Method for culturing industrial cannabis sativa adventitious roots with high yield of cannabinoid and tanshinone

Abstract

The invention relates to a method for culturing industrial cannabinoid and tanshinone high-yield industrial cannabinoid adventitious roots, and belongs to the technical field of adventitious root cultivation. The method comprises the following steps: performing induction treatment on the industrial cannabis sativa adventitious roots by adopting a yeast extract to obtain the industrial cannabis sativa adventitious roots subjected to induction treatment; the yeast extract is low in cost and abundant in raw material supply, and can stimulate industrial cannabis sativa adventitious roots to synthesize more target metabolites as an elicitor; the industrial cannabis sativa adventitious roots subjected to induction treatment are subjected to scale-up culture by utilizing a bioreactor, so that cell proliferation and growth of the adventitious roots are promoted, and the biomass of the adventitious roots is increased; meanwhile, the adventitious roots are promoted to accumulate more target metabolites, and at the moment, the yields of cannabinoid phenol, cannabidiol, tetrahydrotanshinone I, tanshinone IIA and dihydrotanshinone I in the adventitious roots are up to 80.65 mg / L DW, 53.58 mg / L DW, 103.76 mg / L DW, 21.71 mg / L DW and 5.71 mg / L DW respectively. According to the method, the operation steps and the labor intensity are reduced, the defects of low biomass and low cannabinoid yield in the existing industrial cannabinoid adventitious root culture technology are overcome, and the industrial cannabinoid and tanshinone high-yield industrial cannabinoid adventitious roots can be economically and efficiently cultured all the year round.

Description

Technical Field

[0001] The present application relates to the field of adventitious root cultivation, and in particular to a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yields. Background Art

[0002] Industrial hemp (Cannabas satava L.), an annual herb, is rich in active substances, including cannabinoids, flavonoids, phenols, alkaloids, terpenes, etc., and has been widely used in food, drug research and development, and cosmetics. The representative compounds in industrial hemp are cannabinoids. At present, in order to replace the traditional cultivation of industrial hemp, the existing technology shows that the adventitious roots of industrial hemp cultivated in bioreactors can produce cannabinoids, but there are still defects in cannabinoid content and adventitious root biomass, which limits the application of industrial hemp adventitious roots in the commercial production of cannabinoids. Therefore, it is urgent to overcome the shortcomings of the existing adventitious root cultivation technology. Summary of the invention

[0003] The present application provides a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone production to solve the following technical problem: how to simultaneously increase the biomass of industrial hemp adventitious roots and the yield of cannabinoids and tanshinones.

[0004] The present invention provides a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yields, comprising the following steps:

[0005] Using yeast extract to induce the adventitious roots of industrial hemp to obtain the induced adventitious roots of industrial hemp;

[0006] The adventitious roots of industrial hemp after induction treatment were amplified and cultured using a bioreactor;

[0007] Collect the adventitious roots of industrial hemp after amplification and cultivation.

[0008] Optionally, the induction treatment of industrial hemp adventitious roots by using yeast extract to obtain induced industrial hemp adventitious roots specifically includes:

[0009] Under sterile conditions, the adventitious roots of industrial hemp are inoculated into a shake bottle containing a liquid culture medium for cultivation to obtain suspended cultured adventitious roots of industrial hemp;

[0010] The yeast extract is added to the suspension cultured industrial hemp adventitious roots and the culture is continued for 48 hours to 96 hours to obtain the induced industrial hemp adventitious roots.

[0011] Optionally, the yeast extract is added in an amount of 300 mg to 400 mg of yeast extract per liter of liquid culture medium.

[0012] Optionally, the industrial hemp adventitious roots are cultivated using suspension culture technology to obtain an industrial hemp adventitious root suspension culture system, specifically comprising: placing the industrial hemp adventitious roots in a shake bottle containing a liquid culture medium, shaking and culturing in the dark for 30 days to 40 days, to obtain the industrial hemp adventitious root suspension culture system.

[0013] Optionally, the temperature of the shaking dark culture is 20°C to 30°C, and the rotation speed is 90r / min to 110r / min.

[0014] Optionally, the method of using a bioreactor to amplify and culture the adventitious roots of industrial hemp after the induction treatment specifically includes:

[0015] The induced industrial hemp adventitious roots are inoculated into the liquid culture medium in the bioreactor for the first amplification culture;

[0016] The adventitious roots of industrial hemp after the first scale-up culture were used as test materials, inoculated into the bioreactor, and the second scale-up culture was carried out in a fed-batch culture mode.

[0017] Optionally, the inoculation density of the industrial hemp adventitious roots is 10g / L to 15g / L.

[0018] Optionally, the first amplification culture satisfies at least one of the following conditions: the culture temperature is 20°C to 30°C, the culture time is 40d to 50d, and the ventilation rate is 0.06vvm to 0.08vvm.

