CN115094018A - Strain bag based on taxus chinensis raw materials and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of strain packet production, in particular to a strain packet based on taxus chinensis raw materials and a preparation method and application thereof. The strain bag based on the taxus chinensis raw materials is prepared by mixing the taxus chinensis and wheat bran, adding potassium nitrate, potassium hydrogen phosphate, zinc sulfate, copper nitrate and ferric chloride, sterilizing and then bagging. The invention improves the content of taxol generated by endophytic fungi by adding taxus raw materials into the strain bag, and carries out breeding on the endophytic fungi on the basis of the content.
Description
The technical field is as follows:
the invention relates to the technical field of strain packet production, in particular to a strain packet based on taxus chinensis raw materials and a preparation method and application thereof.
Background art:
the leaves, branches, bark and roots of Taxus chinensis contain paclitaxel.
However, because the content of paclitaxel in the taxus plants is low, and the taxus grows slowly under natural conditions and has poor regeneration capability, the number of the taxus is not enough to meet the market demand. At present, the production of paclitaxel mainly comprises artificial synthesis and plant cell culture, the artificial synthesis steps are complicated and difficult, and the precursor substance in semi-synthesis is still extracted from taxus chinensis; plant cell culture yields are low and synthesis costs are high.
Recently, researches show that taxol can be produced by endophytic fungi of taxus chinensis, and the taxol yield of the endophytic fungi can be improved by some substances in the taxus chinensis.
Disclosure of Invention
The invention provides a strain packet based on taxus chinensis raw materials and a preparation method and application thereof, which are used for further researching the characteristic that some substances in taxus chinensis can improve the yield of taxol of endophytic fungi.
The invention provides a preparation method of a strain packet based on taxus chinensis raw materials, which comprises the following steps:
s1: cleaning root, stem and tender leaf of Taxus chinensis, pulverizing, and sieving with 120 mesh sieve;
s2: freeze drying the crushed taxus chinensis raw material;
s3: uniformly mixing 40-60 parts of crushed yew, 40-60 parts of wheat bran, 1-3 parts of potassium nitrate, 0.1-0.5 part of potassium hydrogen phosphate, 0.1-0.5 part of zinc sulfate, 0.1-0.5 part of copper nitrate, 0.1-0.5 part of ferric chloride and 0.1-0.5 part of L-tyrosine to prepare a mixture C;
s4: sterilizing the mixture C and the strain bags;
s5: filling the mixture C into a strain bag under sterile air;
s6: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bag to obtain the strain bag based on the taxus raw material.
Further, the step S3 specifically includes the following steps:
s3.1: mixing 40-60 parts of crushed yew and 40-60 parts of wheat bran in a drum mixer to obtain a mixture A;
s3.2: dissolving 1-3 parts of potassium nitrate, 0.1-0.5 part of potassium hydrogen phosphate, 0.1-0.5 part of zinc sulfate, 0.1-0.5 part of copper nitrate, 0.1-0.5 part of ferric chloride and 0.1-0.5 part of L-tyrosine in a proper amount of deionized water, and uniformly stirring to obtain a mixture B;
s3.3: injecting the mixture B into the mixture A, and uniformly stirring;
s3.4: and (4) freeze-drying the material mixed in the step (S3.3), and controlling the content of free water to be below 25% to prepare a mixture C.
Further, in step S3.2, 0.1-0.5 part of sodium benzoate is added to the mixture B.
Further, the step S4 specifically includes the following steps:
s4.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettle filled with the mixture C to rotate continuously;
s4.2: continuously introducing a gas mixed by ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags;
further, the ratio of ethylene oxide to nitrogen in step S4.2 is 1: 9.
Further, the step S5 specifically includes the following steps
S5.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
s5.2: mixture C was lightly pressed while allowing the dry ice to sublime and vaporize, creating gaps between the mixtures.
The invention also provides a strain packet based on the taxus chinensis raw material, which is prepared by the preparation method of the strain packet based on the taxus chinensis raw material.
The invention also provides an application of the strain packet based on the taxus chinensis raw material.
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FIG. 1 is a table showing the composition of raw materials of examples of the present invention and comparative examples.
FIG. 2 is a graph of the paclitaxel production scale of example 1 of the present invention and comparative example 1;
the invention has the following advantages:
1. the invention improves the content of taxol generated by endophytic fungi by adding taxus raw materials into the strain bag, and carries out breeding on the endophytic fungi on the basis of the content.
