CN111690706B - Gibberellin A produced by fermentation of riboflavin waste liquid 3 Is a method of (2) - Google Patents
Gibberellin A produced by fermentation of riboflavin waste liquid 3 Is a method of (2) Download PDFInfo
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- CN111690706B CN111690706B CN202010751430.2A CN202010751430A CN111690706B CN 111690706 B CN111690706 B CN 111690706B CN 202010751430 A CN202010751430 A CN 202010751430A CN 111690706 B CN111690706 B CN 111690706B
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Abstract
The invention discloses a method for producing gibberellin A by fermenting riboflavin waste liquid 3 The key point of the technical scheme is fermentation culture, seed liquid is inoculated into fermentation culture medium prepared from treated riboflavin waste liquid and essential nutrient substances to ferment, and gibberellin A is produced by using the riboflavin waste liquid to ferment 3 Solves the problem of waste liquid treatment in the production process of the riboflavin, and simultaneously reduces gibberellin A 3 The production cost of the product.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing gibberellin A by fermenting riboflavin waste liquid 3 Is a method of (2).
Background
Gibberellin A 3 Is an endogenous plant regulating substance, has remarkable effects of regulating and controlling the stem extension, seed germination, bolting and fruit setting of plants and the like, and is widely applied to the field of planting and producing economic crops. Industrial gibberellin A 3 The common vine chamber gibberella liquid is produced by fermentation, the fermentation culture medium mainly comprises organic nitrogen and carbon sources such as corn starch, peanut cake powder and the like, and the production cost is high. How to optimize the culture medium and reduce the production cost is all gibberellin A 3 The problem that the manufacturing enterprises have to face.
Riboflavin, also known as vitamin B 2 Is a water-soluble vitamin necessary for human and animals, has remarkable effect on inhibiting digestive tract diseases and nervous system diseases (such as Parkinson's disease, migraine, multiple sclerosis, etc.), and is widely applied in the fields of clinical treatment, feed industry, cosmetic industry, food industry, etc. The riboflavin is mainly produced by a microbial fermentation method, a large amount of waste liquid is generated in the extraction stage, and along with the increasing of environmental protection requirements, how to treat the waste liquid can reach the discharge requirement is an urgent problem to be solved for riboflavin production enterprises. As the riboflavin waste liquid contains carbon and nitrogen sources, inorganic salts and residual mycelium which are not utilized in the process of partial fermentation, the riboflavin waste liquid has high viscosity and high treatment difficulty, and the production cost of the riboflavin is high.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for producing gibberellin A by fermenting riboflavin waste liquid 3 In the method, gibberellin A is produced by fermenting riboflavin waste liquid 3 Solves the problems in the production process of the riboflavinThe problem of waste liquid treatment is solved, and simultaneously, the gibberellin A is reduced 3 The production cost of the product.
The technical aim of the invention is realized by the following technical scheme:
gibberellin A produced by fermentation of riboflavin waste liquid 3 The method comprising the following preparation steps: fermenting and culturing, inoculating the seed solution into a fermentation medium prepared from the treated riboflavin waste liquid and essential nutrients for fermentation.
The method for treating the riboflavin waste liquid is characterized in that the riboflavin waste liquid is placed in a fermentation tank, the temperature is kept at 70+/-5 ℃ for 30-60min to inactivate mycelia, the mycelia are obtained by centrifugation for 10-30min under the condition of 5000-8000rpm, and the obtained light phase is taken to be adjusted to pH 5.5-6.5 by a pH regulator, so that the treated riboflavin waste liquid is obtained.
By adopting the technical scheme, the treated riboflavin waste liquid is used as gibberellin A 3 Not only is beneficial to reducing the treatment cost of the riboflavin waste liquid, but also can fully utilize the organic nitrogen source and the carbon source in the riboflavin waste liquid, and reduce the amount of other nutrient substances in the fermentation medium, thereby effectively reducing gibberellin A 3 Meanwhile, experiments in the application prove that when the application uses the riboflavin waste liquid as the component of the fermentation medium, gibberellin A is finally obtained 3 The yield of gibberellin A is higher than that of gibberellin A produced by normal fermentation 3 Is a yield of (2).
