Method for improving fermentation production of beta-carotene by blakeslea trispora
By monitoring the RQ value and adjusting the ratio of positive and negative bacteria during the fermentation process of Boulardii trispora, the problem of decreased β-carotene production caused by changes in the ratio of positive and negative bacteria was solved, and a significant increase in β-carotene production was achieved. .
Patent Information
- Application Number
- CN202411703274.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-01-24
AI Technical Summary
During the fermentation of Brasstridium trisporin, the proportion of positive and negative bacteria is prone to change, resulting in a decrease in the yield of β-carotene.
By monitoring the RQ value during the fermentation process, and adjusting the ratio of the number of positive and negative spores of Boulardii trispora when it drops to 0.9-1 for the first time, for example, adjust it to 1: (9.5-10.5), and adjust it when When appropriate, add the seed liquid again to keep the ratio of positive and negative bacteria at an optimal level.
The production of β-carotene is effectively increased. Compared with the control ratio without adjustment, the production ratio of β-carotene can be increased by 14.23% to 19.44%.
Smart Images
Figure CN119351509A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for improving the production of beta-carotene by fermentation of Blakeslea trispora, and belongs to the technical field of microbial fermentation. Background Art
[0002] β-Carotene is an orange-yellow terpenoid compound and the most widely found natural pigment in daily life. This fat-soluble compound is an important precursor for the synthesis of vitamin A and belongs to the same carotenoid category as lycopene, canthaxanthin, and astaxanthin. Carotenoids have a strong ability to scavenge oxygen free radicals, that is, they have antioxidant properties and are effective in cancer prevention and treatment. The application of carotenoids has also gradually expanded from being added to food and feed as pigments to the fields of medicine, daily chemicals, etc., becoming a very important industrial product.
[0003] Blakeslea trispora has obvious advantages such as rapid growth, large biomass, and high β-carotene yield per unit cell. It is an ideal strain for producing natural β-carotene and has long been used in industrial production in Eastern Europe. The fungus belongs to the class Zygomycetes, the order Mucorales, the family Coriolus, and the genus Blakeslea. There are two strains, positive and negative, which can produce well-developed hyphae and are long-tubed unicellular lower fungi. When the positive and negative bacteria are cultured alone, the hyphae are elongated and branched, the fission of the nucleus increases, and the cytoplasm increases. In this process, the two strains can only synthesize a small amount of carotenoids and reproduce asexually to produce sporangia and sporangiospores. In addition, the positive and negative strains can also combine heterothallically and form zygospores through sexual reproduction. Studies have shown that the formation of zygospores is closely related to the synthesis of β-carotene. In the mixed fermentation process of positive and negative bacteria, the large-scale production of trisporic acid (also named β factor), a sex hormone substance, plays an important role in promoting the synthesis of β-carotene by Blakeslea trispora. When positive and negative bacteria exist at the same time, trisporic acid is produced in large quantities. Positive bacteria cultured alone can also synthesize trace amounts (0.1%) of trisporic acid, while negative bacteria cultured in pure form hardly synthesize it. However, after trisporic acid is added, negative bacteria are more sensitive than positive bacteria. The effect of trisporic acid on fermentation is comprehensive, not just affecting carotenoid anabolism. β-carotene is cleaved to produce three fragment compounds: C18, C15, and C7, among which trisporic acid compounds belong to the C18 family, containing 18-19 carbon atoms, including a main carbon chain containing 14 carbon atoms. The current research conclusions on the synthesis of trisporic acid are: the last common product of positive and negative bacteria is trisporol; the oxidation of trisporol to trisporol methyl ester only occurs in positive bacteria; the reaction of trisporone to trisporol and trisporol methyl ester to trisporic acid is fast in negative bacteria. Due to these physiological and metabolic characteristics, the ratio of the number of positive and negative bacteria in the fermentation of β-carotene by Blakeslea trispora is very important for the accumulation of products.
[0004] However, in the actual fermentation process, the ratio of positive and negative bacteria often changes after they are mixed and cultured, which results in a large difference between the ratio of positive and negative bacteria in the middle and late stages of fermentation and the ratio at the time of initial inoculation, which ultimately affects the yield of β-carotene at the end of fermentation. Summary of the invention
[0005] The purpose of the present invention is to provide a method for regulating the ratio of the number of positive and negative bacteria in a fermentation process to increase the yield of beta-carotene. By re-adding positive or negative bacteria seed liquid, the ratio of the number of positive and negative bacteria in the fermentation liquid is always at a relatively optimal level, and the beta-carotene yield is finally increased, thereby solving the problem of deviation in the ratio of the number of positive and negative bacteria in the fermentation process in the prior art.
