CN111690706A - Method for producing gibberellin A by fermentation of riboflavin waste liquid3Method (2) - Google Patents
Method for producing gibberellin A by fermentation of riboflavin waste liquid3Method (2) Download PDFInfo
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Abstract
The invention discloses a method for producing gibberellin A by fermentation of riboflavin waste liquid3The method belongs to the technical field of microbial fermentation, and the key point of the technical scheme is fermentation culture, the seed liquid is inoculated into a fermentation culture medium prepared by treated riboflavin waste liquid and essential nutrient substances for fermentation, and the gibberellin A is produced by utilizing the fermentation of the riboflavin waste liquid3Not only solves the problem of waste liquid treatment in the production process of riboflavin, but also achieves the aim of reducing gibberellin A3The production cost of (a).
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing gibberellin A by fermentation of riboflavin waste liquid3The method of (1).
Background
Gibberellin A3Is an endogenous plant regulating substance, has obvious effects on regulating and controlling the elongation of stems of plants, seed germination, bolting and fruit setting and the like, and is widely applied to the field of planting and production of economic crops. Industrial gibberellin A3The red-eared mold is usually produced by liquid fermentation, and a fermentation culture medium of the red-eared mold mainly comprises organic nitrogen carbon sources such as corn starch, peanut cake powder and the like, so that the production cost is high. How to optimize the culture medium and reduce the production cost is that all gibberellin A3A problem that the manufacturing enterprise must face.
Riboflavin also known as vitamin B2The vitamin is a water-soluble vitamin necessary for human and animals, has obvious effect on inhibiting digestive tract diseases and nervous system diseases (such as Parkinson's disease, migraine, multiple sclerosis and the like), and is widely applied to the fields of clinical treatment, feed industry, cosmetic industry, food industry and the like. Riboflavin is mainly produced by a microbial fermentation method, a large amount of waste liquid is generated in the extraction stage, and with the increasing environmental protection requirement, how to treat the waste liquid can meet the emission requirement, which is a problem to be solved urgently in riboflavin production enterprises. The riboflavin waste liquid contains carbon nitrogen source and inorganic substance which are not used up in partial fermentationSalt and residual mycelium cause high viscosity of the riboflavin waste liquid, great treatment difficulty and high production cost of the riboflavin.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for producing gibberellin A by fermentation of riboflavin waste liquid3The method for producing gibberellin A by fermentation of riboflavin waste liquid3Not only solves the problem of waste liquid treatment in the production process of riboflavin, but also achieves the aim of reducing gibberellin A3The production cost of (a).
The technical purpose of the invention is realized by the following technical scheme:
method for producing gibberellin A by fermentation of riboflavin waste liquid3The method comprises the following preparation steps: and (4) fermentation culture, namely inoculating the seed liquid into a fermentation culture medium prepared from the treated riboflavin waste liquid and essential nutrients for fermentation.
The invention is further set as the method for treating the riboflavin waste liquid comprises the steps of placing the riboflavin waste liquid in a fermentation tank, carrying out heat preservation for 30-60min to inactivate mycelium under the condition that the temperature is 70 +/-5 ℃, then carrying out centrifugation for 10-30min under the condition of 5000 plus-8000 rpm to obtain a light phase containing the mycelium, and adjusting the pH of the obtained light phase to 5.5-6.5 by using a pH regulator to obtain the treated riboflavin waste liquid.
By adopting the technical scheme, the treated riboflavin waste liquid is used as gibberellin A3The fermentation culture solution is beneficial to reducing the treatment cost of the riboflavin waste liquid, can fully utilize organic nitrogen sources and carbon sources in the riboflavin waste liquid, and reduces the amount of other nutrient substances in the fermentation culture medium, thereby effectively reducing the gibberellin A content3The experiment proves that when the riboflavin waste liquid is used as the component of the fermentation culture medium, the gibberellin A finally obtained is3The yield of the method is higher than that of the method for producing the gibberellin A by normal fermentation3The yield of (a).
In addition, after the riboflavin waste liquid is treated, the step of adjusting the pH of the fermentation medium is not needed to be added in the fermentation process, so that the gibberellin fermentation can be carried out under the condition of natural pH when other components are added into the fermentation medium, and convenience is brought to the operation.
The invention is further provided that the pH regulator is a 20-30% sodium hydroxide solution.
