Mutagenesis development fermentation culture method of yeast with high ergosterol content
Through the combination of ultraviolet light mutagenesis, purification culture and amplification culture, the screening medium components and amplification culture conditions were optimized, and the problem of low yeast ergosterol content was solved, and efficient and low-cost yeast ergosterol production was achieved.
Patent Information
- Application Number
- CN202510567968.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-05
AI Technical Summary
The prior art is difficult to increase the content of yeast ergosterol efficiently and at low cost, and genetic engineering methods have problems of poor stability and high cost.
The combination of ultraviolet light mutagenesis, purification culture, mutation screening and amplification culture was adopted to optimize the screening medium components and amplification culture conditions. Through multiple combinations of physical ultraviolet mutagenesis, pure culture optimization, and amplification culture, synergistic effects were exerted to improve the content of yeast ergosterol.
It has achieved efficient and low-cost increase in the yeast ergosterol content, obtained high ergosterol yeast raw materials, and enhanced the growth advantages of yeast and ergosterol yield.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to high-ergosterol yeast, and in particular to a method for induced development and fermentation culture of high-ergosterol yeast. Background Art
[0002] As an important biologically active substance, ergosterol is widely used in medicine, food, cosmetics and other fields. Since ergosterol is also an important component of fungal cell membranes, the source of ergosterol is mainly obtained through fermentation synthesis of microorganisms such as yeast. However, the ergosterol content of wild yeast is low, and the output of traditional fermentation technology is difficult to meet the growing market demand. Although some studies have been conducted to increase the content by implanting corresponding gene fragments through genetic engineering, they face problems such as complex technology, high cost, safety, and poor stability caused by iterative breakthroughs in yeast.
[0003] In order to overcome the above difficulties, the present application combines the characteristics of ergosterol production that the raw yeast does not need to be kept alive or the strain characteristics need not be purified, and only needs to obtain high-ergosterol yeast fermentation products, and has developed a production technology for high-ergosterol yeast that is efficient, low-cost and safe. Summary of the Invention
[0004] In order to develop efficient, low-cost and safe high-ergosterol yeast, a mutagenesis and fermentation culture method for high-ergosterol yeast was provided.
[0005] The above-mentioned object of the present invention is achieved through the following technical solutions: A method for induced development and fermentation culture of high ergosterol content yeast, comprising purification culture, ultraviolet light mutagenesis, mutation screening, and amplification culture; The ultraviolet light mutagenesis step is as follows: Pour the suspension containing the purified bacteria into a sterilized culture dish and spread it evenly; Place the culture dish 30-40 cm below the UV lamp and stir it to ensure that the bacteria in the suspension are evenly exposed to the UV light. Take out a culture dish every 10 seconds and mark it. Place the sample irradiated for 10-60 seconds in an incubator away from light. The samples irradiated for 10 to 60 seconds were diluted and inoculated into sterilized PAD agar medium, and cultured at a temperature of 28 to 30°C for 48 to 72 hours until colonies grew to obtain the induced strains; The mutation screening steps are as follows: Prepare screening culture medium, sterilize it, and cool it for use; The induced strains irradiated for 10 to 60 seconds at different time periods were selected and inoculated into the screening culture medium respectively, and placed in a shaker at 28 to 30°C and a rotation speed of 160 to 220 r / min for 18 to 24 hours. Preliminary screening was carried out based on the bacterial growth rate, cumulative total amount, and metabolic production capacity of high ergosterol to obtain dominant strains with high growth rate, high ergosterol content, and high ergosterol production.
[0006] By adopting the above technical scheme, through the combination of physical ultraviolet mutagenesis, pure culture selection, and amplified culture, a synergistic effect is exerted, the practical application significance of induction is broadened, and the acquisition of high ergosterol content yeast is increased, thereby obtaining high ergosterol content yeast raw materials.
[0007] Optional: The screening culture medium components are 5wt% glucose, 1.5wt% corn steep liquor, 0.1wt% KH2PO4, 0.2wt% K2HPO4, 0.0014wt% FeSO4·7H2O, 0.0012wt% MgSO4·5H2O, 0.0018wt% CuSO4·5H2O, and the rest is water.
[0008] By adopting the above technical solution, optimizing the screening culture medium components, adjusting the ratio of carbon and nitrogen sources and adding specific trace elements, yeast with high ergosterol in the cell membrane is given a growth advantage, the screening effect is enhanced, and the resulting yeast raw material has a higher ergosterol content and yield.