[0006] The invention provides a method for improving the yield of beta-carotene produced by fermentation of Blakeslea trispora. The method comprises inoculating positive Blakeslea trispora bacteria and negative Blakeslea trispora bacteria into a fermentation medium at a spore number ratio of 1:(7.5-8.5) to produce beta-carotene by fermentation.
[0007] In one embodiment, the fermentation medium contains: corn starch, corn steep liquor, soybean meal, glucose, potassium dihydrogen phosphate, vitamin B1, and soybean oil.
[0008] In one embodiment, the method further regulates the ratio of the spore numbers of positive Blakeslea trispora to negative Blakeslea trispora to 1:(9.5-10.5) when the RQ value (Respiratory Quotient) drops to 0.9-1 for the first time during the fermentation process.
[0009] In one embodiment, the method further regulates the ratio of the spore counts of positive Blakeslea trispora to negative Blakeslea trispora to 1:(9.5-10.5) when the RQ value drops to 0.92-1 for the first time during the fermentation process.
[0010] In one embodiment, the positive strain of Blakeslea trispora is Blakeslea trispora ATCC 14271(+), and the negative strain of Blakeslea trispora is ATCC 14272(-).
[0011] The present invention also provides a method for producing beta-carotene by fermentation, wherein the number of positive and negative spores of Blakeslea trispora is 1: (7.5-8.5) and the number of spores is 4×10 5 ~6×10 5 / L of inoculum is inoculated into a fermentation medium, and cultured at 26-30°C and under the condition of dissolved oxygen greater than 25% to ferment and produce β-carotene.
[0012] In one embodiment, the obtained positive and negative spores of Blakeslea trispora are inoculated into a fermenter containing 10 L of fermentation medium at a ratio of 1:8 with an inoculation amount of 10%, and cultured at 28° C., dissolved oxygen greater than 25%, and pH=7.
[0013] In one embodiment, the method comprises adding seed liquid again when the RQ value drops to 0.9-1 for the first time during the fermentation process, controlling the number of positive and negative spores of Blakeslea trispora in the fermentation liquid at 1:10, and continuing the culture.
[0014] In one embodiment, the positive strain of Blakeslea trispora is Blakeslea trispora ATCC 14271(+), and the negative strain of Blakeslea trispora is ATCC 14272(-).
[0015] In one embodiment, the method for preparing the seed solution of Blakeslea trispora is: inoculating positive Blakeslea trispora or negative Blakeslea trispora into a seed culture medium, and culturing at 180-220 rpm and 26-30° C. for 48 hours to obtain the seed solution.
[0016] In one embodiment, the seed culture medium (per L) contains: 30-40 g corn starch, 40-50 g corn steep liquor, 10-20 g glucose, 0.5-1.5 g potassium dihydrogen phosphate, 0.01-0.02 g vitamin B1, and 2-4 g soybean oil.
[0017] In one embodiment, during the fermentation process, glucose at a concentration of 500 g / L is added at a constant rate of 12 to 13 g / h.
[0018] In one embodiment, the fermentation medium (per L) contains: 220-260 g corn starch, 180-220 g corn steep liquor, 350-450 g soybean powder, 80-120 g glucose, 8-12 g potassium dihydrogen phosphate, 0.1-0.2 g vitamin B1, 450-550 g soybean oil, and the pH is adjusted to 7.
[0019] In the present invention, the fermentation time is not particularly limited, and can be 96 to 144 hours, preferably 120 hours. Those skilled in the art can select a suitable fermentation time to terminate the fermentation according to actual conditions.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] (1) The present invention is easy to operate and has obvious effects. The inventors found that by detecting and adjusting the ratio of the number of positive and negative bacteria during the fermentation process, the fermentation performance of Blakeslea trispora was improved and the yield of β-carotene was increased;
[0022] (2) The present invention significantly improves the β-carotene yield by optimizing the secondary inoculation time and the ratio of positive and negative bacteria in the secondary inoculation. Compared with the control group without adjustment, the β-carotene yield ratio can be increased by 14.23% to 19.44%. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a graph showing the changes in RQ during the fermentation process of Example 1.
[0024] Figure 2 This is a graph showing the change in the ratio of positive and negative bacteria during the fermentation process of Comparative Example 3. DETAILED DESCRIPTION
[0025] Strain Information:
[0026] Blakeslea trispora ATCC 14271 (+) and Blakeslea trispora ATCC 14272 (-) were purchased from the American Type Culture Collection (ATCC).
