Method for producing carotenoid

Abstract

The invention provides a method for producing carotenoid. The method for producing the carotenoid from the yeast comprises the following steps: performing fermentation culture on a yeast strain, adding D-galactose for induction when the growth of thalli enters a stagnation period, and after the induction is finished, jointly adjusting the pH value of a system in the fermentation process to 5.6-6.2 by using citric acid-ammonia water in a product synthesis stage, wherein the supplementing amount of a nitrogen source is 1.0-2.5 g / L based on the total nitrogen amount. According to the method provided by the invention, the synthesis efficiency of the carotenoid can be effectively improved, and the overall content of the carotenoid in dry thalli can be obviously improved. On the basis, the method provided by the invention can further improve the proportion content of astaxanthin in the product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for producing carotenoids. Background Technology

[0002] Carotenoids belong to the terpenoid family of compounds and are commonly found in yellow, orange-red, or red pigments in animals, higher plants, fungi, and algae. They mainly contain carotene and lutein. Carotene includes β-carotene, γ-carotene, and lycopene, while lutein includes astaxanthin, cryptoxanthin, zeaxanthin, and capsanthin. Carotenoids have significant antioxidant functions, effectively inhibiting free radical activity and reducing damage to cellular genetic material and cell membranes. They also enhance the body's immune defense capabilities, delay aging, and prevent other chronic diseases. Astaxanthin, in particular, is a chain-breaking antioxidant with extremely strong antioxidant capacity. It has multiple physiological effects, including inhibiting tumor development, enhancing immunity, and scavenging free radicals in the body. It also shows good therapeutic effects on skin cancer caused by ultraviolet radiation.

[0003] Astaxanthin can be obtained through chemical synthesis, biological extraction, or production using algae, bacteria, yeast, etc. Current research focuses on producing astaxanthin-containing carotenoids through fermentation of organisms. However, the low unit content of astaxanthin in biological fermentation currently limits the overall yield, which is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0004] Initially, the applicant did not pay attention to pH changes and their impact on the fermentation of yeast for carotenoid production. In subsequent research, the applicant noticed that the pH of the system decreased when the fermentation broth was discharged. Therefore, the applicant promptly adjusted the pH to around 5.8 using conventional sodium hydroxide in the later stages of fermentation. Further research into various process parameters revealed that the pH of the fermentation broth tended to increase in the early stages of product synthesis. Based on this, the present invention developed a combined citric acid-ammonia adjustment scheme. The conventional understanding in the prior art is to use ammonia to adjust the pH of the fermentation system, but this usually has a significant impact on biomass. Furthermore, as an inorganic nitrogen source, it negatively affects product yield and composition. It is usually used in combination with other nitrogen sources, including amino acids. However, this invention found that using a combination of citric acid and ammonia to adjust the pH of the system did not significantly affect the yield; on the contrary, it significantly increased the overall content of carotenoids in the dry cells.

[0005] The present invention provides a method for producing carotenoids from yeast, comprising the following steps:

[0006] Yeast strains were fermented and cultured. When the cell growth entered the stasis phase, D-galactose was added for induction. After induction, citric acid-ammonia water was used to maintain the pH of the fermentation system at 5.6-6.2 during the product synthesis stage. Ammonia water can also be used as a nitrogen source supplement, with the amount of nitrogen supplemented being 2.0-3.8 g / L.

[0007] In a preferred embodiment of the present invention, the method further includes the following step: adding a feed carbon source during the product synthesis stage. The total amount of the feed carbon source added is preferably 100-300 g / L.

[0008] In the fermentation process of engineered yeast that produces carotenoids, most commonly used natural carbon sources, such as glucose, are recognized by the promoters of carotenoid metabolic pathways, which hinders the synthesis of carotenoid products. Therefore, ethanol is usually used as a feed during the product synthesis stage. However, due to its molecular structure, ethanol has a certain toxic effect on cells and is difficult to use as a carbon source for a long time at a high concentration. At the same time, its utilization efficiency as a carbon source is low, and it is difficult to participate in pathways such as glycolysis and the tricarboxylic acid cycle. As a result, some key intermediate products and energy substances in the metabolic pathway cannot be replenished, making it difficult to increase the yield of carotenoids and also making it difficult to increase the content of products such as astaxanthin at the end of metabolism.