[0009] Optional: The amplified culture is sequentially seed culture and fermentation culture, and the seed culture steps are as follows: The dominant strain selected from the mutation screening was selected as the strain, inoculated into PAD agar medium, and cultured at a temperature of 28-30°C for 48-72 hours; Select induced bacterial strains cultured on PAD agar medium at different UV irradiation time periods, select single colonies and inoculate into sterilized YPD liquid medium, culture at 28-30°C and shaking at 160-200 rpm for 18 hours to obtain seed bacterial solution; A sterile culture solution is added to a seed cultivation tank to fill 60-70% of its volume, wherein the sterile culture solution comprises 5 wt% glucose, 1.5 wt% corn steep liquor, 0.1 wt% KH2PO4, 0.2 wt% K2HPO4, 0.0014 wt% FeSO4·7H2O, 0.0012 wt% MgSO4·5H2O, 0.0012 wt% ZnSO4·5H2O, 0.0018 wt% CuSO4·5H2O, and the remainder is water; The seed culture liquid is inoculated according to 2wt% of the sterilized culture liquid in the seed culture tank, the culture temperature is 30-33°C, the pH is 4.5-5.5, the pressure is 0.04-0.05MPa, the dissolved oxygen value is maintained at ≥20%, and the culture time is 18-24h to obtain the seed culture liquid.
[0010] By adopting the above technical solution, the strain community obtained by seed cultivation in the amplified cultivation has more stable iteration of the biological characteristics of high ergosterol content in the cell membrane during the reproduction process, and the yeast raw material finally obtained has higher ergosterol content and yield.
[0011] Optional: Fermentation culture steps are as follows: The culture medium in the fermentation culture tank is inoculated with seed culture medium, the inoculation amount of the seed culture medium is 10-15wt% of the culture medium, the culture temperature is 30-35℃, the pH is 4.5-5.5, the pressure is 0.04-0.05MPa, and the culture time is 28-30h.
[0012] By adopting the above technical solution, the yeast reproduces quickly during the fermentation culture process, the biological characteristics of high ergosterol content in the cell membrane are iteratively stable, and the ergosterol yield in the obtained yeast is higher.
[0013] Optional: During fermentation culture, the dissolved oxygen value of the culture solution in the fermentation culture tank is ≥15%.
[0014] By adopting the above technical solution, the yeast reproduction speed is fast during the fermentation culture process, and the yield of ergosterol of the obtained yeast raw material is higher.
[0015] Optional: The fermentation process is divided into initial growth phase, logarithmic growth phase, stable phase and decay phase according to the yeast growth situation; During the fermentation process, the culture medium is supplemented with sugar to control the sugar content of the culture medium. Sugar content in the initial growth period ≤1g / L; Sugar content during the logarithmic growth period is 1-2 g / L; Sugar content during the stable period: 1±0.1g / L; Stop supplementing sugar during the decline period.
[0016] By adopting the above technical solution, the yeast reproduces quickly during the fermentation culture process, reproduces rapidly in the logarithmic growth phase, and maintains efficient metabolic output of ergosterol in the stable phase, and the obtained yeast raw material has a higher ergosterol content and yield.
[0017] Optional: Fermentation cultivation is divided into initial growth phase, logarithmic growth phase, stable phase and decay phase according to the yeast growth during the fermentation process; During the initial growth phase and logarithmic growth phase, the pH of the culture medium was adjusted to 4.5-5.0; During the stable and decay phases, the pH of the culture medium was adjusted to 5.0-5.5.
[0018] By adopting the above technical solution, as microorganisms accumulate in the early stage of fermentation, glucose consumption increases, and the pH changes rapidly during the logarithmic growth period of yeast accumulation. The pH is controlled within the range of 4.5 to 5.0 by adjusting ammonia water, which is not only beneficial to the accumulation of yeast, but also to the metabolic production of ergosterol, resulting in a higher ergosterol yield in the resulting yeast.
[0019] Optional: Use ammonia solution to adjust the pH during the initial growth phase and logarithmic growth phase, and use NaOH solution to adjust the pH during the stationary phase and decay phase.
[0020] By adopting the above technical solution, ammonia water is used to adjust the pH in the early stage. Ammonia water is easily metabolized, which can reduce the impact of the electrolyte balance inside and outside the cell membrane on ergosterol metabolism, and increase the content and yield of ergosterol in the yeast raw material.
[0021] In summary, this application has at least the following beneficial effects: By combining physical ultraviolet mutagenesis, pure culture selection, and amplified culture, a synergistic effect is exerted, the practical application significance of induction is broadened, and the acquisition of yeast with high ergosterol content is increased, thereby obtaining yeast raw materials with high ergosterol content. DETAILED DESCRIPTION
[0022] raw material PAD agar culture was prepared using commercially available PAD agar medium powder.