[0027] Detection method:
[0028] Wet weight test of bacteria: accurately weigh a certain amount of fermentation liquid, record its mass as G1, centrifuge at 4000rpm for 10min, discard the supernatant, weigh the solid mass as G2, and calculate the wet weight W (%) as follows:
[0029]
[0030] Detection of bacterial spores: Detect the number of spores of positive and negative bacteria according to the following steps:
[0031] (1) extracting the genome of Blakeslea trispora from the fermentation broth using a fungal genomic DNA extraction kit;
[0032] (2) using software to design primers for sexP gene sequence, sexM gene sequence, and rnhA gene sequence;
[0033] (3) using instruments for real-time QPCR amplification and analysis;
[0034] (4) The sexP gene and sexM gene contents were compared with the rnhA gene content respectively to obtain the ratio of the sexP gene to the rnhA gene content and the ratio of the sexM gene to the rnhA gene content, and the sexP gene to the sexM gene ratio was calculated based on this ratio, which is the ratio of the number of positive bacteria to the number of negative bacteria in the fermentation broth.
[0035] Detection of β-carotene content:
[0036] Accurately weigh 0.02 g of dry cells of Blakeslea trispora (wet cells are obtained by centrifuging the fermentation broth and drying the wet cells to constant weight), break the cell wall by acid-heat method, extract with tetrahydrofuran, and detect the yield of β-carotene by high performance liquid chromatography. The high performance liquid chromatography detection method is as follows: (1) Chromatographic conditions: wavelength: 455 nm; flow rate: 1 mL / min; injection volume: 10 μL; column temperature: 30°C; (2) Mobile phase configuration and condition detection: the mobile phase is acetonitrile: water (volume ratio 9:1), and the ethyl acetate gradient elution.
[0037] RQ value detection: Use a mass spectrometer to detect the O2 and CO2 components in the air and fermentation tail gas, and calculate CER and OUR. The RQ calculation method is:
[0038]
[0039] Example 1
[0040] (1) Plate culture: 80 g of peeled potatoes were cut into pieces, added with 500 ml of deionized water and boiled for 30 min. After cooling, the mixture was filtered with gauze. 5 g of glucose and 5 g of agar were added to the filtered clear liquid, and the mixture was sterilized at 121° C. for 20 min to obtain PDA medium. The spore suspensions of positive and negative bacteria of Blakeslea trispora were spread on the PDA plate culture medium, respectively, and the plates were cultured in a 28° C. incubator for 4 to 6 days to obtain positive and negative bacteria plates of Blakeslea trispora.
[0041] (2) Seed culture: Weigh 35 g corn starch and 45 g corn steep liquor, add 800 ml deionized water, stir continuously and heat to 80° C., keep for 20 min, cool and add 15 g glucose, 1 g potassium dihydrogen phosphate, 0.015 g vitamin B1, and 3 g soybean oil, make up to 1 L, mix well and adjust the pH to 7 with 30% NaOH, and sterilize at 121° C. for 20 min after packaging to obtain seed culture medium; pick one ring of mycelium from each of the positive and negative bacterial plates of Blakeslea trispora, inoculate into the seed culture medium, seal with a cotton plug, and culture at 200 rpm and 28° C. for 48 h to obtain seed solution.
[0042] (3) Fermentation culture: 240 g corn starch, 200 g corn steep liquor, 400 g soybean powder were weighed, 8 L deionized water was added, and the mixture was heated to 80° C. with constant stirring for 20 min. After cooling, 100 g glucose, 10 g potassium dihydrogen phosphate, 0.15 g vitamin B1, and 500 g soybean oil were added, and the volume was fixed to 10 L. After mixing evenly, the pH was adjusted to 7 with 30% NaOH, and the mixture was sterilized at 121° C. for 30 min to obtain a fermentation medium. The positive and negative spores of Blakeslea trispora obtained were inoculated into a fermenter containing 10 L fermentation medium at an inoculum amount of 10% according to a quantitative ratio of 1:8, and the mixture was cultured at 28° C. and under the condition that the dissolved oxygen was greater than 25%. During the culture process, 500 g / L glucose was added at a constant rate of 12 to 13 g / h. When the RQ value dropped to 0.92 for the first time during the fermentation process, the ratio of the number of positive and negative bacteria spores in the fermentation liquid was detected. At this time, the ratio of the number of positive and negative bacteria spores was 1:4.7. By adding an appropriate amount of negative bacteria seed liquid for the second time, the ratio of the number of positive and negative bacteria was adjusted to 1:10, and the culture was continued to 120 hours. The specific steps of the second inoculation are: centrifuge 100g of fermentation liquid at 4000rpm for 10min, and measure the wet weight of the bacteria to be 41.22g; and obtain the ratio of the number of positive and negative bacteria in the wet weight according to the QPCR test results; according to the above ratio, the required second inoculation of negative bacteria seed liquid is calculated to be 1.6L.
