium sp.A2 slant strain with 5mL sterile water, sucking the strain liquid with a big-end suction pipe, inoculating into a seed shake flask, and performing seed culture; inoculating the cultured Fusarium sp.A2 seed liquid into potato glucose agar culture liquid (PDA) culture medium of Plectranthus niveus callus at an inoculation amount of 10%, and standing in dark at 27 deg.C for 20d. Meanwhile, a white incense wood suspension cell control group which is not cultured together with the fungus and a strain A2 hypha control group which is cultured in a Potato Dextrose Agar (PDA) culture medium for 7 days are arranged.
TABLE 1 composition of the culture media
(5) Extraction and identification of products
Crushing the fermentation liquor by a high-pressure homogenizer, adding 95% ethanol into the whole fermentation liquor after cell crushing, soaking overnight, centrifuging to remove precipitates, evaporating the extracting solution to dryness by a rotary evaporator, dissolving by 1.5mL of chloroform, and passing through a 0.22-micron microporous membrane for later use. And (3) setting an agilawood control group, dissolving the extract agilawood extract with chloroform, filtering the extract with a microporous filter membrane for later use, and detecting and identifying components by using an Shimadzu gas chromatography-mass spectrometer (a gas chromatography end GC-2010PLUS [ with an AOC-5000PLUS automatic sample injector; a mass spectrometry end GC-MS TQ 8030).
The instrument parameters are shown in table 2:
| temperature at sample inlet | 250℃ |
| Interface temperature | 280℃ |
| Temperature of detector | 230℃ |
| Split ratio | 2:1 |
| Flow rate of carrier gas | 22.9cm/s |
| Purge flow rate | 3mL/min |
And (4) carrying out qualitative determination by adopting mass spectrum database retrieval of an NIST14 mass spectrum database, and carrying out relative quantification by adopting a chromatographic peak area normalization method. (see FIG. 1) it can be seen that there are eaglewood-characteristic substances including benzyl acetone, linalool, cumyl alcohol, anisyl acetone, dihydrocarvone, aquilaria aldehyde, etc.
(6) Preparation of agilawood balsam
Preparing the extract agilawood paste into agilawood paste, wherein the weight components of the raw materials are shown in the table 3:
a control group (placebo balm) was set, and the weight components of the raw materials are shown in table 4:
heating the component B to 70 ℃ in a closed stainless steel kettle under the condition of continuously stirring; the dissolved C component was slowly added. The excess alkalinity or acidity was determined by titration of samples. The alkalinity was adjusted to make the stearic acid 5% excess. Adding A, D phase; poured into a mold at 55 ℃.
In the embodiment, the agilawood paste contains the agilawood extract, is added into cosmetics for application, is applied to the outer surface of the skin, has the effects of improving sleep, relieving anxiety, and the like, and can well relieve the symptoms of tension, anxiety and insomnia.
Taking the agilawood balm obtained by the preparation method as an experimental sample for human body efficacy evaluation, recruiting 200 volunteers, screening 50 persons with nervous and anxious emotions or sleep disorder as tested subjects, wherein the persons are 25-50 years old and randomly divided into 2 groups, namely a balm base material placebo group without agilawood addition (placebo balm group for short) and an agilawood balm group with agilawood addition (agilawood balm group for short), providing the placebo balm and the agilawood balm of the embodiment for the volunteers to use, and then respectively scoring the user experience from 3 indexes of fragrance comfort, sleep improvement condition and anxiety relieving emotion, wherein each index is fully divided into 10 groups, and the higher score represents the higher satisfaction degree. The SPSS25.0 statistical analysis software is used for carrying out difference analysis on the common balsam group embodiment groups, and the results show that the comfort balsam group and the agilawood balsam group have no statistical difference in fragrance comfort degree, but have extremely significant difference in performance in the aspects of improving sleep and relieving anxiety, so that the agilawood ointment has the effects of improving sleep, relieving stress and anxiety, and the results are shown in Table 5.
Table 5 customer satisfaction results table (n = 10)
| Index (I) | Comfort balsam | Agilawood balsam |
| Fragrance comfort | 6.21 | 8.34 |
| Improving sleep | 2.01 | 5.89* |
| Relieving anxiety | 4.65 | 8.31** |
Note: the comparison between placebo and agilawood balm groups indicates that the difference is significant (P <)
0.05 ); * Indicates that the difference has a very significant meaning (P < 0.01).