[0019] Optionally, the second scale-up culture is carried out in a fed-batch mode, specifically comprising: adding feed medium to the bioreactor after a period of initial culture, and then carrying out fed-batch culture;

[0020] The second amplified culture satisfies at least one of the following conditions: the culture temperature is 20°C to 30°C, the ventilation rate is 0.06vvm to 0.08vvm, the initial culture time is 45d to 55d, the addition amount of the feed culture medium is 25% to 35% of the initial culture medium, and the feed culture time is 30d to 40d.

[0021] Optionally, the liquid culture medium and the feed culture medium include: 0.75×MS-MS culture medium, 2 mg / L to 3 mg / L IBA and 25 g / L to 35 g / L sucrose, and the pH is 5 to 6.

[0022] The above technical solution provided by this application has the following advantages compared with the prior art:

[0023] The present application relates to a method for cultivating industrial hemp adventitious roots with high yield of cannabinoids and tanshinones, and belongs to the field of adventitious root cultivation technology. The method includes inducing the industrial hemp adventitious roots with yeast extract to obtain the induced industrial hemp adventitious roots; the yeast extract has low cost and abundant raw material supply, and can be used as an inducer to stimulate the industrial hemp adventitious roots to synthesize more target metabolites; the induced industrial hemp adventitious roots are amplified and cultured with a bioreactor, which promotes the cell proliferation and growth of the adventitious roots and increases the biomass of the adventitious roots; at the same time, the adventitious roots are promoted to accumulate more target metabolites, and the yields of cannabinol, cannabidiol, tetrahydrotanshinone I, tanshinone IIA and dihydrotanshinone I in the adventitious roots are as high as 80.65, 53.58, 103.76, 21.71 and 5.71 mg / LDW respectively. The method saves operation steps and labor intensity, overcomes the defects of low biomass and low cannabinoid yield in the existing industrial hemp adventitious root culture technology, and can realize the economical and efficient cultivation of industrial hemp adventitious roots with high cannabinoid and tanshinone yields throughout the year. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0026] Figure 1 A schematic flow chart of a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yields according to some embodiments of the present application;

[0027] Figure 2 It is the precursor ion mass spectrum and MRM chromatogram of the standard in the UPLC-QqQ-MS / MS detection example, including cannabinoid compounds (CBG, CBD and CBN) and tanshinone compounds (tanshinone ⅡA, tetrahydrotanshinone Ⅰ and dihydrotanshinone Ⅰ).

[0028] Figure 3 Schematic diagram of the effect of yeast extract (YE) treatment concentration and treatment time on the growth of adventitious roots of industrial hemp (a) and the accumulation of cannabinoids and tanshinone (b) in the embodiment. Data represent mean ± standard deviation (n = 3). Mean ± standard deviation that does not share the same lowercase letter is significantly different (p < 0.05)

[0029] Figure 4Schematic diagram of the effect of yeast extract (YE) on the content (a) and yield (b) of cannabinoids and tanshinones in the adventitious roots of industrial hemp cultured in a bioreactor, and the observation of the morphological structure of the adventitious roots (c) in the examples.

[0030] Figure 5 Schematic diagram of the effect of initial culture time on the biomass (a and b) of adventitious roots of industrial hemp cultured in bioreactors and its cannabinoid and tanshinone content (c) and yield (d) in the examples. Data represent mean ± standard deviation (n = 3). Mean ± standard deviations that do not share the same lowercase letter are significantly different (p < 0.05)

[0031] Figure 6 Schematic diagram of the effect of feed medium volume on adventitious root biomass (a and b) of industrial hemp cultured in bioreactors and its cannabinoid and tanshinone content (c) and yield (d) in the examples. Data represent mean ± standard deviation (n = 3). Mean ± standard deviation that does not share the same lowercase letter is significantly different (p < 0.05)

[0032] Figure 7 Schematic diagram of the effect of feed culture time on the biomass (a and b) of adventitious roots of industrial hemp cultured in bioreactors and its cannabinoid and tanshinone content (c) and yield (d) in the examples. Data represent mean ± standard deviation (n = 3). Mean ± standard deviations that do not share the same lowercase letter are significantly different (p < 0.05)

[0033] Figure 8 It is a schematic diagram comparing the effects of untreated and treated yeast extract and fed-batch culture mode on the growth state of adventitious roots of industrial hemp in the embodiments.

[0034] Fig. 9 It is a schematic diagram of the effects of the continuous culture mode without and with yeast extract addition in the comparative bioreactor in the embodiment, and the fed-batch culture mode on the yield of cannabinoids and tanshinones in the adventitious roots of industrial hemp. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0037] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions, refers to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, if the mass ratio of substance A, substance B and substance C is 1:2:3, then substance A, substance B and substance C should correspond to the proportional numbers in the proportional formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0039] The present invention provides a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yields, comprising the following steps:

[0040] Using yeast extract to induce the adventitious roots of industrial hemp to obtain the induced adventitious roots of industrial hemp;

[0041] The adventitious roots of industrial hemp after induction treatment were amplified and cultured using a bioreactor;

[0042] Collect the adventitious roots of industrial hemp after amplification and cultivation.