In addition, after the riboflavin waste liquid is treated, the step of adjusting the pH of the fermentation medium is not needed in the fermentation process, so that gibberellin fermentation can be carried out under the condition of natural pH when other components in the fermentation medium are added, and convenience is brought to operation.
The invention further provides that the pH regulator is a 20-30% sodium hydroxide solution.
Through adopting above-mentioned technical scheme, after this application pH regulator adopted sodium hydroxide solution, can reach the treatment to riboflavin waste liquid, also can not influence the nitrogen content in the riboflavin waste liquid simultaneously to provide the guarantee for later fermentation.
The invention further provides that the riboflavin waste liquid is waste liquid generated in the extraction stage in the process of producing the riboflavin by fermentation.
The invention further provides that the fermentation culture is preceded by a seed culture, the seed culture being divided into two stages:
primary seed culture: taking the gibberella cappa mutant strain into a shake flask filled with seed culture solution, and culturing for 40-60h at the temperature of 250rmp and 30+/-1 ℃ to obtain primary seed solution; the gibberella canescens mutant strainFusarium fujikuroi GA-347) Purchased from China center for type culture collection, wherein the mutant strain of the gibberella canis @Fusarium fujikuroi GA-347) Deposited in China center for type culture Collection, address: mail code 430072, deposit number of university of Wuhan in Wuhan, china: cctccc NO: m2019378, date of preservation 2019, 5, 20;
secondary seed culture: inoculating the first-stage seed solution into a second-stage seed tank at a volume ratio of 0.5-1.0%, and culturing at 30+ -1deg.C and dissolved oxygen of 20-70% under natural pH for 40-60 hr to obtain second-stage seed solution.
The invention further provides that in the fermentation culture, the secondary seed liquid is inoculated into a fermentation culture medium according to the volume ratio of 5-10%, and the fermentation liquid is obtained after the fermentation culture for 7-8 days under the conditions of 30+/-1 ℃ and dissolved oxygen of 20-60% and natural pH.
The invention further provides that the culture solution for the two-stage culture comprises the following components: corn starch 20-30g/L, sucrose 5-10g/L, peanut powder 20-30g/L, KH 2 PO 4 1-2g/L、MgSO 4 1-2g/L。
The invention is further arranged that the fermentation medium comprises 400-600g/L of riboflavin waste liquid, 50-120g/L of starch and 5-10g/L, KH of peanut powder 2 PO 4 0.2-0.5g/L、K 2 SO 4 0.2-0.5g/L、MgSO 4 0.1-0.2g/L。
In summary, the invention has the following beneficial effects:
1. the application uses the riboflavin waste liquid asProduction of gibberellin A 3 The main components of the fermentation 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 gibberellin A 3 After that, gibberellin A is also effectively reduced 3 Is not limited in the production cost;
2. as the residual sugar contained in the riboflavin waste liquid can meet the requirement of carbon source in the single colony early growth process, the hydrolysis steps of gibberellin A3 fermentation medium starch and rice flour are avoided, and gibberellin A is simplified 3 The production process of the (2) saves the cost;
3. the yield of gibberellin A3 in the present application is up to 2458mg/L, whereas gibberellin A is present in the prior art 3 The yield of (C) was 2123mg/L.
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FIG. 1 is a high performance liquid chromatogram of a standard sample;
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 by industrial production, wherein the riboflavin waste liquid is the riboflavin waste liquid obtained by about 1% of residual carbon content when the production of the riboflavin is terminated.
The concentration of the standard sample after 50-fold dilution was 65. Mu.g/mL.
The chromatogram of the standard sample is shown in figure 1, gibberellin A 3 The integration results of (2) are shown in Table 1.
TABLE 1 gibberellin score results table for standard culture
| Peak #) | Retention time | Area of | Height | Area percent | Theoretical plate #) |
| 1 | 6.033 | 1395930 | 126308 | 100.000 | 7017.251 |
| Totals to | —— | 1395930 | 126308 | 100.000 | —— |
The yield calculation formula of the comparative example is: control yield= (control peak area/standard peak area) x concentration after dilution of standard x dilution fold.
Since the ratio of peak area to yield is proportional, the ratio of peak area to yield is: the yield calculation formula for example 1 is: example yield= (example sample peak area/control peak area) ×control yield.
