CN110295205B - Application of gluconate in improving co-production of polymalic acid and heavy oil by aureobasidium pullulans - Google Patents
Application of gluconate in improving co-production of polymalic acid and heavy oil by aureobasidium pullulans Download PDFInfo
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Abstract
The invention relates to an application of gluconate in improving aureobasidium pullulans to co-produce polymalic acid and heavy oil, belongs to the technical field of fermentation, and particularly relates to a method for improving the aureobasidium pullulans to co-produce polymalic acid and heavy oil by using gluconate, wherein the method specifically comprises the following steps: firstly, inoculating aureobasidium pullulans to a seed culture medium, and performing shake culture to obtain an activated seed solution; then inoculating the obtained activated seed liquid into a fermentation culture medium containing 10-50g/L of gluconate for fermentation culture. In the method, the carbon metabolic flow and the cofactor flow direction of the polymalic acid and heavy oil synthesis are adjusted by adding the gluconate, so that the synthesis of two metabolites is improved. Furthermore, the addition of gluconate in the amount defined in the present invention does not inhibit the growth of aureobasidium pullulans cells. The method has the advantages of low fermentation cost, high yield, strong technical economy and the like, and can be applied to industrial actual amplification production of two metabolites of polymalic acid and heavy oil.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to application of gluconate in improving aureobasidium pullulans and co-producing polymalic acid and heavy oil.
Background
Aureobasidium pullulans (Aureobasidium pullulans) is a kind of yeast fungi, which has both mycelial and yeast-like forms. The strain exists widely in nature. Aureobasidium pullulans can be fermented to generate various metabolites such as Polymalic acid, pullulan polysaccharide, heavy oil, melanin and the like, wherein Polymalic acid (PMA) is a water-soluble aliphatic polyester compound, and a novel polyester compound formed by intermolecular esterification of a monomer malic acid with a repeating structural unit has the properties of excellent biodegradability, high water solubility, biocompatibility and the like; heavy oil is also an extracellular oil produced by the metabolism of aureobasidium pullulans, is called Heavy oil (Heavy oils) because of its large specific gravity and is located at the lower part of the fermentation broth, and research results show that the Heavy oil has various physiological activities such as anti-tumor.
Generally, polymalic acid and heavy oil belong to metabolites of two different synthetic pathways, and the existing patents report more on the improvement of the synthesis of a single metabolite. Therefore, a method for effectively increasing the yield of two metabolites, polymalic acid and heavy oil, is urgently needed.
Disclosure of Invention
In view of this, the present invention aims to provide an application of gluconate in improving co-production of polymalic acid and heavy oil by aureobasidium pullulans and a method for improving co-production of polymalic acid and heavy oil by aureobasidium pullulans by using gluconate, in view of the defects in the existing fermentation production process technology of aureobasidium pullulans polymalic acid and heavy oil, and by combining a biosynthesis pathway of polymalic acid and heavy oil.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the application of gluconate in improving the co-production of polymalic acid and heavy oil by Aureobasidium pullulans is disclosed.
2. A method for improving co-production of polymalic acid and heavy oil by Aureobasidium pullulans through gluconate comprises the following steps:
(1) Seed culture
Inoculating aureobasidium pullulans to a seed culture medium, and performing shake culture to obtain an activated seed solution;
(2) Fermentation culture
Inoculating the activated seed liquid obtained in the step (1) into a fermentation culture medium containing 10-50g/L of gluconate for fermentation culture.
Preferably, in step (1), the Aureobasidium pullulans is inoculated into the seed culture medium in an inoculum size of 5 to 20% v/v.