By adopting the technical scheme, after the pH regulator adopts the sodium hydroxide solution, the treatment on the riboflavin waste liquid can be achieved, and meanwhile, the nitrogen content in the riboflavin waste liquid can not be influenced, so that the later fermentation is guaranteed.
The invention is further configured that the riboflavin waste liquid is waste liquid generated in an extraction stage in the process of producing riboflavin by fermentation.
The invention is further configured that the fermentation culture comprises seed culture before the fermentation culture, and the seed culture comprises two stages of culture:
first-order seed culture: taking the red-eared mold mutant strain to a shake flask filled with a seed culture solution, and culturing for 40-60h under the conditions of 250rmp and 30 +/-1 ℃ to obtain a primary seed solution; a mutant strain of Gibberella fujikuroi of the present application (A)Fusarium fujikuroi GA-347) Purchased from China center for type culture Collection, wherein, the mutant of Gibberella fujikuroi: (Fusarium fujikuroi GA-347) Deposited in China center for type culture Collection, address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019378, date of deposit 2019, 5 months and 20 days;
secondary seed culture: inoculating the first-stage seed solution to a second-stage seed tank at a volume ratio of 0.5-1.0%, and culturing at 30 + -1 deg.C, dissolved oxygen of 20-70%, and natural pH for 40-60h to obtain second-stage seed solution.
The invention is further set that in the fermentation culture, the secondary seed liquid is inoculated in the fermentation culture medium according to the volume proportion of 5-10%, and the fermentation culture medium is cultured for 7-8 days under the conditions of 30 +/-1 ℃, dissolved oxygen of 20-60% and natural pH value, so as to obtain the fermentation liquid.
The invention is further configured that the culture solution for the two-stage culture comprises the following components: 20-30g/L of corn starch, 5-10g/L of cane sugar and 20-30g/L, KH of peanut powder2PO41-2g/L、MgSO41-2g/L。
The invention is further set that the fermentation medium comprises 600g/L of riboflavin waste liquid 400-2PO40.2-0.5g/L、K2SO40.2-0.5g/L、MgSO40.1-0.2g/L。
In conclusion, the invention has the following beneficial effects:
1. the application utilizes the riboflavin waste liquid as the production of gibberellin A3The main components of the fermentation culture medium not only reduce the discharge amount of the riboflavin waste liquid, save the energy consumed by sewage treatment and reduce the production cost of the riboflavin, but also utilize the riboflavin waste liquid to ferment and produce the gibberellin A3Later, the gibberellin A is effectively reduced3The production cost of (2);
2. because residual sugar contained in the riboflavin waste liquid can meet the demand of the carbon source in the early growth process of the single colony, the hydrolysis step of gibberellin A3 fermentation culture medium starch and rice flour is omitted, and the gibberellin A is simplified3The production process saves the cost;
3. the yield of the gibberellin A3 in the application is 2458mg/L at most, while the gibberellin A in the existing production3The yield of (5) was 2123 mg/L.
Drawings
FIG. 1 is a high performance liquid chromatogram of a standard;
FIG. 2 is a high performance liquid chromatogram of example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of the comparative example.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The riboflavin waste liquid used in the application is the riboflavin waste liquid obtained in industrial production, wherein the riboflavin waste liquid is the riboflavin waste liquid obtained when the residual carbon content is about 1% when the riboflavin production is stopped.
The concentration of the standard sample after 50-fold dilution was 65. mu.g/mL.
The standard chromatogram is shown in FIG. 1, gibberellin A3The integration results of (a) are shown in table 1.
TABLE 1 Table of integration results of standard gibberellins
| Peak # | Retention time | Area of | Height | Area% | Theoretical plate # |
| 1 | 6.033 | 1395930 | 126308 | 100.000 | 7017.251 |
| Total of | —— | 1395930 | 126308 | 100.000 | —— |
The yield of the comparative example was calculated by the following formula: yield of control = (control peak area/standard peak area) × concentration after dilution of standard × dilution factor.
Since the peak area ratio is proportional to the yield ratio, therefore: the yield calculation formula for example 1 is: example yield = (example sample peak area/control peak area) × control yield.