[0043] In the above embodiment, yeast extract is used as an inducer because yeast extract has low cost and abundant raw material supply. As an inducer, it can stimulate the adventitious roots of industrial hemp to synthesize more target metabolites; the adventitious roots of industrial hemp after induction treatment are amplified and cultured using a bioreactor, which promotes the cell proliferation and growth of the adventitious roots and increases the biomass of the adventitious roots; at the same time, the adventitious roots are promoted to accumulate more target metabolites. At this time, the yields of cannabinol, cannabidiol, tetrahydrotanshinone I, tanshinone IIA and dihydrotanshinone I in the adventitious roots are as high as 80.65, 53.58, 103.76, 21.71 and 5.71 mg / L DW, respectively. This method saves operating steps and labor intensity, overcomes the defects of low biomass and low cannabinoid yield in the existing industrial hemp adventitious root culture technology, and can achieve economical and efficient cultivation of industrial hemp adventitious roots with high yield of cannabinoids and tanshinones throughout the year.

[0044] As an optional embodiment, the method of inducing the adventitious roots of industrial hemp with yeast extract to obtain the induced adventitious roots of industrial hemp specifically includes:

[0045] Under sterile conditions, the adventitious roots of industrial hemp are inoculated into a shake bottle containing a liquid culture medium for cultivation to obtain suspended cultured adventitious roots of industrial hemp;

[0046] The yeast extract is added to the suspension cultured industrial hemp adventitious roots and the culture is continued for 48 hours to 96 hours to obtain the induced industrial hemp adventitious roots.

[0047] As an optional embodiment, the yeast extract is added in an amount of 300 mg to 400 mg of yeast extract per liter of liquid culture medium.

[0048] In the above embodiment, the reason for controlling the addition amount of yeast extract is that when the addition amount of yeast extract is 300mg to 400mg per liter of liquid culture medium, the adventitious roots greatly promote the accumulation of cannabinoids and tanshinones. When the addition amount of yeast extract is lower than 300mg / L, the concentration of yeast extract in the culture medium is low and cannot fully promote the synthesis of target metabolites by adventitious roots. When the addition amount of yeast extract is higher than 400mg / L, the concentration of yeast extract in the culture medium is higher, and high-concentration yeast extract contains a large amount of nutrients such as amino acids, vitamins, minerals, etc., which may lead to overnutrition; at the same time, certain components in yeast extract may inhibit the production of target metabolites by adventitious roots.

[0049] Exemplarily, the amount of yeast extract added per liter of liquid culture medium can be set to 300 mg, 310 mg, 32 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg or 400 mg.

[0050] As an optional embodiment, the industrial hemp adventitious roots are cultivated using suspension culture technology to obtain an industrial hemp adventitious root suspension culture system, specifically comprising: placing 20g / L-40g / L industrial hemp adventitious roots into a shake bottle containing a liquid culture medium, shaking and culturing in the dark for 30d to 40d, to obtain an industrial hemp adventitious root suspension culture system.

[0051] In the above embodiment, the adventitious roots of industrial hemp are first subjected to suspension culture. At this time, due to the small number of adventitious roots, the inoculation density is 20g / L-40g / L. By way of example, the inoculation density of the adventitious roots of industrial hemp during suspension culture can be set to 20g / L, 22g / L, 24g / L, 25g / L, 26g / L, 28g / L, 30g / L, 32g / L, 34g / L, 35g / L, 36g / L, 38g / L or 40g / L.

[0052] As an optional embodiment, the temperature of the shaking dark culture is 20° C. to 30° C., and the rotation speed is 90 r / min to 110 r / min.

[0053] In the above embodiment, the temperature for shaking dark culture of industrial hemp adventitious roots can be set to 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, preferably 25±2°C; the rotation speed can be set to 90r / min, 95r / min, 100r / min, 105r / min or 110r / min.

[0054] As an optional embodiment, the method of using a bioreactor to amplify and culture the adventitious roots of industrial hemp after the induction treatment specifically includes:

[0055] The induced industrial hemp adventitious roots are inoculated into the liquid culture medium in the bioreactor for the first amplification culture;

[0056] The adventitious roots of industrial hemp after the first scale-up culture were used as test materials, inoculated into the bioreactor, and the second scale-up culture was carried out in a fed-batch culture mode.

[0057] In the above embodiment, during the first amplification culture, the induced industrial hemp adventitious roots are inoculated into a bioreactor containing a liquid culture medium. After the adventitious roots are cultured in a light-proof environment for 40 to 50 days, the biomass of the adventitious roots and the content of their target metabolites are significantly increased, and the liquid culture medium is absorbed and exhausted by the adventitious roots.

[0058] During the second amplification culture, a fed-batch culture mode is adopted, a certain amount of adventitious roots after the first amplification culture is inoculated into the bioreactor, and the second amplification culture is continued under dark conditions.

[0059] Adding the culture solution into the bioreactor in batches can provide a continuous and stable environment for the growth of adventitious roots.