Preferably, in the step (1), the seed culture medium comprises 40-100g/L of glucose, 1-5g/L of ammonium nitrate and KH 2 PO 4 0.005-0.3g/L、ZnSO 4 0.005-0.3g/L、MgSO 4 0.005-0.3g/L, 0.05-0.3g/L corn steep liquor and CaCO 3 10-50g/L。
Preferably, in the step (1), the shake culture specifically comprises: inoculating the aureobasidium pullulans into a shake flask filled with the seed culture medium, and performing shake culture for 36-96h under the conditions that the temperature is 23-30 ℃ and the rotating speed of a shaking table is 180-300 rpm.
Preferably, in the step (2), the gluconate is at least one of sodium gluconate, potassium gluconate, calcium gluconate and zinc gluconate.
Preferably, in the step (2), the activated seed solution obtained in the step (1) is inoculated into a fermentation medium containing 10-50g/L of gluconate according to the inoculation amount of 5-20% v/v.
Preferably, in the step (2), the fermentation medium comprises 50-200g/L of sugar, 1-10g/L of nitrogen source and KH 2 PO 4 0.005-0.3g/L、ZnSO 4 0.005-0.3g/L、MgSO 4 0.005-0.3g/L, 0.05-3g/L corn steep liquor and CaCO 3 10-50g/L。
Preferably, the sugar is, but not limited to, at least one of xylose, arabinose, sucrose, fructose, or glucose.
Preferably, the nitrogen source is, but not limited to, one of ammonium sulfate, ammonium chloride, potassium nitrate or sodium nitrate.
Preferably, in step (2), the fermentation culture is one of a shake flask fermentation culture or a fermenter fermentation culture.
Preferably, the shake flask fermentation culture is specifically as follows: inoculating the activated seed solution into a shake flask filled with the fermentation culture medium, and culturing for 96-150h at the temperature of 23-30 ℃ and the rotation speed of a shaking table of 180-300 rpm; the fermentation culture of the fermentation tank specifically comprises the following steps: inoculating the activated seed solution into a fermentation tank filled with the fermentation culture medium, and culturing for 60-200h under the conditions that the temperature is 23-30 ℃, the rotating speed is 300-1000rpm, and the aeration ratio is 1.8-1.
The invention has the beneficial effects that: the invention provides application of gluconate in improving co-production of polymalic acid and heavy oil by aureobasidium pullulans.in the method for co-production of the polymalic acid and the heavy oil by using the aureobasidium pullulans.the gluconate is added to increase the content of an intermediate Xylulose-5-phosphate (Xylulose-5-P) and the supply of a cofactor NADPH in an HMP pathway, and further increase the flow of a common precursor Acetyl coenzyme A (Acetyl-CoA) synthesized by the polymalic acid and the heavy oil, so that a directional regulation strategy with simple operation is established, carbon metabolic flow and cofactor flow regulation of the synthesis of the polymalic acid and the heavy oil are realized, and the synthesis of two metabolites is improved. Furthermore, the addition of gluconate in the amount defined in the present invention does not inhibit the growth of Aureobasidium pullulans cells. The method has the advantages of low fermentation cost, high yield, strong technical economy and the like, and can be applied to industrial actual scale-up production of two metabolites of polymalic acid and heavy oil.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Detailed Description
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
(1) Seed culture
Inoculating aureobasidium pullulans (A. Pullulans CCTCC M2012223) into a 500mL shake flask filled with 50mL of seed culture medium according to the inoculation amount of 10% v/v, and performing shake culture for 48h under the conditions that the temperature is 25 ℃ and the rotating speed of a shaking table is 220rpm to obtain activated seed liquid, wherein the seed culture medium comprises 60g/L of glucose, 3g/L of ammonium nitrate and KH 2 PO 4 0.2g/L、ZnSO 4 0.1g/L、MgSO 4 0.15g/L, 2g/L corn steep liquor and CaCO 3 20g/L；
(2) Fermentation culture
Inoculating the activated seed solution obtained in the step (1) into a 500mL shake flask containing 50mL fermentation medium containing 10g/L sodium gluconate according to the inoculation amount of 10% v/v, and culturing at 25 ℃ and 220rpm of a shaking table for 120 hours, wherein the fermentation medium comprises 90g/L xylose mother liquor (58.5 g/L xylose, 19.8g/L arabinose, 11.7g/L glucose), 3g/L ammonium sulfate and KH 2 PO 4 0.2g/L、ZnSO 4 0.15g/L、MgSO 4 0.2g/L, 1g/L corn steep liquor and CaCO 3 20g/L。
Comparative example 1
The fermentation medium was not supplemented with 10g/L sodium gluconate, and the remaining steps and conditions were the same as in example 1.