[0009] This invention discovers two more effective optimization schemes during the feeding process: one is to directly use fructose as the feeding carbon source. This invention found that using fructose does not affect the metabolic pathways of carotenoids and has high utilization, effectively increasing the proportion of astaxanthin in the product. The other scheme is to use a mixture of glucose and ethanol as the feeding carbon source, controlling the glucose content and supplementation amount to partially replace ethanol. This avoids glucose's obstruction of metabolic pathways and compensates for the shortcomings of ethanol as a carbon source, also effectively increasing the proportion of astaxanthin in the product. In a more preferred embodiment of this invention, the mass ratio of ethanol to glucose is (1.5-3.5):1.

[0010] Meanwhile, during the process of optimizing the carbon source for feeding, the applicant found that after using monosaccharides (such as glucose, fructose, etc.) to partially or completely replace ethanol, the impact on the pH of the fermentation broth was reduced. However, when the amount of citric acid-ammonia water introduced into the fermentation broth was kept within the aforementioned range, it could not only significantly increase the overall content of carotenoids in the dry cells, but also be more conducive to increasing the yield of astaxanthin.

[0011] During bacterial culture, carbon sources are sometimes added as feed during the normal growth phase of the bacteria. However, it is important to emphasize that the feed carbon sources containing glucose and ethanol or fructose, as described in this invention, are preferably added during the product synthesis stage.

[0012] In a preferred embodiment of the present invention, the total amount of the supplemental carbon source added is 100-300 g / L based on the volume of the culture system. In a specific embodiment of the present invention, the supplemental carbon source can be added either in a single addition or by a fed-batch method. When a fed-batch method is used, the addition rate can be 5-20 mL / h.

[0013] In a preferred embodiment of the present invention, during the cell growth stage, when the nitrogen source content (here, nitrogen source content refers to total nitrogen) in the fermentation medium is below 1 g / L, nitrogen source is added. The amount of nitrogen source added to the fermentation culture system is 2.5-6 g / L, based on total nitrogen. In specific embodiments of the present invention, the choice of nitrogen source for supplementation during the growth stage is not overly restricted. For example, ammonia water can be selected, or other nitrogen sources can be added or directly selected. Other nitrogen source raw materials can be nitrogen sources commonly used in the art. In the present invention, other nitrogen sources preferably also include yeast powder.

[0014] In a preferred embodiment of the present invention, citric acid and ammonia are used to adjust the pH of the system to 5.6-6.2 during the entire fermentation process.

[0015] In a specific embodiment of the present invention, the yeast strain can be a *Saccharomyces cerevisiae* strain capable of producing carotenoids. In this invention, the yeast strain is fermented and cultured using any fermentation medium, as long as the growth requirements of the yeast strain are met. The type of fermentation medium does not affect the effect brought about by the core inventive point of the present invention. In a specific embodiment of the present invention, the initial nitrogen content in the fermentation medium can be maintained at 2.5-4 g / L (based on total nitrogen), and the carbon source can be maintained at 10-20 g / L (based on total carbon). In a specific embodiment of the present invention, an initial fermentation medium comprising 1.5-3% carbon source (wherein the carbon source can be glucose, fructose, etc.) and 0.5-4% nitrogen source (the nitrogen source can be yeast extract and tryptone, where yeast extract can be 0.5-1% and tryptone can be 1-3%) can be used to describe the present invention in detail.

[0016] In the present invention, the culture conditions of the fermentation medium can be a fermentation temperature of 30°C, an aeration rate of 2.5 vvm, a DO control of 40%, and a stirring speed range of 400-600 rpm.

[0017] This invention effectively improves synthesis efficiency by controlling the carbon source in the feed added during the product synthesis stage, requiring only 150-180 hours of fermentation to achieve the desired fermentation effect.

[0018] The method of the present invention effectively increases the content of astaxanthin in the product per unit time. Another object of the present invention is to provide carotenoids obtained by the above method.