[0060] As an optional embodiment, during the scale-up culture, the inoculation density of the industrial hemp adventitious roots is 10 g / L to 15 g / L.

[0061] In the above embodiment, the reason for controlling the inoculation density of the adventitious roots of industrial hemp is that when the inoculation density of the adventitious roots of industrial hemp is greater than 15g / L, the nutrient supply of the liquid culture medium is insufficient, affecting the growth of the adventitious roots. When the inoculation density of the adventitious roots of industrial hemp is less than 10g / L, it causes a waste of liquid culture medium. Exemplarily, the inoculation density of the adventitious roots of industrial hemp can be set to 10g / L, 11g / L, 12g / L, 13g / L, 14g / L or 15g / L.

[0062] As an optional embodiment, the first amplification culture satisfies at least one of the following conditions: the culture temperature is 20° C. to 30° C., the culture time is 40 days to 50 days, and the ventilation rate is 0.06 vvm to 0.08 vvm.

[0063] In the above embodiment, the temperature of the first amplification culture can be set to 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, preferably 25±2°C; the culture time can be adjusted according to the growth of the adventitious roots. For example, the culture time can be 40d, 41d, 42d, 43d, 44d, 45d, 46d, 47d, 48d, 49d or 50d; the ventilation rate can be set to 0.06vvm, 0.07vvm or 0.08vvm.

[0064] As an optional embodiment, the second scale-up culture is carried out in a fed-batch mode, specifically comprising: adding feed medium to the bioreactor after a period of initial culture, and then carrying out fed-batch culture;

[0065] The second amplified culture satisfies at least one of the following conditions: the culture temperature is 20°C to 30°C, the ventilation rate is 0.06vvm to 0.08vvm, the initial culture time is 45d to 55d, the addition amount of the feed culture medium is 25% to 35% of the volume of the initial culture medium, and the feed culture time is 30d to 40d.

[0066] In the above embodiment, the temperature and ventilation rate of the second amplification culture are the same as those of the first amplification culture. The main difference is that an initial culture is performed for 45 days to 55 days. This time period can be set according to the actual consumption of the culture medium, such as an initial culture of 45 days, 48 ​​days, 50 days, 53 days or 55 days. The addition amount of the feed medium is 25% to 35% of the volume of the initial culture medium. When the addition amount of the feed medium is less than 25%, the biomass of the adventitious roots increases with the increase of the addition amount, and reaches a maximum at 25%, but most of the accumulated target metabolites at this time are not in the optimal state; when the addition amount of the feed medium is higher than 35%, the biomass of the adventitious roots and the target metabolites are not significantly improved, so the feed medium can be set to 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the volume of the initial culture medium; the feed culture time can be set to 30, 31d, 32d, 33d, 34d, 35d, 36d, 37d, 38d, 39d or 40d according to actual conditions.

[0067] As an optional embodiment, the liquid culture medium and the feed culture medium may be conventional culture medium, or may include: 0.75×MS-MS culture medium, 2 mg / L-3 mg / L IBA and 25 g / L-35 g / L sucrose, with a pH of 5-6.

[0068] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually measured in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are measured in accordance with common international standards, conventional conditions or conditions recommended by the manufacturer.

[0069] Example 1

[0070] This embodiment provides a method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone production, comprising the following steps:

[0071] 20 g / L of industrial hemp adventitious roots were placed in a shake bottle containing liquid culture medium and cultured in the dark at 25°C and 110 r / min for 40 days;

[0072] The liquid culture medium includes 3 / 4MS medium, 2 mg / L IBA and 25 g / L sucrose, and the pH is 5.

[0073] Add 300 mg / L yeast extract to the liquid culture medium of the shake flask in the previous step, continue to suspend and culture the adventitious roots for 96 hours, and use the yeast extract to induce the adventitious roots of industrial hemp to obtain the induced adventitious roots of industrial hemp;

[0074] Under sterile conditions, the adventitious roots of industrial hemp after induction were inoculated into the bioreactor for the first scale-up culture, with an inoculation density of 10 g / L and a culture time of 40 days, and the adventitious roots were collected;

[0075] The adventitious roots of industrial hemp after the first amplification culture were used as the test materials for the second amplification culture. 10 g / L of adventitious roots were inoculated into the bioreactor. After the initial culture for 45 days, the feed medium was added to the bioreactor, and then the feed culture was carried out for 30 days.

[0076] Collect the adventitious roots of industrial hemp after the second amplification culture.