Polymalic acid yield, heavy oil yield and cell growth conditions
The yield of the polymalic acid in example 1 is 46.8g/L, which is 5.2% higher than that in comparative example 1; the heavy oil yield in example 1 is 16.2g/L, which is improved by 20.9 percent compared with that in comparative example 1; the amount of Aureobasidium pullulans cells in example 1 was 16.8g/L, which is similar to that in comparative example 1, and indicates that there was no tendency for the cells to grow downward in example 1.
Example 2
(1) Seed culture
Inoculating aureobasidium pullulans (A.pullulans CCTCC M2012223) into a 500mL shake flask filled with 50mL seed culture medium according to the inoculation amount of 15% v/v, and performing shake culture for 60h under the conditions that the temperature is 30 ℃ and the rotation speed of a shaking table is 300rpm to obtain activated seed liquid, wherein the seed culture medium comprises 80g/L of glucose, 5g/L of ammonium nitrate and KH 2 PO 4 0.15g/L、ZnSO 4 0.3g/L、MgSO 4 0.3g/L, 3g/L corn steep liquor and CaCO 3 40g/L；
(2) Fermentation culture
Inoculating the activated seed solution obtained in the step (1) into a 500mL shake flask containing 40mL fermentation medium containing 20g/L potassium gluconate according to the inoculation amount of 15% v/v, and culturing at 30 ℃ and 300rpm of a shaking table for 150h, wherein the fermentation medium comprises 120g/L xylose mother liquor (60 g/L xylose, 45g/L arabinose, 15g/L glucose), 6g/L ammonium chloride and KH 2 PO 4 0.15g/L、ZnSO 4 0.3g/L、MgSO 4 0.3g/L, 3g/L corn steep liquor and CaCO 3 40g/L。
Comparative example 2
The fermentation medium was supplemented with no 20g/L potassium gluconate, and the procedure and conditions were the same as in example 2.
Polymalic acid yield, heavy oil yield and cell growth conditions
The yield of the polymalic acid in example 2 is 48.9g/L, which is improved by 9.9% compared with that in comparative example 2; the heavy oil yield in example 2 is 17.4g/L, which is improved by 29.8 percent compared with that in comparative example 2; the amount of Aureobasidium pullulans cells in example 2 was 17.7g/L, which is similar to that in comparative example 2, and indicates that there was no tendency of the cells in example 2 to grow downward.
Example 3
(1) Seed culture
Inoculating aureobasidium pullulans (A. Pullulans CCTCC M2012223) into a 500mL shake flask filled with 50mL of seed culture medium according to the inoculation amount of 20% v/v, and performing shake culture for 72h under the conditions that the temperature is 25 ℃ and the rotating speed of a shaking table is 250rpm to obtain activated seed liquid, wherein the seed culture medium comprises 80g/L of glucose, 3g/L of ammonium nitrate and KH 2 PO 4 0.25g/L、ZnSO 4 0.15g/L、MgSO 4 0.2g/L, 0.1g/L corn steep liquor and CaCO 3 30g/L；
(2) Fermentation culture
Inoculating the activated seed liquid obtained in the step (1) into a 500mL shake flask filled with 40mL fermentation medium containing 25g/L calcium gluconate according to the inoculation amount of 20% v/v, and culturing at 23 ℃ and 180rpm of a shaking table for 96h, wherein the fermentation medium comprises 200g/L xylose mother liquor (80 g/L sucrose, 50g/L fructose, 70g/L glucose), 8g/L ammonium chloride and KH 2 PO 4 0.3g/L、ZnSO 4 0.2g/L、MgSO 4 0.1g/L, 0.2g/L corn steep liquor and CaCO 3 30g/L。
Comparative example 3
The fermentation medium was not supplemented with 25g/L calcium gluconate, and the remaining steps and conditions were the same as in example 3.