2. Potassium nitrate, potassium hydrogen phosphate, zinc sulfate, copper nitrate and ferric chloride are dissolved in water and mixed with the mixture A, so that trace elements are provided for endophytic fungi, and the growth of the endophytic fungi is promoted;
3. the sodium benzoate is added to induce the endophytic fungi to generate more paclitaxel, so that the paclitaxel content is conveniently detected;
4. the sterilization method disclosed by the invention is used for sterilizing the mixture C and the strain bags, so that the influence of external strains on the growth of endophytic fungi is avoided, and the mixture C is sterilized more thoroughly through the rotation of the reaction kettle;
5. according to the invention, through sublimation and dissipation of the dry ice, gaps are formed in the strain bag, so that the growth of endophytic fungi is facilitated.
The specific implementation mode is as follows:
the present invention will be further described with reference to specific embodiments for making the objects, technical solutions and advantages of the present invention more apparent, but the present invention is not limited to these examples. It should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment. In the invention, all parts and percentages are mass units, and the adopted equipment, raw materials and the like can be purchased from the market or are commonly used in the field. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1
A preparation method of a strain packet based on taxus chinensis raw materials comprises the following steps:
s1: cleaning roots, stems and tender leaves of taxus chinensis, crushing, and sieving with a 120-mesh sieve;
s2: freeze-drying the crushed taxus raw material, and facilitating subsequent mixing after drying;
s3.1: mixing 60 parts of crushed taxus chinensis and 60 parts of wheat bran in a drum mixer to obtain a mixture A;
s3.2: dissolving 3 parts of potassium nitrate, 0.2 part of potassium hydrogen phosphate, 0.2 part of zinc sulfate, 0.2 part of copper nitrate and 0.2 part of ferric chloride in a proper amount of deionized water, uniformly stirring to obtain a mixture B, and adding 0.5 part of sodium benzoate into the mixture B, wherein the potassium nitrate, the potassium hydrogen phosphate, the zinc sulfate, the copper nitrate and the ferric chloride are used for providing trace elements, so that the influence of lack of the trace elements on the growth of strains is avoided, and the sodium benzoate can induce endophytic fungi to produce more taxol;
s3.3: injecting the mixture B into the mixture A, and uniformly stirring, wherein the liquid mixture B can be mixed with the mixture A;
s3.4: freeze-drying the material mixed in the step S3.3, and controlling the free water content to be below 25% to prepare a mixture C;
s4.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettles filled with the mixture C to rotate continuously;
s4.2: continuously introducing a gas mixed by ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags, wherein the ratio of the ethylene oxide to the nitrogen is 1:9, and the mixture C and the strain bags are sterilized to avoid the influence of external strains on the growth of endophytic fungi;
s5.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
s5.2: slightly pressing the mixture C, and simultaneously sublimating and gasifying the dry ice to form a plurality of gaps among the mixtures, wherein the gaps can prevent hyphae from being blocked in the growth process of the endophytic fungi;
s6: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bag to obtain the strain bag based on the taxus raw material.
Example 2
A preparation method of a strain packet based on taxus chinensis raw materials comprises the following steps:
s1: cleaning root, stem and tender leaf of Taxus chinensis, pulverizing, and sieving with 120 mesh sieve;
s2: freeze-drying the crushed taxus raw material, and facilitating subsequent mixing after drying;
s3.1: mixing 60 parts of crushed taxus chinensis and 60 parts of wheat bran in a drum mixer to obtain a mixture A;
s3.2: dissolving 3 parts of potassium nitrate, 0.2 part of potassium hydrogen phosphate, 0.2 part of zinc sulfate, 0.2 part of copper nitrate and 0.2 part of ferric chloride in a proper amount of deionized water, uniformly stirring to obtain a mixture B, and adding 0.1 part of sodium benzoate into the mixture B, wherein the potassium nitrate, the potassium hydrogen phosphate, the zinc sulfate, the copper nitrate and the ferric chloride are used for providing trace elements, so that the influence of lack of the trace elements on the growth of strains is avoided, and the sodium benzoate can induce endophytic fungi to produce more taxol;
s3.3: injecting the mixture B into the mixture A, and uniformly stirring, wherein the liquid mixture B can be mixed with the mixture A;
s3.4: freeze-drying the material mixed in the step S3.3, and controlling the free water content to be below 25% to prepare a mixture C;
s4.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettle filled with the mixture C to rotate continuously;
s4.2: continuously introducing a gas mixed by ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags, wherein the ratio of the ethylene oxide to the nitrogen is 1:9, and the mixture C and the strain bags are sterilized to avoid the influence of external strains on the growth of endophytic fungi;
s5.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
s5.2: slightly pressing the mixture C, and simultaneously sublimating and gasifying the dry ice to form a plurality of gaps among the mixtures, wherein the gaps can prevent hyphae from being blocked in the growth process of the endophytic fungi;
s6: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bag to obtain the strain bag based on the taxus raw material.