Example 1
Gibberellin A produced by fermentation of riboflavin waste liquid 3 Comprises the following steps:
(1) Treatment of riboflavin waste liquid
Placing the riboflavin waste liquid in a fermentation tank, preserving heat for 45min at 70 ℃ to inactivate mycelia, centrifuging for 20min by a horizontal screw centrifuge at 6500rpm to obtain an upper light phase containing mycelia, and taking the upper light phase to adjust the pH 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 A 3
a. Seed culture: the culture solution for two-stage seed culture comprises the following components: corn starch 25g/L, sucrose 7.5g/L, peanut flour 25g/L, KH 2 PO 4 1.5g/L、MgSO 4 1.5g/L, natural pH;
primary seed culture: 250mL of culture solution is filled in 1000mL of shaking flask, and gibberellin A is selected for production 3 Filling the gibberella cappa mutant strain into a shake flask, and culturing for 48 hours at the temperature of 250rmp and 30+/-1 ℃ to obtain primary seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shaking bottle, inoculating the primary seed solution into a secondary seed tank at a volume ratio of 1.0%, and culturing for 40h under the conditions of 30+/-1 ℃ and dissolved oxygen of 40-50% and natural pH to obtain secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 120g/L of starch and 10g/L, KH of peanut powder 2 PO 4 0.5g/L、K 2 SO 4 0.5g/L、MgSO 4 0.2g/L, 400g/L of riboflavin waste liquid obtained in the step (1), and natural pH;
inoculating the secondary seed solution into a fermentation medium according to a volume ratio of 5% in a 3t fermentation tank, and culturing for 8 days under the conditions of 30+/-1 ℃ and dissolved oxygen of 30-40% and natural pH to obtain a fermentation liquid;
c. product detection
Adding 4.75mL of distilled water into 0.25mL of fermentation liquor, shaking strongly for 10min, centrifuging at 4000rpm for 10min, and taking supernatant for high performance liquid chromatography under the following conditions: hypersil BDS C18 reversed phase chromatographic column (150 mm. Times.4.6 mm), mobile phase is methanol-water solution, volume ratio of methanol to water is 40:60, flow rate is 0.80mL/min; the wavelength of the ultraviolet detector is 230nm, and the sample injection volume is 20 mu L. The chromatogram is shown in figure2, gibberellin A 3 The integration results of (2) are shown in Table 2, gibberellin A is obtained 3 The yield of (2) is: 2458mg/L.
TABLE 1 gibberellin integration results Table
| Peak #) | Retention time | Area of | Height | Area percent | 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 |
| Totals to | —— | 1334742 | 124807 | 100.000 | —— |
The 3# peak in FIG. 2 is the gibberellin A as the target product 3 The 1# peak, the 2# peak and the 4# peak are all impurity peaks, but the 1# peak, the 2# peak and the gibberellin A are due to 3 Structural differences in comparison to peak 4# and gibberellin A 3 The structural difference of (2) is relatively large, so impurities of the No. 1 peak and the No. 2 peak are easy to remove in the subsequent purification process, thereby not affecting gibberellin A 3 And the 4# peak with gibberellin A 3 The structure difference of the (4) is small, and the removal is difficult, so that the 4# peak is considered as a main impurity peak affecting the subsequent extraction purity, and the content of the 4# peak is small in the method, so that convenience is provided for the subsequent purification process.