Polymalic acid yield, heavy oil yield and cell growth
The yield of the polymalic acid in example 3 is 50.9g/L, which is 11.4% higher than that in comparative example 3; the heavy oil yield in example 3 is 16.5g/L, which is improved by 24.7 percent compared with that in comparative example 3; the amount of aureobasidium pullulans cells in example 3 was 17.5g/L, which is similar to that in comparative example 3, and indicates that there was no tendency for the cells to grow downward in example 3.
Example 4
(1) Seed culture
Inoculating aureobasidium pullulans (A.pullulans CCTCC M2012223) into a 500mL shake flask filled with 40mL seed culture medium according to the inoculation amount of 5% v/v, and performing shake culture for 96h under the conditions that the temperature is 27 ℃ and the rotation speed of a shaking table is 180rpm to obtain activated seed liquid, wherein the seed culture medium comprises 40g/L of glucose, 1g/L of ammonium nitrate and KH 2 PO 4 0.3g/L、ZnSO 4 0.005g/L、MgSO 4 0.3g/L, 0.05g/L corn steep liquor and CaCO 3 20g/L；
(2) Fermentation culture
Inoculating the activated seed solution obtained in the step (1) into a 5L fermentation tank filled with 3L fermentation medium containing 30g/L sodium gluconate according to the inoculation amount of 20% v/v, and culturing for 168h under the conditions that the temperature is 25 ℃, the rotating speed is 400rpm, and the aeration ratio is 1.3, wherein the fermentation medium comprises 124g/L of xylose mother liquor (65 g/L of xylose, 32g/L of arabinose, 27g/L of glucose), 3g/L of potassium nitrate and KH 2 PO 4 0.2g/L、ZnSO 4 0.15g/L、MgSO 4 0.2g/L, 1g/L corn steep liquor and CaCO 3 20g/L。
Comparative example 4
30g/L sodium gluconate was not added to the fermentation medium, and the remaining steps and conditions were the same as in example 4.
Polymalic acid yield, heavy oil yield and cell growth conditions
The yield of the polymalic acid in example 4 is 82.8g/L, which is 11.4% higher than that of comparative example 4; the heavy oil yield in example 4 is 28.3g/L, which is improved by 34.6 percent compared with that in comparative example 4; the amount of Aureobasidium pullulans cells in example 4 was 20.7g/L, which is similar to that in comparative example 4, and indicates that there was no tendency for the cells to grow downward in example 4.
Example 5
(1) Seed culture
Inoculating aureobasidium pullulans (A. Pullulans CCTCC M2012223) into a 500mL shake flask filled with 60mL of seed culture medium according to the inoculation amount of 10% v/v, and performing shake culture for 36h under the conditions that the temperature is 23 ℃ and the rotating speed of a shaking table is 240rpm to obtain activated seed liquid, wherein the seed culture medium comprises 100g/L glucose, 3g/L ammonium nitrate and KH 2 PO 4 0.005g/L、ZnSO 4 0.2g/L、MgSO 4 0.005g/L, 1g/L corn steep liquor and CaCO 3 10g/L；
(2) Fermentation culture
Inoculating the activated seed solution obtained in the step (1) into a 5L fermentation tank filled with 2.5L of fermentation medium containing 50g/L of zinc gluconate according to the inoculation amount of 10% v/v, and culturing for 120h under the conditions that the temperature is 30 ℃, the rotating speed is 800rpm, and the aeration ratio is 1 2 PO 4 0.3g/L、ZnSO 4 0.3g/L、MgSO 4 0.1g/L, 3g/L corn steep liquor and CaCO 3 40g/L。
Comparative example 5
The fermentation medium was not supplemented with 50g/L zinc gluconate, and the remaining steps and conditions were the same as in example 5.