Example 1
Method for producing gibberellin A by fermentation of riboflavin waste liquid3The method comprises the following steps:
(1) treatment of riboflavin waste liquor
Putting the riboflavin waste liquid into a fermentation tank, preserving heat for 45min at the temperature of 70 ℃ to inactivate mycelium, centrifuging for 20min by a horizontal screw centrifuge at 6500rpm to obtain an upper layer light phase containing the mycelium, and adjusting the pH of the upper layer light phase to 6.0 +/-0.2 by using a sodium hydroxide solution with the mass fraction of 30% for later use;
(2) fermentation production of gibberellin A3
a. Seed culture: the two-stage culture is carried out, and the culture solution of the two-stage seed culture comprises the following components: corn starch 25g/L, cane sugar 7.5g/L, peanut powder 25g/L, KH2PO41.5g/L、MgSO41.5g/L, natural pH;
first-order seed culture: charging 250mL of culture medium into 1000mL of shake flask, and picking to produce gibberellin A3The red-eared mold mutant strain is filled into a shake flask and cultured for 48 hours under the conditions of 250rmp and 30 +/-1 ℃ to obtain first-class seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shake flask, inoculating the primary seed solution into a secondary seed tank according to the volume proportion of 1.0%, and culturing for 40h under the conditions of the temperature of 30 +/-1 ℃, dissolved oxygen of 40-50% and natural pH to obtain a secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 120g/L of starch and 10g/L, KH of peanut powder2PO40.5g/L、K2SO40.5g/L、MgSO40.2g/L, 400g/L of the riboflavin waste liquid obtained in the step (1) and natural pH;
inoculating the secondary seed solution into a fermentation medium in a 3t fermentation tank in a volume ratio of 5%, and culturing for 8 days at 30 + -1 deg.C, dissolved oxygen of 30-40%, and natural pH to obtain a fermentation liquid;
c. product detection
Take 0.25mLAdding 4.75mL of distilled water into the fermentation liquid, strongly shaking for 10min, centrifuging at 4000rpm for 10min, collecting the supernatant, and performing high performance liquid chromatography with Hypersil BDSC18 reversed phase chromatographic column (150 mm × 4.6.6 mm), methanol-water solution as mobile phase, methanol-water volume ratio of 40:60, flow rate of 0.80mL/min, ultraviolet detector wavelength of 230nm, sample injection volume of 20 μ L, chromatogram shown in FIG. 2, and gibberellin A3The integration results are shown in Table 2, giving gibberellin A3The yield of (A) is: 2458 mg/L.
TABLE 1 gibberellin integration results Table
| Peak # | Retention time | Area of | Height | Area% | Theoretical plate # |
| 1 | 4.735 | 133544 | 14666 | 10.005 | 5958.443 |
| 2 | 4.997 | 109779 | 11019 | 8.225 | 5829.390 |
| 3 | 6.055 | 1056141 | 95991 | 79.127 | 7148.329 |
| 4 | 6.740 | 35277 | 3132 | 2.643 | 8224.769 |
| Total of | —— | 1334742 | 124807 | 100.000 | —— |
The peak 3# in FIG. 2 is the target product gibberellin A3The 1# peak, the 2# peak and the 4# peak are impurity peaks, but the 1# peak, the 2# peak and gibberellin A3Compared with the 4# peak and gibberellin A3Has larger structural difference, so the impurities of the 1# peak and the 2# peak are easy to remove in the subsequent purification process, thereby the gibberellin A is not influenced3And the 4# peak is purified from gibberellin A3The structure difference of (2) is little, hardly gets rid of, so the 4# peak is the main impurity peak that influences follow-up extraction purity for the affirmation, and the content of 4# peak is few in this application, provides convenient for follow-up purification process.