Example 3
A preparation method of a strain packet based on taxus chinensis raw materials comprises the following steps:
s1: cleaning roots, stems and tender leaves of taxus chinensis, crushing, and sieving with a 120-mesh sieve;
s2: freeze-drying the crushed taxus raw material, and facilitating subsequent mixing after drying;
s3.1: mixing 60 parts of crushed taxus chinensis and 60 parts of wheat bran in a drum mixer to obtain a mixture A;
s3.2: dissolving 3 parts of potassium nitrate, 0.2 part of potassium hydrogen phosphate, 0.2 part of zinc sulfate, 0.2 part of copper nitrate, 0.2 part of ferric chloride and 0.2 part of L-tyrosine in a proper amount of deionized water, uniformly stirring to obtain a mixture B, and adding 0.3 part of sodium benzoate into the mixture B, wherein the potassium nitrate, the potassium hydrogen phosphate, the zinc sulfate, the copper nitrate and the ferric chloride are used for providing trace elements, so that the influence of lack of the trace elements on the growth of strains is avoided, and the sodium benzoate can induce endophytic fungi to produce more taxol;
s3.3: injecting the mixture B into the mixture A, and uniformly stirring, wherein the liquid mixture B can be mixed with the mixture A;
s3.4: freeze-drying the material mixed in the step S3.3, and controlling the free water content to be below 25% to prepare a mixture C;
s4.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettle filled with the mixture C to rotate continuously;
s4.2: continuously introducing a gas mixed by ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags, wherein the ratio of the ethylene oxide to the nitrogen is 1:9, and the mixture C and the strain bags are sterilized to avoid the influence of external strains on the growth of endophytic fungi;
s5.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
s5.2: slightly pressing the mixture C, and simultaneously sublimating and gasifying the dry ice to form a plurality of gaps among the mixtures, wherein the gaps can prevent hyphae from being blocked in the growth process of the endophytic fungi;
s6: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bag to obtain the strain bag based on the taxus raw material.
Example 4
An application of a strain packet based on taxus chinensis raw materials comprises the following steps:
(1) putting the strain bag based on the taxus chinensis raw material prepared in the embodiment 1-3 in a low-light and humid environment, and cutting the bag opening;
(2) spraying water at proper time, and culturing for 3 weeks;
(3) adding a proper amount of deionized sterile water into the culture, and filtering to obtain endophytic fungi liquid;
(4) dividing the endophytic fungi liquid into 10 parts, determining the taxol content in each part of endophytic fungi liquid through immunoassay, marking the part with the highest taxol content, and breeding.
Comparative example 1
T1.1: mixing 120 parts of wheat bran in a drum mixer to obtain a mixture A;
t1.2: dissolving 3 parts of potassium nitrate, 0.2 part of potassium hydrogen phosphate, 0.2 part of zinc sulfate, 0.2 part of copper nitrate, 0.2 part of ferric chloride and 0.2 part of L-tyrosine in a proper amount of deionized water, uniformly stirring to obtain a mixture B, and adding 0.5 part of sodium benzoate into the mixture B, wherein the potassium nitrate, the potassium hydrogen phosphate, the zinc sulfate, the copper nitrate and the ferric chloride are used for providing trace elements, so that the influence of lack of the trace elements on the growth of strains is avoided, and the sodium benzoate can induce endophytic fungi to produce more taxol;
t1.3: injecting the mixture B into the mixture A, and uniformly stirring, wherein the liquid mixture B can be mixed with the mixture A;
t1.4: freeze-drying the material mixed in the step T1.3, and controlling the content of free water to be below 25% to prepare a mixture C;
t2.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettle filled with the mixture C to rotate continuously;
t2.2: continuously introducing a mixed gas of ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags, wherein the ratio of ethylene oxide to nitrogen is 1:9, and the mixture C and the strain bags are sterilized to avoid the influence of external strains on the growth of endophytic fungi;
t3.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
t3.2: slightly pressing the mixture C, and simultaneously sublimating and gasifying the dry ice to form a plurality of gaps among the mixtures, wherein the gaps can prevent hyphae from being blocked in the growth process of the endophytic fungi;
t4: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bag to obtain the strain bag.