[0077] UPLC-QqQ-MS / MS was used to test the content of cannabinoids (CBD, CBG, CBN) and tanshinones (tanshinone ⅡA, dihydrotanshinone Ⅰ and tetrahydrotanshinone Ⅰ) in the adventitious roots of industrial hemp. The specific test method is as follows:

[0078] Preparation of samples to be tested:

[0079] The standards were accurately weighed and dissolved in UPLC-MS grade methanol to prepare solutions, which were then serially diluted to obtain standard solutions of corresponding concentrations. After grinding the freeze-dried adventitious root material from the experimental group, 0.1 g was weighed and mixed with 3 mL of methanol-dichloromethane (9:1, v / v), and then extracted by ultrasound at 0°C for 60 min and centrifuged at 12000 rpm for 8 min. The supernatant was then freeze-dried with 0.5 mL of UPLC-MS grade methanol for 12 h and centrifuged (12000 rpm, 8 min) to obtain the extraction solution. Subsequently, the standard solution and sample solution were filtered with a 0.22 μm PVDF membrane and transferred to an autosampler vial for UPLC-MS / MS analysis.

[0080] Instruments and analytical methods

[0081] Agilent 1290 ultra-high performance liquid chromatograph and Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, USA) and autosampler were used for analysis. The ion source was set to electrospray ionization (ESI) and operated in positive or negative ion mode for all analytes. Agilent Mass Hunter workstation software (version B.07.00) was used as the data system. Chromatographic separation was performed at 30°C using an Agilent ZORBAX Eclipse Plus C18 column (50 mm, 2.1 mm ID, 1.8 μm). The mobile phase flowed at a rate of 0.4 mL / min and consisted of a linear gradient of 0.1% (v / v) formic acid in water (a) and acetonitrile (B): 0–1.0 min, 5% B v / v; 1.0–5.0 min, 5–95% B v / v; 5.0–6.0 min, 95% B v / v; 6.0–7.0 min, 95–5% B (v / v). The column was flushed for 3 min before each injection. The optimized ESI conditions were set as follows: gas temperature of 350 °C, gas flow rate of 10 L / min, nebulizer pressure of 50 psi, and cell acceleration voltage of 4 V. Agilent software was used to optimize the specific MRM parameters for cannabinoid and tanshinone standards, including the selection of precursor / product ion combinations, fragmentation voltages, and collision energies (Tables 1 and Figure 2 ).

[0082] Table 1 Optimization of multiple reaction monitoring (MRM) parameters for cannabinoid and tanshinone standards

[0083]

[0084] Calibration curves were established based on the peak areas of six concentrations of cannabinoids (CBD, CBG, CBN) and tanshinones (tanshinone ⅡA, dihydrotanshinone Ⅰ and tetrahydrotanshinone Ⅰ) in standard solutions, and their linearity was studied using unweighted least squares linear regression. The standard curves were y = 0.126x ± 0.061 (R 2 =0.9993), y=4.194x+0.191(R 2 =0.9989), y=3.15x±0.021(R 2 =0.9998), y=0.132x+0.093(R 2 =0.9991), y=0.715x+0.024(R 2 =0.9992) and y = 3.319x + 0.693 (R 2 =0.9996). The production of cannabinoids and tanshinone in adventitious roots was calculated using the formula: content × dry weight / volume = production.

[0085] Experimental Example 1

[0086] This experimental example is used to compare the effects of different addition amounts of yeast extract on the adventitious roots of industrial hemp, and includes the following steps:

[0087] 25 g / L of industrial hemp adventitious roots were inoculated into 6 groups of shake flasks containing liquid culture medium and cultured in the dark at 25°C and 110 r / min for 30 days;

[0088] Yeast extracts with concentrations of 0, 100, 200, 300, 400 and 500 mg / L were added to the liquid culture medium of the shake flask in the previous step, and the adventitious roots were suspended and cultured for 72 hours to obtain the induced adventitious roots of industrial hemp;

[0089] The above process was repeated ten times, and the adventitious roots of industrial hemp after induced treatment were collected and tested.

[0090] The test results are shown in Table 2 and Figure 3 As shown:

[0091] Table 2 Effect of yeast extract treatment concentration on adventitious root growth of industrial hemp cultured in shake flasks

[0092] Treatment concentration (mg / L) Fresh weight / FW(g / L) Dry weight / DW(g / L) 0 11.33±0.05 1.19±0.02 50 11.41±0.07 1.23±0.02 100 11.26±0.03 1.17±0.01 200 11.19±0.02 1.11±0.01 300 11.27±0.04 1.18±0.01 400 11.35±0.03 1.21±0.01

[0093] Note: The values ​​are expressed as mean ± standard deviation (n = 10).

[0094] From the data in Table 2, it can be seen that after the adventitious roots of industrial hemp were treated with different concentrations of yeast extract, the fresh weight and dry weight of the adventitious roots were not significantly affected. Figure 3It can be known that for the accumulation of cannabinoids and tanshinones in adventitious roots, as the yeast extract treatment concentration increases to 300 mg / L, the output of cannabinoid compounds and tanshinone compounds in adventitious roots increases to the highest, among which tetrahydrotanshinone I, tanshinone IIA and CBD have more obvious improvement effects, which increase by 1.8 times, 1.8 times and 1.5 times, respectively. When the yeast extract treatment concentration exceeds 400 mg / L, the accumulation of the target metabolites decreases instead. Therefore, the appropriate treatment concentration of yeast extract in the present invention is 300 mg / L to 400 mg / L.