[0043] The results showed that by adding seed liquid when the RQ value first dropped to 0.92 during the fermentation process, the ratio of positive and negative bacteria was adjusted to 1:10, so that the trisporic acid content in the fermentation broth after 120 hours of fermentation was 121 mg / L. At the end of fermentation, the β-carotene production reached 6.88 g / L.
[0044] Example 2
[0045] On the basis of Example 1, the ratio of the number of positive and negative bacteria after the secondary inoculation was adjusted to 1:9.5, and the trisporic acid content and β-carotene content after the fermentation were detected according to the same method as Example 1. The results are shown in Table 1.
[0046] Example 3
[0047] On the basis of Example 1, the ratio of the number of positive and negative bacteria after the secondary inoculation was adjusted to 1:10.5, and the trisporic acid content and β-carotene content after the fermentation were detected according to the same method as Example 1. The results are shown in Table 1.
[0048] Example 4
[0049] On the basis of Example 1, the time of the secondary inoculation was adjusted to when the RQ value dropped to 0.9 for the first time during the fermentation process, the final number ratio of positive and negative bacteria was adjusted to 1:9.5, and the trisporic acid content and β-carotene content after the fermentation were detected according to the same method as Example 1. The results are shown in Table 1.
[0050] Example 5
[0051] On the basis of Example 1, the time of the secondary inoculation was adjusted to when the RQ value dropped to 1 for the first time during the fermentation process, the final number ratio of positive and negative bacteria was adjusted to 1:10, and the trisporic acid content and β-carotene content after the fermentation were detected according to the same method as Example 1. The results are shown in Table 1.
[0052] Table 1 Fermentation results of Examples 2-5
[0053]
[0054] Comparative Example 1
[0055] Based on Example 1, adjustments were made when the RQ value dropped to 1.05 for the first time during the fermentation process. The results are shown in Table 2.
[0056] Comparative Example 2
[0057] On the basis of Example 1, the RQ value was adjusted when it first dropped to 0.85 during the fermentation process. The trisporic acid content and β-carotene content after the fermentation were detected in the same manner as in Example 1. The results are shown in Table 2.
[0058] Comparative Example 3
[0059] On the basis of Example 1, the ratio of positive bacteria to negative bacteria was not adjusted during the fermentation process. The trisporic acid content and β-carotene content after the fermentation were detected in the same manner as in Example 1. The results are shown in Table 2.
[0060] Table 2 Fermentation results of comparative examples
[0061]
[0062] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for increasing the yield of β-carotene produced by fermentation of Blakeslea trispora, characterized in that: The fermentation medium was inoculated with positive Blakeslea trispora and negative Blakeslea trispora at a spore number ratio of 1: (7.5-8.5).
2. The method according to claim 1, characterized in that The fermentation medium contains corn starch, corn steep liquor, soybean powder, glucose, potassium dihydrogen phosphate, vitamin B1 and soybean oil.
3. The method according to claim 1 or 2, characterized in that: The method also regulates the number of positive and negative spores of Blakeslea trispora when the RQ value drops to 0.9-1.0 for the first time during the fermentation process, and continues the fermentation.
4. The method according to claim 3, characterized in that The regulation is to adjust the ratio of the spore numbers of positive Blakeslea trispora bacteria and negative Blakeslea trispora bacteria to 1:(9.5-10.5).
5. A method for producing β-carotene by fermentation, characterized in that: The method comprises the following steps: inoculating positive and negative spores of Blakeslea trispora into a fermentation medium at a ratio of 1:(7.5-8.5), and culturing the medium at 26-30°C and under the condition that the dissolved oxygen is greater than 25%.
6. The method according to claim 5, characterized in that When the RQ value drops to 0.9-1 for the first time during the fermentation process, the number of positive and negative spores of Blakeslea trispora in the fermentation liquid is adjusted to 1:(9.5-10.5).
7. The method according to claim 5, characterized in that The positive bacteria spores and the negative bacteria spores of Blakeslea trispora are inoculated into a fermentation medium at a ratio of 1:(7.5-8.5), and cultured at 26-30° C. and under the condition that the dissolved oxygen is greater than 25%. When the RQ value drops to 0.9-1 for the first time during the fermentation process, the positive bacteria spores and the negative bacteria spores of Blakeslea trispora in the fermentation liquid are adjusted to 1:(9.5-10.5).
8. The method according to any one of claims 5 to 7, characterized in that: Glucose was fed during the culture.
9. The method according to any one of claims 5 to 8, characterized in that: The fermentation medium contains corn starch, corn steep liquor, soybean powder, glucose, potassium dihydrogen phosphate, vitamin B1 and soybean oil.
10. Use of the method according to any one of claims 1 to 9 in producing β-carotene or products containing β-carotene.