[0023] YPD liquid medium: 2 wt% glucose, 2 wt% peptone, 1 wt% yeast extract, and 95 wt% water.
[0024] Corn steep liquor is a commercially available chemically pure product.
[0025] Glucose, KH2PO4, K2HPO4, FeSO4·7H2O, MgSO4·5H2O, CuSO4·5H2O, and ZnSO4·5H2O were all commercially available products of pharmaceutical grade analytical grade.
[0026] Ammonia solution, concentration of 10wt%; NaOH solution, concentration of 16wt%; Example 1 A method for induced development and fermentation culture of high-ergosterol yeast comprises the following steps: (1) Purification and culture After activating the refrigerated liquid bacteria, streak the activated bacteria onto a PAD agar plate and culture in a constant temperature incubator at 30°C for 64 hours. A single colony with good growth was selected from the PAD agar medium and inoculated into the YPD liquid medium. The culture was placed in a shaker at a temperature of 30°C and a rotation speed of 160 rpm / min for 20 hours, and the yeast concentration reached 12-13 g / L to obtain a qualified suspension containing the initial strain. Take the suspension containing the initial strain and inoculate it into YPD liquid medium and culture it at 30℃ until OD 650 =0.8, and obtain the sampled bacterial solution; Take 50 ml of the sampled bacterial solution and centrifuge it at 4500 r / min for 7 min, remove the supernatant, and collect the initial bacterial strain; Wash the initial bacterial strain repeatedly three times with sterile saline, take a loop of the initial bacterial strain with an inoculating loop, and prepare 10 ml of bacterial strain suspension stock solution with sterile saline, take 1 ml of the bacterial strain suspension stock solution and prepare 5 ml of suspension containing purified bacterial strain with sterile saline, and prepare a total of 6 suspensions containing purified bacterial strains.
[0027] (2) Ultraviolet light mutagenesis Turn on the UV lamp in advance and irradiate for 30 minutes to preheat; Pour 6 suspensions containing purified bacteria into sterilized culture dishes and spread them evenly. Place the culture dish on a magnetic stirrer 35 cm below the UV lamp and turn on the magnetic stirrer to ensure that the cells in the cell suspension can be evenly irradiated with UV light. Take out a culture dish every 10 seconds and mark it. Place the sample irradiated for 10 to 60 seconds in an incubator away from light. The six samples were diluted separately, with the dilution gradient being 10 -1 , 10 -2 , 10 -3 , 10 -4 , take 10 -4 , 10 -5 100 μL of the diluted sample was evenly spread on sterilized PAD agar medium and cultured at 30°C for 64 h until colonies grew to obtain the mutagenic strain; (3) Mutation screening A screening medium is prepared, sterilized, and cooled for use, wherein the screening medium comprises 5 wt% glucose, 1.5 wt% corn steep liquor, 0.1 wt% KH2PO4, 0.2 wt% K2HPO4, 0.0014 wt% FeSO4·7H2O, 0.0012 wt% MgSO4·5H2O, 0.0018 wt% CuSO4·5H2O, and the remainder is water; The mutagenic strains irradiated for 10 to 60 seconds were selected and inoculated into the screening medium respectively. The culture was shaken at 30°C and 200 rpm for 24 hours. Samples were taken at 18 and 24 hours to detect the yeast content and ergosterol content. Then, the bacterial solution of the mutagenic strain cultured for 24 hours was diluted and inoculated into sterilized PAD agar medium, and cultured at a culture temperature of 30°C for 64 hours until colonies grew to obtain the second-round recurring mutagenic strain. The second-round recurring mutagenic strains irradiated for 10 to 60 seconds at different time periods were selected again and inoculated into the screening medium respectively. The culture was shaken at 30°C with a speed of 200 r / min for 24 hours. Samples were taken at 18 hours and 24 hours during the culture process to detect the yeast content and ergosterol content. According to the yeast content and ergosterol content of the first mutagenized strain and the second round of recurrent mutagenized strain after propagation in the screening medium, the suspension containing the purified strain obtained by initial strain culture was gradually diluted to 10 -4 The strain was then inoculated with a screening medium and cultured under shaking at 30°C, with a rotation speed of 200 r / min for 24 hours. During the culture process, samples were taken at 18 hours and 24 hours to detect the yeast content and ergosterol content as a baseline control. For comparison, a strain with high yeast content and high ergosterol content was selected as the dominant strain QTA005. The results of the 24-hour shake flask culture of the first mutagenized strain, the second round of replicated mutagenized strain, and the initial strain in Example 1 are shown in Table 1 below. (4) Scale-up culture The scale-up culture is followed by seed cultivation and fermentation culture.