[0019] The method provided by this invention can effectively improve the synthesis efficiency of carotenoids and significantly increase the overall content of carotenoids in dry bacterial cells. Furthermore, the method can further increase the proportion of astaxanthin in the product. This method accelerates the metabolism of the entire carotenoid pathway, increases the product content per unit cell in the fermentation product while shortening the fermentation time, reduces the difficulty of product separation and purification while meeting the total yield target, and simultaneously meets the downstream application requirements for dry bacterial cell content. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] Since the strain involved in CN110923159B has already achieved significant advantages in other regulatory aspects, this invention mainly uses the strain involved in CN110195023B, which is still under further research, as an example to illustrate the purpose and effects of this invention. Specifically, in the specific embodiments of this invention, the yeast strain used is a *Saccharomyces cerevisiae* strain, specifically the *Saccharomyces cerevisiae* strain with accession number CGMCCNO.17913, which is disclosed in patent CN110195023B. In the embodiments of this invention, any *Saccharomyces cerevisiae* strain capable of producing carotenoids can be used in this invention. The specific embodiments of this invention only use the strain involved in CN110195023B as an example to illustrate the purpose and effects of this invention, and do not imply that only this strain can achieve this invention.

[0022] In this invention, unless otherwise specified, "%" refers to a percentage by mass. The fermentation scale in this invention is a 7L tank.

[0023] Example 1

[0024] Primary seed culture: Yeast strain CGMCC No.17913 stored at -80℃ was activated by streaking on SC-URA plates. After activation, a single colony of the strain was inoculated into SC-URA medium and cultured at 30℃ and 250rpm for 24h until the OD600 was approximately 5-8.

[0025] Secondary seed culture: The primary seeds were transferred at an inoculation rate of 4% to 250mL shake flasks (5 flasks) containing 50mL SC-URA medium, and cultured at 30℃ and 250rpm for 12h until the OD600 was approximately 5-8.

[0026] Fermentation tank inoculation: Secondary seed was inoculated at a rate of 10% (400 mL) into a 7 L fermenter containing 3.6 L of fermentation medium to start fermentation; the initial liquid volume of the fermenter was 4 L, and the initial medium consisted of 2% glucose (carbon source), 1% yeast extract (nitrogen source), and 1.5% tryptone (nitrogen source), so that the nitrogen content in the medium was 3.74 g / L.

[0027] Fermentation tank parameter settings: fermentation temperature is 30℃, aeration rate is set to 2.5vvm, DO is controlled at 40%, stirring speed range is 400-600rpm, and yeast powder is used as the initial nitrogen source.

[0028] Yeast powder was added starting at 12 hours, at a nitrogen content of 4 g / L (i.e., 20% yeast powder aqueous solution added at a rate of 15 mL / h). Addition was stopped during induction. At 48 hours, the fermentation temperature was controlled at 20℃ and D-galactose was added for induction, which lasted for 5 hours. After induction, ethanol and glucose were added in a 2:1 mass ratio during product synthesis. Glucose was prepared at a concentration of 10% and added at a rate of 10 mL / h, while ethanol (pure ethanol) was added at a rate of 2 mL / h. Citric acid and ammonia were introduced during fermentation to maintain the pH of the system between 5.6 and 6.0. Ammonia was added in a fed-batch manner at a concentration of 25% and an addition rate of 18 mL / L. Fermentation continued until 160 hours had elapsed.

[0029] The dried bacterial cells obtained contained 8.8 mg / g of carotenoids and 33% of astaxanthin (accounting for 415 mg / L of total carotenoids).

[0030] Example 2

[0031] The method in this embodiment is the same as that in embodiment 1, except that:

[0032] Fermentation tank inoculation: Secondary seed was inoculated at a rate of 10% (400 mL) into a 7 L fermenter containing 3.6 L of fermentation medium to start fermentation; the initial liquid volume of the fermenter was 4 L, and the initial medium consisted of 3% glucose (carbon source), 0.5% yeast extract (nitrogen source), and 2% tryptone (nitrogen source), so that the nitrogen content in the medium was 3.2 g / L.

[0033] Fermentation tank parameter settings: fermentation temperature is 30℃, aeration rate is set to 2.5vvm, DO is controlled at 40%, stirring speed range is 400-600rpm, and yeast powder is used as the initial nitrogen source.