Example 2
Gibberellin A produced by fermentation of riboflavin waste liquid 3 Comprises the following steps:
(1) Treatment of riboflavin waste liquid
Placing the riboflavin waste liquid in a fermentation tank, preserving heat for 30min at 65 ℃ to inactivate mycelia, centrifuging for 10min by a horizontal screw centrifuge at 8000rpm to obtain an upper light phase containing mycelia, and adjusting the pH of the upper 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 A 3
a. Seed culture: the culture solution for two-stage seed culture comprises the following components: corn starch 20g/L, sucrose 10g/L, peanut powder 20g/L, KH 2 PO 4 1g/L、MgSO 4 2g/L, natural pH;
primary seed culture: 250mL of culture solution is filled in 1000mL of shaking flask, and gibberellin A is selected for production 3 The single colony of the seed is put into a shake flask and cultured for 60 hours under the conditions of 180rmp and 30+/-1 ℃ to obtain primary seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shaking bottle, inoculating the primary seed solution into a secondary seed tank according to the volume ratio of 0.5%, and culturing for 60h under the conditions of the temperature of 30+/-1 ℃ and the dissolved oxygen of 20-40% and the natural pH value to obtain secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 80g/L starch, 8g/L, KH peanut powder 2 PO 4 0.3g/L、K 2 SO 4 0.3g/L、MgSO 4 0.15g/L, 500g/L of riboflavin waste liquid obtained in the step (1);
inoculating the secondary seed solution into a fermentation medium according to the proportion of 10% in a 3t fermentation tank, and culturing for 7 days under the conditions of 30+/-1 ℃ and dissolved oxygen of 40-60% and natural pH to obtain a fermentation liquid;
c. product detection
Adding 4.75mL of distilled water into 0.25mL of fermentation liquor, shaking strongly for 10min, centrifuging at 4000rpm for 10min, and taking supernatant for high performance liquid chromatography under the following conditions: hypersil BDS C18 reversed phase chromatographic column (150 mm. Times.4.6 mm), mobile phase is methanol-water solution, volume ratio of methanol to water is 40:60, flow rate is 0.80mL/min; the wavelength of the ultraviolet detector is 230nm, and the sample injection volume is 20 mu L. The detection results are as follows: gibberellin A 3 The yield of (2) is: 2210mg/L, the detection result was the same as in example 1, and no other impurity peaks appeared, so that no liquid chromatogram was attached.
Example 3
Gibberellin A produced by fermentation of riboflavin waste liquid 3 Comprises the following steps:
(1) Treatment of riboflavin waste liquid
Placing the riboflavin waste liquid in a fermentation tank, preserving heat for 60min at 75 ℃ to inactivate mycelia, centrifuging for 30min by a horizontal screw centrifuge at 5000rpm to obtain an upper light phase containing mycelia, and adjusting the pH of the upper 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 A 3
a. Seed culture: the culture solution for two-stage seed culture comprises the following components: 30g/L corn starch, 5g/L sucrose and 30g/L, KH peanut powder 2 PO 4 2g/L、MgSO 4 1g/L, natural pH;
primary seed culture: 250mL of culture solution is filled in 1000mL of shaking flask, and gibberellin A is selected for production 3 The single colony of the seed is put into a shake flask and cultured for 40 hours under the conditions of 280rmp and 30+/-1 ℃ to obtain primary seed liquid;
secondary seed culture: filling 100L of culture solution into a 250L shaking bottle, inoculating the primary seed solution into a secondary seed tank according to the volume ratio of 1.0%, and culturing for 40h under the conditions of the temperature of 30+/-1 ℃ and the dissolved oxygen of 50-70% and the natural pH value to obtain secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: starch 50g/L, peanut powder 5g/L, KH 2 PO 4 0.2g/L、K 2 SO 4 0.2g/L、MgSO 4 0.1g/L of riboflavin waste liquid obtained in the step (1) 600g/L;
inoculating the secondary seed solution into a fermentation medium according to the volume ratio of 10% in a 3t fermentation tank, and culturing for 8 days under the conditions of 30+/-1 ℃ and dissolved oxygen of 20-30% and natural pH to obtain a fermentation liquid;
c. product detection
Adding 4.75mL distilled water into 0.25mL fermentation broth, shaking strongly for 10min, centrifuging at 4000rpm for 10min, collecting supernatantThe liquid is subjected to high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: hypersil BDS C18 reversed phase chromatographic column (150 mm. Times.4.6 mm), mobile phase is methanol-water solution, volume ratio of methanol to water is 40:60, flow rate is 0.80mL/min; the wavelength of the ultraviolet detector is 230nm, and the sample injection volume is 20 mu L. The detection results are as follows: gibberellin A 3 The yield of (2) is: 2279mg/L, the liquid chromatography test result of example 3 was the same as that of example 1, except that no new impurity peak was present, so that no liquid chromatography was attached.