T5: the strain packets based on the taxus chinensis raw material obtained in example 1 and the strain packets prepared in step T4 were cultured for 3 weeks, and then the cultures were tested for the content of paclitaxel by immunoassay, respectively, as shown in fig. 2, and more paclitaxel was produced by the endophytic fungi cultured in the strain packets to which the taxus chinensis raw material was added.
The above embodiments are merely preferred embodiments of the present invention, and any simple modification, modification and substitution changes made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (8)
1. A preparation method of a strain packet based on a taxus chinensis raw material is characterized by comprising the following steps:
s1: cleaning roots, stems and tender leaves of taxus chinensis, crushing, and sieving with a 120-mesh sieve;
s2: freeze drying the crushed Chinese yew raw material;
s3: uniformly mixing 40-60 parts of crushed yew, 40-60 parts of wheat bran, 1-3 parts of potassium nitrate, 0.1-0.5 part of potassium hydrogen phosphate, 0.1-0.5 part of zinc sulfate, 0.1-0.5 part of copper nitrate, 0.1-0.5 part of ferric chloride and 0.1-0.5 part of L-tyrosine to prepare a mixture C;
s4: sterilizing the mixture C and the strain bags;
s5: filling the mixture C into a strain bag under sterile air;
s6: inoculating the taxol-producing endophytic fungi into the mixed material, and sealing the strain bags to obtain the strain bag based on the taxus raw material.
2. The method for preparing a bacterial seed bag based on taxus chinensis raw materials as claimed in claim 1, wherein the step S3 specifically comprises the following steps:
s3.1: mixing 40-60 parts of crushed yew and 40-60 parts of wheat bran in a drum mixer to obtain a mixture A;
s3.2: dissolving 1-3 parts of potassium nitrate, 0.1-0.5 part of potassium hydrogen phosphate, 0.1-0.5 part of zinc sulfate, 0.1-0.5 part of copper nitrate, 0.1-0.5 part of ferric chloride and 0.1-0.5 part of L-tyrosine in a proper amount of deionized water, and uniformly stirring to obtain a mixture B;
s3.3: injecting the mixture B into the mixture A, and uniformly stirring;
s3.4: and (4) freeze-drying the material mixed in the step (S3.3), and controlling the free water content to be below 25% to prepare a mixture C.
3. The method for preparing a seed bag based on taxus chinensis raw material according to claim 2, wherein 0.1-0.5 part of sodium benzoate is added to the mixture B in the step S3.2.
4. The method for preparing a strain bag based on taxus raw materials as claimed in claim 1, wherein the step S4 comprises the following steps:
s4.1: placing the mixture C and the strain bags in different reaction kettles respectively, and enabling the reaction kettle filled with the mixture C to rotate continuously;
s4.2: and continuously introducing a gas mixed by ethylene oxide and nitrogen into the reaction kettle to sterilize the mixture C and the strain bags.
5. The method for preparing a seed bag based on taxus chinensis raw material as claimed in claim 4, wherein the ratio of ethylene oxide to nitrogen in step S4.2 is 1: 9.
6. The method for preparing strain packets based on taxus chinensis raw materials as claimed in claim 1, wherein the step S5 specifically comprises the following steps
S5.1: when the mixture C is bagged, simultaneously filling dry ice into the mixture C at intervals;
s5.2: mixture C was lightly pressed while allowing the dry ice to sublime and vaporize, creating gaps between the mixtures.
7. A strain bag based on Taxus chinensis raw materials, which is prepared by the method for preparing a strain bag based on Taxus chinensis raw materials as claimed in claims 1-6.
8. Use of a seed bag based on taxus raw material according to claim 7.
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2022
- 2022-07-18 CN CN202210839879.3A patent/CN115094018A/en active Pending
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| CN101318863A (en) * | 2008-07-04 | 2008-12-10 | 福建省农业科学院农业工程技术研究所 | Process for cultivating edible mushroom with waste slag from yew processing |
| CN105400842A (en) * | 2015-12-31 | 2016-03-16 | 浙江省林业科学研究院 | Method for increasing yield of paclitaxel in endophytic fungus fermentation product |
| CN106520854A (en) * | 2016-08-19 | 2017-03-22 | 重庆市碚圣医药科技股份有限公司 | Chinese yew solid state fermentation medium, preparation method thereof and Chinese yew solid state fermentation method |
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