Example 2
Method for producing gibberellin A by fermentation of riboflavin waste liquid3The method comprises the following steps:
(1) treatment of riboflavin waste liquor
Putting the riboflavin waste liquid into a fermentation tank, preserving heat for 30min to inactivate mycelium under the condition that the temperature is 65 ℃, then centrifuging for 10min under the condition of 8000rpm by a horizontal screw centrifuge to obtain an upper layer light phase containing the mycelium, and adjusting the pH of the upper layer light phase to 5.5 +/-0.2 by using a sodium hydroxide solution with the mass fraction of 20-30% for later use;
(2) fermentation production of gibberellin A3
a. Seed culture: the two-stage culture is carried out, and the culture solution of the two-stage seed culture comprises the following components: 20g/L of corn starch, 10g/L of cane sugar and 20g/L, KH of peanut powder2PO41g/L、MgSO42g/L, natural pH;
first-order seed culture: charging 250mL of culture medium into 1000mL of shake flask, and picking to produce gibberellin A3The single colony is filled into a shake flask and cultured for 60 hours under the conditions of 180rmp and 30 +/-1 ℃ to obtain first-class seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shake flask, inoculating the primary seed solution into a secondary seed tank according to the volume proportion of 0.5%, and culturing for 60h under the conditions of the temperature of 30 +/-1 ℃, the dissolved oxygen of 20-40% and the natural pH value to obtain a secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 80g/L of starch and 8g/L, KH of peanut powder2PO40.3g/L、K2SO40.3g/L、MgSO40.15g/L, 500g/L of riboflavin waste liquid obtained in the step (1);
inoculating the secondary seed solution into a fermentation medium in a 3t fermentation tank in a proportion of 10%, and culturing for 7 days at 30 + -1 deg.C, dissolved oxygen of 40-60%, and natural pH to obtain a fermentation liquid;
c. product detection
Adding 0.25mL fermentation liquid into 4.75mL distilled water, strongly shaking for 10min, centrifuging at 4000rpm for 10min, collecting supernatant, and performing high performance liquid chromatography under the conditions of high performance liquid chromatographyHypersil BDSC18 reversed phase chromatographic column (150 mm × 4.6.6 mm), methanol-water solution as mobile phase, methanol-water volume ratio of 40:60, flow rate of 0.80mL/min, ultraviolet wavelength of 230nm and injection volume of 20 μ L, and gibberellin A as detection result3The yield of (A) is: 2210mg/L, the same detection result as in example 1, no other impurity peak was observed, and therefore, no liquid chromatogram was attached.
Example 3
Method for producing gibberellin A by fermentation of riboflavin waste liquid3The method comprises the following steps:
(1) treatment of riboflavin waste liquor
Putting the riboflavin waste liquid into a fermentation tank, preserving heat for 60min to inactivate mycelium under the condition that the temperature is 75 ℃, then centrifuging for 30min under the condition of 5000rpm by a horizontal screw centrifuge to obtain an upper layer light phase containing the mycelium, and adjusting the pH of the upper layer light phase to 6.5 +/-0.2 by using a sodium hydroxide solution with the mass fraction of 30% for later use;
(2) fermentation production of gibberellin A3
a. Seed culture: the two-stage culture is carried out, and the culture solution of the two-stage seed culture comprises the following components: 30g/L of corn starch, 5g/L of cane sugar and 30g/L, KH of peanut powder2PO42g/L、MgSO41g/L, natural pH;
first-order seed culture: charging 250mL of culture medium into 1000mL of shake flask, and picking to produce gibberellin A3The single colony is filled into a shake flask and cultured for 40h under the conditions of 280rmp and 30 +/-1 ℃ to obtain first-class seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shake flask, inoculating the primary seed solution into a secondary seed tank according to the volume proportion of 1.0%, and culturing for 40h under the conditions of the temperature of 30 +/-1 ℃, dissolved oxygen of 50-70% and natural pH to obtain a secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 50g/L of starch and 5g/L, KH of peanut powder2PO40.2g/L、K2SO40.2g/L、MgSO40.1g/L, 600g/L of the riboflavin waste liquid obtained in the step (1);
inoculating the secondary seed solution into a fermentation medium in a 3t fermentation tank in a volume ratio of 10%, and culturing for 8 days at 30 + -1 deg.C, dissolved oxygen of 20-30%, and natural pH to obtain a fermentation liquid;
c. product detection
Adding 0.25mL of fermentation liquid into 4.75mL of distilled water, strongly shaking for 10min, centrifuging at 4000rpm for 10min, collecting the supernatant, and performing high performance liquid chromatography with a Hypersil BDSC18 reversed phase chromatographic column (150 mm × 4.6.6 mm) at a methanol-water solution flow rate of 40:60 and a flow rate of 0.80mL/min, an ultraviolet detector wavelength of 230nm and a sample injection volume of 20 μ L3The yield of (A) is: 2279mg/L, the liquid chromatography detection result of example 3 was the same as that of example 1, and no new impurity peak was observed, so that no liquid chromatogram was attached.
Comparative example
The existing production of gibberellin A3The method of (3) is different from the method of example 1 in that no riboflavin fermentation waste liquid is treated, and the fermentation culture solution comprises the following components: 160g/L of starch and 16g/L, KH of peanut powder2PO40.4g/L、K2SO40.4g/L、MgSO40.2g/L, natural pH, wherein the starch hydrolysis is controlled until the reducing sugar content in the fermentation medium is 1-2%. The chromatogram is shown in FIG. 3, gibberellin A3The integration results are shown in Table 3, giving gibberellin A3The yield of (A) is: 2123 mg/L.