[0095] Experimental Example 2

[0096] This experimental example is used to compare the effects of different yeast extract treatment times on the adventitious roots of industrial hemp, and includes the following steps:

[0097] 25 g / L of industrial hemp adventitious roots were inoculated into 6 groups of shake flasks containing liquid culture medium and cultured in the dark at 25°C and 110 r / min for 30 days;

[0098] Adding yeast extract with a concentration of 300 mg / L to the liquid culture medium of the shake flask in the previous step, continuing to culture the adventitious roots in suspension for 0 h, 12 h, 24 h, 48 h, 96 h and 192 h to obtain the induced adventitious roots of industrial hemp;

[0099] The adventitious roots of industrial hemp after induction treatment were collected and tested. The test results are shown in Table 3 and Figure 3 As shown:

[0100] Table 3 Effect of yeast extract treatment time on adventitious root growth of industrial hemp cultured in shake flasks

[0101] Processing time (h) Fresh weight / FW(g / L) Dry weight / DW(g / L) 0 10.95±0.01 1.09±0.01 12 10.97±0.01 1.10±0.01 24 10.99±0.01 1.11±0.02 48 10.94±0.01 1.09±0.01 96 11.02±0.02 1.12±0.03 192 11.04±0.02 1.10±0.01

[0102] Note: The values ​​are expressed as mean ± standard deviation (n = 10).

[0103] From the data in Table 3, it can be seen that the biomass of industrial hemp adventitious roots did not change significantly during the treatment with yeast extract; Figure 3 It can be seen that the production of cannabinoids and tanshinones in adventitious roots reached the highest level at 48h and 96h of yeast extract treatment, among which the three tanshinone compounds increased more significantly at 96h of treatment, namely tanshinone IIA (1.12 times), tetrahydrotanshinone Ⅰ (1.33 times) and dihydrotanshinone Ⅰ (1.48 times).

[0104] Experimental Example 3

[0105] This experimental example is used to compare the growth and accumulation of target metabolites of industrial hemp adventitious roots in a bioreactor before and after yeast extract induction treatment, and includes the following steps:

[0106] 25 g / L of industrial hemp adventitious roots were inoculated into 6 shake flasks containing liquid culture medium and cultured in the dark at 25°C and 110 r / min for 30 days;

[0107] Yeast extract at a concentration of 300 mg / L was added to three of the shake flasks to induce adventitious roots. After continued cultivation for 96 hours, the induced adventitious roots were collected. The adventitious roots in the other three shake flasks were not induced. After continued cultivation for 96 hours, the uninduced adventitious roots were collected.

[0108] Under sterile conditions, the industrial hemp adventitious roots induced by yeast extract collected in the previous step were inoculated into a bioreactor containing 3 / 4MS medium, 3mg / LIBA and 35g / L sucrose liquid medium for the first scale-up culture. At the same time, the untreated adventitious roots were inoculated into the bioreactor for culture as a control. The inoculation density was 15g / L. During the culture process, the aeration rate was set to 0.08vvm, and three replicates were set for each group. After 40 days of dark culture, the adventitious roots were collected for investigation and observation of the morphological structure of the adventitious roots.

[0109] The collected fresh adventitious roots were fixed in 2% (v / v) glutaraldehyde for 12 hours, and the supernatant was discarded after centrifugation. After that, the adventitious roots were washed three times with buffer solution, and then dehydrated with ethanol (30%, 50%, 70%, 90% and 100%) respectively. The adventitious roots were centrifuged (4°C, 12000r / min, 5min) and freeze-dried. The surface of the adventitious roots was sprayed with a conductive film for 2min using an ion sputtering instrument. The morphological structure of the adventitious roots was observed using a scanning electron microscope. The results are as follows: Figure 4 (c) Observation Figure 4 (c) The morphological structure of adventitious roots showed that yeast extract reduced the number of cortical cells in the adventitious roots cultured in the bioreactor compared with the control group, which reduced the diameter of the central column and changed the structure of the adventitious roots.

[0110] The biomass of adventitious roots was investigated, and the data obtained are shown in Table 4. From the data in Table 4, it can be seen that the biomass of the adventitious roots in the first amplified culture after the induction treatment is significantly higher than that of the control. Yeast extract treatment promotes the rapid growth of adventitious roots cultured in the bioreactor, but because too much culture medium is consumed during the growth of the adventitious roots, the growth of the adventitious roots is restricted and aging occurs. Therefore, the adventitious roots are brown-yellow, and the differentiated lateral roots are short and hard. The target metabolites were calculated, and the results are as follows Figure 4 (a) and Figure 4As shown in (b), it can be seen from the figure that compared with the control group, the production of cannabinoids and tanshinone in the induced adventitious roots was also significantly increased.

[0111] Table 4 Effect of yeast extract on adventitious root growth of industrial hemp cultured in bioreactor

[0112]

[0113] Note: The values ​​are expressed as mean ± standard deviation (n = 3).