Polymalic acid yield, heavy oil yield and cell growth
The yield of the polymalic acid in example 5 is 88.5g/L, which is improved by 14.8 percent compared with that in comparative example 5; the heavy oil yield in example 5 is 30.7g/L, which is improved by 38.6 percent compared with that in comparative example 5; the amount of Aureobasidium pullulans cells in example 5 was 20.2g/L, which is similar to that in comparative example 5, and indicates that there was no tendency for the cells to grow in example 5.
The source of the Aureobasidium pullulans in the invention can also be Aureobasidium strains preserved or naturally separated by mechanisms such as American Ministry of agriculture culture Collection (NRRL), american Type Culture Collection (ATCC), china general microbiological culture Collection center (CGMCC) and the like.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. The application of the gluconate in improving the yield of aureobasidium pullulans co-production polymalic acid and heavy oil.
2. The method for improving the yield of co-production of polymalic acid and heavy oil of Aureobasidium pullulans by using gluconate is characterized by comprising the following steps of:
(1) Seed culture
Inoculating aureobasidium pullulans to a seed culture medium, and performing shake culture to obtain an activated seed solution;
(2) Fermentation culture
Inoculating the activated seed liquid obtained in the step (1) into a fermentation culture medium containing 10-50g/L of gluconate for fermentation culture.
3. The method according to claim 2, wherein in the step (1), the aureobasidium pullulans is inoculated into a seed medium in an inoculum size of 5 to 20% v/v.
4. The method of claim 2, wherein in step (1), the seed culture medium comprises 40-100g/L glucose, 1-5g/L ammonium nitrate, KH 2 PO 4 0.005-0.3g/L、ZnSO 4 0.005-0.3g/L、MgSO 4 0.005-0.3g/L, 0.05-0.3g/L corn steep liquor and CaCO 3 10-50g/L。
5. The method according to claim 2, wherein in step (1), the shake culture is specifically: inoculating the aureobasidium pullulans into a shake flask filled with the seed culture medium, and performing shake culture for 36-96h under the conditions that the temperature is 23-30 ℃ and the rotating speed of a shaking table is 180-300 rpm.
6. The method of claim 2, wherein in step (2), the gluconate salt is at least one of sodium gluconate, potassium gluconate, calcium gluconate, and zinc gluconate.
7. The method of claim 2, wherein in step (2), the activated seed solution obtained in step (1) is inoculated into a fermentation medium containing 10-50g/L of gluconate in an amount of 5-20% v/v.
8. The method of claim 2, wherein in step (2), the fermentation medium comprises 50-200g/L of sugar, 1-10g/L of nitrogen source, KH 2 PO 4 0.005-0.3g/L、ZnSO 4 0.005-0.3g/L、MgSO 4 0.005-0.3g/L, 0.05-3g/L corn steep liquor and CaCO 3 10-50g/L。
9. The method of claim 2, wherein in step (2), the fermentation culture is one of a shake flask fermentation culture or a fermentor fermentation culture.
10. The method according to claim 9, wherein the shake flask fermentation culture is in particular: inoculating the activated seed solution into a shake flask filled with the fermentation culture medium, and culturing for 96-150h at the temperature of 23-30 ℃ and the rotation speed of a shaking table of 180-300 rpm; the fermentation culture of the fermentation tank specifically comprises the following steps: inoculating the activated seed solution into a fermentation tank filled with the fermentation culture medium, and culturing for 60-200h under the conditions that the temperature is 23-30 ℃, the rotation speed is 300-1000rpm, and the aeration ratio is 1.
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