TABLE 3 gibberellin integration results Table
| Peak # | Retention time | Area of | Height | Area% | Theoretical plate # |
| 1 | 4.737 | 93448 | 10441 | 8.136 | 6147.529 |
| 2 | 5.001 | 85528 | 8704 | 7.447 | 6032.989 |
| 3 | 6.064 | 911916 | 83291 | 79.398 | 7215.675 |
| 4 | 6.750 | 57646 | 5090 | 5.019 | 8090.448 |
| Total of | —— | 1148538 | 107525 | 100.000 | —— |
As is clear from tables 3 and 2, the relative peak area of the peak 3# in example 1 is similar to the relative peak area (%) of the peak 3# in the comparative example, indicating that gibberellin A obtained in the present application3Is close to that in the comparative example, and in addition, the 4# peak influences the subsequent extraction of gibberellin A3The relative peak area of the 4# peak in the application is far lower than that of the 4# peak in the comparison example, which indicates that the content of the impurities obtained in the application is less than that in the comparison example, and the gibberellin A obtained in the application is3The purity of the gibberellin A is better than that of the gibberellin A in the control example3And the peak area of the 3# peak of the present application is much higher than that of the 3# peak of the comparative example, so that gibberellin A in the present application3The yield of (A) was also higher than that in the comparative example, and it can be also demonstrated that the riboflavin waste liquid was used as gibberellin A3After fermentation culture of nutrient components, gibberellin A can be ensured3The yield and the purity of the riboflavin waste liquid are reduced, and the treatment cost of the riboflavin waste liquid is reduced.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (8)
1. Method for producing gibberellin A by fermentation of riboflavin waste liquid3The method is characterized by comprising the following preparation steps: and (4) fermentation culture, namely inoculating the seed liquid into a fermentation culture medium prepared from the treated riboflavin waste liquid and essential nutrients for fermentation.
2. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution3The method of (1), wherein the method of treating the riboflavin waste liquid is as follows：
Putting the riboflavin waste liquid into a fermentation tank, preserving the heat for 30-60min to inactivate mycelium under the condition that the temperature is 70 +/-5 ℃, centrifuging for 10-30min under the condition of 5000 plus-8000 rpm to obtain a light phase containing the mycelium, and adjusting the pH of the obtained light phase to 5.5-6.5 by using a pH regulator to obtain the treated riboflavin waste liquid.
3. The method for producing gibberellin A according to claim 2, wherein the fermentation of the riboflavin waste solution is performed3The method of (2), characterized by: the pH regulator is 20-30% sodium hydroxide solution.
4. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution3The method of (2), characterized by: the riboflavin waste liquid is waste liquid generated in an extraction stage in the process of producing riboflavin by fermentation.
5. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution3The method of (2), characterized by: the method comprises seed culture before fermentation culture, wherein the seed culture comprises two-stage culture:
first-order seed culture: picking the red-eared mold mutant strain into a shake flask filled with seed culture solution, and culturing for 40-60h under the conditions of 180-280rmp and 30 +/-1 ℃ to obtain first-level seed solution;
secondary seed culture: inoculating the first-stage seed solution to a second-stage seed tank at a volume ratio of 0.5-1.0%, and culturing at 30 + -1 deg.C, dissolved oxygen of 20-70%, and natural pH for 40-60h to obtain second-stage seed solution.
6. The method for producing gibberellin A according to claim 5, wherein the fermentation of the riboflavin waste solution is performed3The method of (2), characterized by: in the fermentation culture, the secondary seed liquid is inoculated in a fermentation culture medium according to the volume proportion of 5-10%, and cultured for 7-8 days under the conditions of 30 +/-1 ℃, dissolved oxygen of 20-60% and natural pH to obtain fermentation liquid.
7. According to claim 5The method for producing gibberellin A by fermenting riboflavin waste liquid3The method is characterized in that the culture solution of the two-stage culture comprises the following components: 20-30g/L of corn starch, 5-10g/L of cane sugar and 20-30g/L, KH of peanut powder2PO41-2g/L、MgSO41-2g/L。
8. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution3The method of (2), characterized by: the fermentation culture medium comprises 400-600g/L of riboflavin waste liquid, 50-120g/L of starch and 5-10g/L, KH of peanut powder2PO40.2-0.5g/L、K2SO40.2-0.5g/L、MgSO40.1-0.2g/L。
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