[0114] Experimental Example 4

[0115] This experimental example is used to compare the effects of different initial culture times on the adventitious roots of industrial hemp in a fed-batch culture mode, and includes the following steps:

[0116] Under sterile conditions, 10 g / L of industrial hemp adventitious roots after the first amplification culture induction treatment were inoculated into four groups of bioreactors containing 2 L of liquid culture medium. After initial culture for 30 days, 40 days, 50 days and 60 days, 25% of the initial culture medium volume was added as feed medium, and then the industrial hemp adventitious roots were collected after continuing to culture for 20 days.

[0117] The liquid culture medium and feed culture medium had the same composition, including 3 / 4MS culture medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0118] The biomass and target metabolite content of the collected industrial hemp adventitious roots were investigated. Figure 5 As shown. As the initial culture time increased from 30 days to 50 days, the fresh weight (124.51 ± 0.5 g / L) and dry weight (12.84 ± 0.2 g / L) of industrial hemp adventitious roots increased to the maximum value, and the yields of cannabinoids and tanshinones also showed similar trends. Compared with the adventitious roots collected in the previous group, the yields of CBD (1.42 times) and tetrahydrotanshinone I (1.63 times) increased significantly; when the initial culture time was extended to 60 days, the biomass of the adventitious roots gradually decreased, and the accumulated cannabinoids and tanshinones also began to decrease. Therefore, the initial culture time of 45 days to 55 days is the appropriate initial culture time.

[0119] Experimental Example 5

[0120] This experimental example is used to compare the effects of different feed medium volumes on the adventitious roots of industrial hemp in a fed-batch culture mode, and includes the following steps:

[0121] Under sterile conditions, 10 g / L of industrial hemp adventitious roots after the first amplification culture induction treatment were inoculated into four groups of bioreactors containing 2 L of liquid culture medium. After 50 days of initial culture, 10%, 20%, 30% and 40% of the initial culture medium volume were added to each group of bioreactors as feed medium, respectively. Each group was set up with three replicates, and the adventitious roots were collected after 20 days of fed culture under the same conditions.

[0122] The liquid culture medium and feed culture medium had the same composition, including 3 / 4MS culture medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0123] The collected adventitious root biomass and metabolite content were tested. The results were as follows: Figure 6 As shown in the figure, as the volume of feed medium added to the bioreactor increased from 10% to 20% of the initial culture medium volume, the fresh weight and dry weight of the adventitious roots increased to the maximum value, 136.51±0.02g / L and 13.57±0.03g / L, respectively; when the volume of feed medium increased from 20% to 30%, the biomass of the adventitious roots did not change significantly. The accumulation of cannabinoids and tanshinones in adventitious roots was investigated, and it was found that when the volume of feed medium was 30% of the initial culture medium volume, the yield of CBD and tanshinone in adventitious roots reached the highest, among which the yield of tanshinones was significantly increased compared with the adventitious roots under the previous optimal conditions, which were tetrahydrotanshinone I (1.27 times), tanshinone IIA (1.24 times) and dihydrotanshinone I (1.12 times). Only the yield of CBG reached the highest when the volume of feed medium was 20% of the initial culture medium volume, which was 43.57±0.18mg / L DW. Taking all factors into consideration, in the actual large-scale cultivation of industrial hemp adventitious roots with high cannabinoid and tanshinone production, 25% to 35% of the initial culture medium volume can be selected as the appropriate feed medium volume.

[0124] Experimental Example 6

[0125] This experimental example is used to compare the effects of different feeding culture times on the adventitious roots of industrial hemp in a fed-batch culture mode, and includes the following steps:

[0126] Under sterile conditions, the adventitious roots of industrial hemp after induced treatment were cultured for the first time, and 10 g / L of the collected adventitious roots were inoculated into 4 groups of bioreactors respectively. After the initial culture for 50 days, 30% of the initial culture medium volume was added to each group of bioreactors as feed medium. After continuing to culture under the same conditions for 10 days, 20 days, 30 days, and 40 days, three replicates were set up in each group to collect the adventitious roots.

[0127] The liquid culture medium and feed culture medium had the same composition, including 3 / 4MS culture medium, 2 mg / L IBA and 25 g / L sucrose, with a pH of 5.

[0128] The collected adventitious root biomass and metabolite content were tested. The results were as follows: Figure 7 As shown in the figure, when the feeding culture time was increased to 40 days, the accumulation of cannabinoids and tanshinones in adventitious roots was measured, and it was found that the content and yield of cannabinoids and tanshinones reached the highest level at the 30-day feeding culture time, among which the yields of CBG, CBD and tanshinone ⅡA increased by 1.85 times, 1.85 times and 1.47 times compared with the adventitious roots under the previous optimal conditions, followed by dihydrotanshinone Ⅰ (1.31 times). Therefore, the appropriate feeding culture time is 30 days to 40 days.