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (7)
1. A method for producing agilawood by fermentation by utilizing a plant cell culture technology is characterized by comprising the following steps:
(1) Preparation of white incense wood callus cell induction culture medium
The induction culture medium consists of a B5 basic salt culture medium, 20-30g/L of cane sugar, 6-8g/L of agar, 80-120mg/L of inositol, 2-3.5mg/L of 6-benzylaminopurine, 0.2-0.6mg/L of naphthylacetic acid, 0.4-1.2mg/L of indole-3-acetic acid, 1-1.8g/L of choline and water;
(2) White incense wood callus cell induction preparation method
Taking a stem section newly germinated by a aquilaria sinensis plant, performing surface sterilization treatment on the stem section, then inoculating the stem section into the induction culture medium in the step (1), culturing to obtain aquilaria sinensis embryonic callus, transferring the dispersed embryonic callus cell particles into a tissue culture bottle for subculture, then transferring the tissue culture bottle into a liquid shake flask culture medium, performing shake culture at 26 ℃, at the rotating speed of 115r/min for 15 days, removing 50% of the supernatant of the culture solution, and then supplementing a fresh culture medium for subculture; when the concentration of the dry thallus in the culture solution reaches more than 25g/L, filtering to remove supernatant to obtain the white incense wood callus cells, and drying at 85 ℃ for later use;
the tissue culture bottle culture medium comprises the following components: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.0002g/L of 6-benzylaminopurine, 0.004g/L of alpha-naphthylacetic acid, 2g/L of polyvinylpyrrolidone, 8-10g/L of agar powder and 5.8-6.0 of pH;
the liquid shake flask culture medium comprises the following components: 3.21g/L of B5 culture medium, 30g/L of cane sugar, 2g/L of polyvinylpyrrolidone and 5.8-6.0 of pH;
(3) Strain activation for preparing microorganism from agilawood
Selecting a small amount of mycelium of a bacterial strain Fusarium sp.A2 stored in a low-temperature refrigerator, inoculating the mycelium onto a slant surface of a sterile potato glucose agar culture liquid, and culturing at 28 ℃ for 7 days to activate the strain;
the potato glucose agar culture liquid culture medium comprises the following components: 200g/L of potatoes, 20g/L of glucose and 1000ml of distilled water, wherein the pH is natural, and the potatoes are sterilized for 25min at the temperature of 115 ℃ in advance for later use;
(4) Microbial fermentation culture of white incense wood callus cells
Sucking the aquilaria sinensis suspension cells by using a big-end suction pipe under the aseptic condition, putting the aquilaria sinensis suspension cells into an aquilaria sinensis cell liquid culture medium, washing Fusarium sp.A2 slant strains by using 5mL of sterile water, sucking the bacterial liquid by using the big-end suction pipe, inoculating the bacterial liquid into a seed shake flask, and performing seed culture; inoculating the cultured Fusarium sp.A2 seed liquid into a potato glucose agar culture liquid culture medium according to the inoculation amount of 10%, and standing in the dark at 27 deg.C for 20 days;
the aquilaria sinensis cell liquid culture medium comprises the following components: 20g/L of balsamiferous tree callus cells, 20g/L of glucose and 1g/L of corn steep liquor, wherein the pH value is natural, and the balsamiferous tree callus cells are sterilized at the temperature of 115 ℃ for 25min in advance;
(5) Extraction and identification of products
Crushing the fermentation liquor by using a high-pressure homogenizer, adding 95% ethanol into the whole fermentation liquor after cell crushing, soaking overnight, centrifuging to remove precipitates, evaporating an extracting solution by using a rotary evaporator to obtain an agilawood extract, dissolving the agilawood extract by using 1.5mL of chloroform, filtering through a 0.22-micrometer micropore, and detecting and identifying components;
(6) Preparation of agilawood balsam
And preparing the agilawood paste from the agilawood extract subjected to component identification.
2. The method for producing agilawood by fermentation through plant cell culture technology as claimed in claim 1, wherein the step (2) of inoculating the stem segments into the induction medium of step (1) after performing surface sterilization treatment comprises the following specific steps: washing young and tender stem segments of picked aquilaria sinensis with clear water for about 2h, shearing the young and tender stem segments into small blocks of 1cm by using sterilized scissors on a sterile super-clean bench, soaking the small blocks in 70% ethanol for 30s, soaking the small blocks in 4% sodium hypochlorite solution for 15-20min, and then inoculating the small blocks into a tissue culture bottle containing hormones 6-benzylaminopurine and alpha-naphthylacetic acid to induce callus cells.
3. The method for producing agilawood by fermentation through plant cell culture technology as claimed in claim 1, wherein the step (2) of transferring the dispersed embryogenic callus particles into a tissue culture bottle for subculture comprises the following specific steps: selecting loose callus particles by using forceps on an aseptic super clean bench, dividing the loose callus particles into small pieces, and transferring the small pieces into a tissue culture bottle for subculture.
4. The method for producing agilawood by using the plant cell suspension culture technology as claimed in claim 1, wherein in the step (5), the extracting solution is evaporated to dryness by using a rotary evaporator to obtain an agilawood extract, and then the obtained agilawood extract is detected and identified by using a gas chromatography-mass spectrometer, wherein the instrument parameters comprise the following sample inlet temperature: 250 ℃; the interface temperature is 280 ℃; temperature of the detector: 230 ℃; the split ratio is 2:1; flow rate of carrier gas: 22.9cm/s; purge flow rate: 3mL/min.
5. The method for producing agilawood by using plant cell suspension culture technology as claimed in claim 1, wherein the agilawood paste in step (6) comprises the following components by weight: 75-85 parts of denatured ethanol; 2-4 parts of diethylene glycol; 2-4 parts of glycerol; 4-6 parts of stearic acid; 1-3 parts of agilawood extract; 0-0.5 part of preservative; the balance of water, and the pH value is adjusted to 4.6-6.8.
6. The method for producing agilawood according to claim 5 by using plant cell suspension culture technology, wherein the preservative is one or more of potassium sorbate, sodium benzoate, ethylhexyl glycerol and chlorphenesin.
7. The method for producing agilawood by plant cell suspension culture technology according to claim 1, wherein the agilawood paste in step (6) is prepared by the following steps: under the condition of continuously stirring, in a closed stainless steel kettle, mixing and heating diethylene glycol, glycerol and stearic acid to 70 ℃;
a) Slowly adding water;
b) Sampling and titrating to determine excessive alkalinity or acidity;
c) Adding denatured ethanol and lignum Aquilariae Resinatum extract;
d) The mixture was poured into a mold at 55 ℃.
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