[0034] Yeast powder was added starting at 12 hours, at a nitrogen content of 3.5 g / L (i.e., 20% yeast powder aqueous solution added at a rate of 15 mL / h). Addition was stopped during induction. At 48 hours, the fermentation temperature was controlled at 20°C, and D-galactose was added for induction, which lasted for 5 hours. After induction, fructose was added during the product synthesis stage, with a fructose concentration of 50% added at a rate of 15 mL / h. During fermentation, citric acid and ammonia were introduced to maintain the pH of the system between 5.6 and 6.0. Ammonia was added in a fed-batch manner at a concentration of 25% and an addition rate of 20 mL / L. Fermentation continued until 160 hours.

[0035] The dried bacterial cells obtained contained 9.2 mg / g of carotenoids and 36% of astaxanthin (accounting for 430 mg / L of total carotenoids).

[0036] Example 3

[0037] The method in this embodiment is the same as in Embodiment 1, except that the carbon source for feeding is still ethanol:

[0038] Yeast powder was added in a fed-batch manner starting at 12 hours, with 20% yeast powder added at a rate of 15 mL / h. Addition was stopped at 48 hours of induction. At 48 hours, the fermentation temperature was controlled at 20℃ and D-galactose was added for induction. After 5 hours of induction, ethanol was added at a rate of 6 mL / h for fermentation up to 85 hours. The pH of the fermentation system was controlled at 5.8-6.2 using 10 g / L citric acid. Due to the metabolic characteristics of the strain, there was a tendency for the pH to rise during this process. From 85 to 160 hours, the pH showed a continuous decreasing trend. Therefore, 25% industrial ammonia was used to adjust the pH of the fermentation system to 5.6-6.2, with an ammonia addition rate of 18 mL / L.

[0039] The dried bacterial cells obtained contained 8.2 mg / g of carotenoids, 25% of astaxanthin, and 300 mg / L of astaxanthin.

[0040] Comparative Example 1

[0041] The specific cultivation process is as follows:

[0042] Primary seed culture: Yeast strain CGMCC No.17913 stored at -80℃ was activated by streaking on SC-URA plates. After activation, a single colony of the strain was inoculated into SC-URA medium and cultured at 30℃ and 250rpm for 24h until the OD600 was approximately 5-8.

[0043] Secondary seed culture: The primary seeds were transferred at an inoculation rate of 4% to 250mL shake flasks (5 flasks) containing 50mL SC-URA medium, and cultured at 30℃ and 250rpm for 12h until the OD600 was approximately 5-8.

[0044] Fermentation tank inoculation: Secondary seed was inoculated at a rate of 10% (400 mL) into a 7 L fermenter containing 3.6 L of fermentation medium to start fermentation; the initial liquid volume of the fermenter was 4 L, and the initial medium was 2% glucose (carbon source), 1% yeast extract (nitrogen source), and 1.5% tryptone (nitrogen source), so that the nitrogen content in the medium was 3.74 g / L and the amino nitrogen content was 1.25 g / L.

[0045] Fermentation tank parameter settings: fermentation temperature 30℃, pH controlled at 5.8 using NaOH solution, aeration rate set to 2.5 vvm, DO controlled at 40%, stirring speed range 400-600 rpm, yeast extract as the supplementary nitrogen source, starting from 12h, 224.4g of yeast extract was added via a fed-batch method, i.e., 56.1g / L (total nitrogen approximately 3.6g / L), with the yeast extract prepared to a 30% concentration, maintaining the total nitrogen in the fermentation system at 0.5-1.5g / L, and ensuring no loss of 0. Feeding was stopped at 70h induction. At 70h, the fermentation temperature was controlled to 20℃ and D-galactose was added for induction, with ethanol added at a rate of 6mL / h, fermenting to 215h.

[0046] The dried bacterial cells obtained contained 5.8 mg / g of carotenoids and 24% of astaxanthin (accounting for 24% of the total carotenoids), with an astaxanthin yield of 293 mg / L.