[0129] Experimental Example 7

[0130] This experimental example is used to compare the effects of fed-batch culture mode and conventional amplification culture mode on adventitious root growth, and includes the following steps:

[0131] Under sterile conditions, the adventitious roots of industrial hemp after the induction treatment were respectively cultured for the first time, and 10 g / L of the collected adventitious roots of industrial hemp from the first culture were inoculated into three groups of bioreactors containing 2 L of liquid culture medium, and one group of bioreactors was cultured for 50 days, and 30% of the volume of the initial culture medium was supplemented as feed medium, and then the adventitious roots were collected after continuing to culture for 30 days as the adventitious roots of industrial hemp for the second culture;

[0132] The other two groups took the industrial hemp adventitious roots before and after yeast extract induction treatment and cultured them continuously in the bioreactor for 80 days and collected the adventitious roots as the industrial hemp adventitious roots for conventional amplified culture.

[0133] The liquid culture medium and feed medium used in this experiment had the same composition, including 3 / 4MS medium, 2.5 mg / L IBA and 25 g / L sucrose.

[0134] The growth of adventitious roots collected and the accumulation of target metabolites were investigated. The results are shown in Figure 8 and Fig. 9. The adventitious roots after fed-batch culture differentiated into new lateral roots, and showed obvious elongation and growth. Their biomass was significantly higher than that of the adventitious roots in conventional amplification culture. At the same time, higher cannabinoids and tanshinone substances accumulated in the adventitious roots, among which tetrahydrotanshinone I, CBG and CBD had more significant effects, which were 3.32 times, 3.99 times and 8.53 times of the adventitious roots untreated with yeast extract in conventional amplification culture, and 2.56, 2.84 and 3.76 times of the adventitious roots treated with yeast extract in conventional amplification culture. This proves that the fed-batch culture mode not only promotes the growth, proliferation and differentiation of industrial hemp adventitious roots, but also facilitates the accumulation of target metabolites in the adventitious roots.

[0135] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by the present application.

Claims

1. A method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yields, characterized in that: The following steps are involved: Using yeast extract to induce the adventitious roots of industrial hemp to obtain the induced adventitious roots of industrial hemp; The adventitious roots of industrial hemp after induction treatment were amplified and cultured using a bioreactor; Collect the adventitious roots of industrial hemp after amplification and cultivation.

2. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 1, characterized in that: The method of inducing the adventitious roots of industrial hemp by using yeast extract to obtain the induced adventitious roots of industrial hemp specifically includes: Under sterile conditions, the adventitious roots of industrial hemp are inoculated into a shake bottle containing a liquid culture medium for cultivation to obtain suspended cultured adventitious roots of industrial hemp; The yeast extract is added to the suspension cultured industrial hemp adventitious roots and the culture is continued for 48 hours to 96 hours to obtain the induced industrial hemp adventitious roots.

3. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 1 or 2, characterized in that: The yeast extract is added in an amount of 300 mg to 400 mg of yeast extract per liter of liquid culture medium.

4. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 2, characterized in that: The method of culturing industrial hemp adventitious roots by suspension culture technology to obtain an industrial hemp adventitious root suspension culture system specifically includes: placing the industrial hemp adventitious roots in a shaking bottle containing a liquid culture medium, and culturing in the dark with shaking for 30 days to 40 days to obtain the industrial hemp adventitious root suspension culture system.

5. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 4, characterized in that: The temperature of the shaking dark culture is 20°C to 30°C, and the rotation speed is 90r / min to 110r / min.

6. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 1, characterized in that: The method of using a bioreactor to amplify and culture the adventitious roots of industrial hemp after the induction treatment specifically includes: The induced industrial hemp adventitious roots are inoculated into the liquid culture medium in the bioreactor for the first amplification culture; The adventitious roots of industrial hemp after the first amplification culture were inoculated into the bioreactor, and the second amplification culture was carried out in a fed-batch culture mode.

7. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 6, characterized in that: During amplified cultivation, the inoculation density of the industrial hemp adventitious roots is 10 g / L-15 g / L.

8. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 6, characterized in that: The first amplification culture satisfies at least one of the following conditions: the culture temperature is 20° C. to 30° C., the culture time is 40 days to 50 days, and the ventilation rate is 0.06 vvm to 0.08 vvm.

9. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 6, characterized in that: The second scale-up culture is carried out in a fed-batch mode, specifically comprising: adding feed culture medium to the bioreactor after a period of initial culture, and then carrying out fed-batch culture; The culture temperature of the second amplification culture is 20°C to 30°C, the ventilation rate is 0.06vvm to 0.08vvm; the initial culture time is 45d to 55d, the addition amount of the feed culture medium is 25% to 35% of the initial culture medium, and the feed culture time is 30d to 40d.

10. The method for cultivating industrial hemp adventitious roots with high cannabinoid and tanshinone yield according to claim 4, 6 or 9, characterized in that: The liquid culture medium and the feed culture medium include: 0.75×MS-MS culture medium, 2 mg / L-3 mg / L IBA and 25 g / L-35 g / L sucrose, and the pH value is 5-6.

Images