Comparative example
Present gibberellin A production 3 The process of (2) differs from example 1 in that there is no treatment of riboflavin-free fermentation broth consisting of: 160g/L of starch and 16g/L, KH of peanut powder 2 PO 4 0.4g/L、K 2 SO 4 0.4g/L、MgSO 4 0.2g/L, natural pH, wherein the starch is controlled to hydrolyze to 1-2% of the reducing sugar content in the fermentation medium. The chromatogram is shown in FIG. 3, gibberellin A 3 The integration results of (3) are shown in Table 3, gibberellin A is obtained 3 The yield of (2) is: 2123mg/L.
TABLE 3 gibberellin integration results Table
| Peak #) | Retention time | Area of | Height | Area percent | 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 |
| Totals to | —— | 1148538 | 107525 | 100.000 | —— |
As can be seen from tables 3 and 2, the relative peak area of the 3# peak in example 1 and the relative peak area of the 3# peak in the comparative exampleThe areas (%) are similar, and illustrate gibberellin A obtained by the present application 3 The relative percentage of (2) is close to that of the comparative example, and in addition, the 4# peak is used for influencing the subsequent extraction of gibberellin A 3 The relative peak area of the main impurity peak of purity, which is far lower than that of the 4# peak in the comparative example, shows that the impurity content obtained in the present application is smaller than that in the comparative example, and the gibberellin A obtained in the present application 3 Is superior to gibberellin A in the comparative example in purity 3 And the peak area of the No. 3 peak of the present application is much higher than that of the No. 3 peak of the comparative example, so that gibberellin A in the present application 3 The yield of (C) was also higher than that in the comparative example, and thus it can be said that riboflavin waste was used as gibberellin A 3 After fermenting and culturing the nutrient components, not only gibberellin A can be ensured 3 And simultaneously reduces the treatment cost of the riboflavin waste liquid.
The embodiments of the present invention are all preferred embodiments of the present invention, and are not intended to limit the scope of the present invention in this way, therefore: all equivalent changes in structure, shape and principle of the invention should be covered in the scope of protection of the invention.
Claims (1)
1. Gibberellin A produced by fermentation of riboflavin waste liquid 3 Characterized in that it comprises the following preparation steps:
(1) Treatment of riboflavin waste liquid
Placing the riboflavin waste liquid in a fermentation tank, preserving heat for 45min at 70 ℃ to inactivate mycelia, centrifuging for 20min by a horizontal screw centrifuge at 6500rpm to obtain an upper light phase containing mycelia, and taking the upper light phase to adjust the pH 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 A 3
a. Seed culture: the culture solution for two-stage seed culture comprises the following components: corn starch 25g/L, sucrose 7.5g/L, peanut flour 25g/L, KH 2 PO 4 1.5g/L、MgSO 4 1.5g/L, self-primingThe pH is then determined;
primary seed culture: 250mL of culture solution is filled in 1000mL of shaking flask, and gibberellin A is selected for production 3 The gibberella cappa mutant Fusarium fujikuroi GA-347 is put into a shake flask and cultured for 48 hours under the conditions of 250rmp and 30+/-1 ℃ to obtain primary seed liquid; wherein the gibberella canescens mutant Fusarium fujikuroi GA-347 is purchased from the China center for type culture collection, accession number: cctccc NO: m2019378;
secondary seed culture: filling 100L of culture solution into a 250L shaking bottle, inoculating the primary seed solution into a secondary seed tank at a volume ratio of 1.0%, and culturing for 40h under the conditions of 30+/-1 ℃ and dissolved oxygen of 40-50% and natural pH to obtain secondary seed solution;
b. fermentation culture: the fermentation medium comprises the following components: 120g/L of starch and 10g/L, KH of peanut powder 2 PO 4 0.5g/L、K 2 SO 4 0.5g/L、MgSO 4 0.2g/L, 400g/L of riboflavin waste liquid obtained in the step (1), and natural pH;
inoculating the secondary seed solution into a fermentation medium at a volume ratio of 5% in a 3t fermentation tank, and culturing for 8 days under the conditions of 30+/-1 ℃ and dissolved oxygen of 30-40% and natural pH to obtain a fermentation liquid.
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| 藤仓赤霉菌赤霉素生物合成及代谢调控研究进展;王冰璇等;生物工程学报;第36卷(第2期);第189-200页 * |
| 赤霉素978菌株产赤GA3发酵的影响探讨;雷文等;农业与技术;第35卷(第2期);第2页 * |
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