[0047] Comparative Example 2

[0048] The method used in this comparative example is the same as that in Example 1, the only difference being:

[0049] Yeast powder was added in a fed-batch manner starting at 12 hours, with 20% yeast powder added at a rate of 15 mL / h. Addition was stopped during induction. At 48 hours, the fermentation temperature was controlled at 20℃ and D-galactose was added for induction, which lasted for 5 hours. After induction, ethanol and glucose (2:1 ratio) were added simultaneously, with glucose added at a rate of 10 mL / h at a concentration of 50% and ethanol added at a rate of 2 mL / h. Since the pH did not change significantly during fermentation, no adjustment was made. Simultaneously, 50 g of yeast extract was added once, and fermentation continued until 160 hours.

[0050] The dried bacterial cells obtained contained 6.3 mg / g of carotenoids and 35% of astaxanthin (accounting for 35% of the total carotenoid content), with an astaxanthin yield of 348 mg / L.

[0051] Comparative Example 3

[0052] The method used in this comparative example is the same as that in Example 3, except that the citric acid-ammonia combination used to control pH is replaced with phosphate-sodium hydroxide, and 50g of yeast extract is added once during the product synthesis stage.

[0053] The dried bacterial cells obtained contained 6.2 mg / g of carotenoids, 23% of astaxanthin, and 290 mg / L of astaxanthin.

[0054] Comparative Example 4

[0055] The method used in this comparative example is the same as that in Example 1, the only difference being:

[0056] During fermentation, 1.2 times the amount of yeast powder (based on the initial yeast powder concentration in the culture medium) was added at 12h, 1.0 times the amount of yeast powder was added at 65h, 0.8 times the amount of yeast powder was added at 75h, and 0.3 times the amount of yeast powder was added at 120h. The concentration was maintained at 0.3 times the initial amount until cell growth entered the stasis phase, at which point feeding was stopped. The 1.2 times yeast powder concentration was 400g / L, with a volume of 120mL. Feeding was stopped during induction. When cell growth entered the stasis phase at 70h, the fermentation temperature was controlled at 20℃ and D-galactose was added for induction, which lasted for 5h. After induction, ethanol was added simultaneously at a rate of 6mL / h until fermentation continued for 215h.

[0057] The obtained carotenoid products contained 11 mg / L of lycopene and 420 mg / L of astaxanthin, with a lycopene to astaxanthin ratio of 1:38.2. The dried bacterial cells contained 4 mg / g of carotenoids and 61% of astaxanthin, with an astaxanthin yield of 406 mg / L.

[0058] Finally, the method of this invention is merely a preferred embodiment and is not intended to limit the scope of protection of this invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for producing carotenoids from yeast, characterized in that, Includes the following steps: The yeast strain was fermented and cultured. When the cell growth entered the stasis phase, D-galactose was added for induction. After induction, citric acid-ammonia water was used to maintain the pH of the fermentation system at 5.6-6.2 during the product synthesis stage. The amount of nitrogen source supplemented was 2.0-3.8 g / L based on the total nitrogen content.

2. The method according to claim 1, characterized in that, It also includes the following steps: adding a feed carbon source during the product synthesis stage; the total amount of the feed carbon source added is 100-300 g / L.

3. The method according to claim 2, characterized in that, The feed carbon source is a mixture of glucose and ethanol or fructose.

4. The method according to claim 3, characterized in that, The mass ratio of ethanol to glucose is (1.5-3.5):

1.

5. The method according to any one of claims 1 to 4, characterized in that, Throughout the fermentation process, citric acid and ammonia were used to adjust the pH of the system to 5.6-6.

2.

6. The method according to any one of claims 1 to 4, characterized in that, During the cell growth stage, when the nitrogen source content in the fermentation medium is lower than 1 g / L, nitrogen source is added. The amount of nitrogen source added to the fermentation culture system is 2.5-6 g / L, calculated as total nitrogen.

7. The method according to claim 5, characterized in that, The initial nitrogen content of the fermentation medium is 2.5-4 g / L (total nitrogen), and the initial carbon content is 10-20 g / L (total carbon).

8. The method according to claim 6, characterized in that, The nitrogen source includes yeast powder.

9. The method according to any one of claims 1 to 8, characterized in that, The fermentation time is 150-180 hours.

10. The carotenoids obtained by the method of any one of claims 1 to 9.