[0028] The seed cultivation steps are as follows: The dominant strain QTA005 from mutation screening was selected as the strain, inoculated into PAD agar medium, and placed in a constant temperature incubator. The incubation temperature was 30°C and the incubation time was 64 h; Select induced bacterial strains cultured on PAD agar medium at different UV irradiation time periods, select single colonies and inoculate into sterilized YPD liquid medium, and culture in a shaker at 30°C and 180 rpm for 18 hours to obtain seed bacterial solution; A sterile culture solution was added to the seed culture tank to fill 65% of its volume. The sterile culture solution contained the following ingredients: 5 wt% glucose, 1.5 wt% corn steep liquor, 0.1 wt% KH2PO4, 0.2 wt% K2HPO4, 0.0014 wt% FeSO4·7H2O, 0.0012 wt% MgSO4·5H2O, 0.0012 wt% ZnSO4·5H2O, 0.0018 wt% CuSO4·5H2O, and the remainder was water. The seed culture solution was inoculated at 2 wt% of the culture solution in the seed culture tank, the culture temperature was 32°C, the pH was 5.0, the pressure was 0.045 MPa, the dissolved oxygen value was maintained at 21-22%, and the culture time was 20 hours to obtain the seed culture solution; Fermentation culture, the fermentation culture steps are as follows: The culture medium in the fermentation tank was inoculated with seed culture medium at a rate of 12.5 wt% of the culture medium. The culture temperature was 32°C, the pressure was 0.045 MPa, and the culture time was 30 h. The dissolved oxygen value was dynamically maintained at 16% to 17% by adjusting the gas flux and stirring speed in real time. During the fermentation process, yeast 650 The initial growth period (OD 650 Less than 0.4), logarithmic growth period (OD 650 0.4~1.0), stable period (OD 650 1.0±0.2) and decay period (OD 650 Less than 0.8); During the initial growth phase and logarithmic growth phase, ammonia solution was used to adjust the pH of the culture medium to 4.5-5.0. During the stationary phase and decay phase, NaOH solution was used to adjust the pH of the culture medium to 5.0-5.5. During the fermentation cultivation process, the culture medium is supplemented with sugar to control the sugar content of the culture medium: the sugar content in the initial growth period is ≤1g / L; the sugar content in the logarithmic growth period is 1-2g / L; the sugar content in the stable period is 1±0.1g / L; in the decline period, sugar supplementation is stopped.
[0029] (5) Collection and extraction After the fermentation is completed, the fermentation liquid is centrifuged for the first separation using a disc centrifuge, and the yeast solids obtained are washed and separated once more by adding water; The yeast emulsion obtained after the primary separation and water washing separation is washed three times with purified water to obtain clean centrifuged wet yeast after the yeast is freed of impurities, residual nutrients, etc.; The wet yeast obtained by centrifugal separation is adjusted to a solid concentration of 18 wt%, and spray-dried at high temperature and pyrolyzed into solid powder to obtain a dry yeast product.
[0030] Table 1. Screening test results in mutation screening in Example 1 Therefore, in Example 1, the sample with an induction time of 50 seconds was the QTA002 strain.
[0031] Example 1 During the fermentation cultivation process of (4) amplification cultivation, the yeast content and ergosterol content were detected in real time. The detection results are shown in Table 2 below.
[0032] Table 2. Detection results of fermentation products of Example 1 Comparative Example 1 A yeast mutagenesis fermentation culture method, wherein the suspension containing the purified strain obtained by initial strain culture in step (1) of Example 1 is gradually diluted to 10 -4 The resulting product was then inoculated into a sterilized PAD agar medium and cultured at 30°C for 64 h until colonies grew to obtain an initial strain. The dominant strain QTA002 was replaced by the initial strain as the strain, and amplified culture was performed according to step (4) of Example 1. The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the peak were recorded.
[0033] Example 2 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that: In step (3) mutation screening, the screening culture medium components are glucose 5wt%, corn steep liquor 1.5wt%, KH2PO4 0.08wt%, K2HPO4 0.21wt%, FeSO4·7H2O 0.0010wt%, MgSO4·5H2O 0.0016wt%, CuSO4·5H2O 0.0015wt%, and the remainder is water; The components of the sterilized culture medium in step (4) amplification cultivation are: Glucose 5wt%, corn steep liquor 1.5wt%, KH2PO4 0.08wt%, K2HPO4 0.21wt%, FeSO4·7H2O 0.0010wt%, MgSO4·5H2O 0.0016wt%, ZnSO4·5H2O 0.0012wt%, CuSO4·5H2O 0.0015wt%, and the rest is water.
[0034] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0035] Example 3 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that: In step (3) mutation screening, the screening culture medium components are glucose 5wt%, corn steep liquor 1.0wt%, KH2PO4 0.08wt%, K2HPO4 0.21wt%, FeSO4·7H2O 0.0010wt%, MgSO4·5H2O 0.0016wt%, CuSO4·5H2O 0.0015wt%, and the remainder is water; The sterilized culture medium in the amplified cultivation in step (4) comprises 5 wt% of glucose, 1.0 wt% of corn steep liquor, 0.08 wt% of KH2PO4, 0.21 wt% of K2HPO4, 0.0010 wt% of FeSO4·7H2O, 0.0016 wt% of MgSO4·5H2O, 0.0012 wt% of ZnSO4·5H2O, 0.0015 wt% of CuSO4·5H2O, and the remainder is water; The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0036] Example 4 A method for induced development and fermentation culture of high-ergosterol yeast is disclosed, which differs from Example 1 in that the screening culture medium in step (3) mutation screening comprises 5 wt% glucose, 2.0 wt% corn steep liquor, 0.08 wt% KH2PO4, 0.21 wt% K2HPO4, 0.0010 wt% FeSO4·7H2O, 0.0016 wt% MgSO4·5H2O, 0.0015 wt% CuSO4·5H2O, and the remainder is water.
[0037] The sterilized culture medium in the amplified cultivation in step (4) comprises 5 wt% of glucose, 2.0 wt% of corn steep liquor, 0.08 wt% of KH2PO4, 0.21 wt% of K2HPO4, 0.0010 wt% of FeSO4·7H2O, 0.0016 wt% of MgSO4·5H2O, 0.0012 wt% of ZnSO4·5H2O, 0.0015 wt% of CuSO4·5H2O, and the remainder is water.
[0038] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0039] Example 5 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the culture pH in the seed culture for amplification culture in step (4) is 4.0.
[0040] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0041] Example 6 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the pH of the culture in the seed culture for amplification culture in step (4) is 6.0.
[0042] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0043] Example 7 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the dissolved oxygen in the seed culture for amplification culture in step (4) is 16±1%.
[0044] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0045] Example 8 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the culture pH in the fermentation culture of the amplified culture in step (4) is 4.0.
[0046] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0047] Example 9 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the pH of the culture in the fermentation culture during the amplified culture in step (4) is 6.0.
[0048] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0049] Example 10 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that the dissolved oxygen in the fermentation culture during the amplified culture in step (4) is 8±1%.
[0050] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0051] Example 11 A method for induced development and fermentation of high-ergosterol yeast is different from that of Example 1 in that the sugar content in the fermentation culture of the amplified culture in step (4) is controlled as follows: Sugar content in the initial growth period ≤1g / L; Sugar content during logarithmic growth period: 1 ± 0.1 g / L; Sugar content during the stable period: 1±0.1g / L; Stop supplementing sugar during the decline period.
[0052] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0053] Example 12 A method for induced development and fermentation of high-ergosterol yeast is different from that of Example 1 in that the sugar content in the fermentation culture of the amplified culture in step (4) is controlled as follows: Sugar content during the initial growth period is 1-2 g / L; Sugar content during the logarithmic growth period is 1-2 g / L; Sugar content during the stable period: 1±0.1g / L; Stop supplementing sugar during the decline period.
[0054] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0055] Example 13 A method for induced development and fermentation of high-ergosterol yeast is different from that of Example 1 in that the pH of the culture medium in the fermentation culture in the amplified culture in step (4) is 4.5-5.0.
[0056] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0057] Example 14 A method for induced development and fermentation of high-ergosterol yeast is different from that of Example 1 in that the pH of the culture medium in the fermentation culture of the amplified culture in step (4) is controlled as follows: During the initial growth phase and the logarithmic growth phase, the pH of the culture solution is adjusted to 5.0-5.5; During the stable phase and the decay phase, the pH of the culture medium is adjusted to 4.5-5.0.
[0058] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0059] Example 15 A method for induced development and fermentation of high-ergosterol yeast is different from that of Example 1 in that ammonia water is used to adjust the pH in the fermentation culture during the amplified culture in step (4).
[0060] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0061] Example 16 A method for induced development and fermentation culture of high-ergosterol yeast is different from that of Example 1 in that a NaOH solution is used to adjust the pH during the fermentation culture in step (4) of amplified culture.
[0062] The ergosterol content and yield during the fermentation process were detected, and the ergosterol yield peak and the ergosterol content at the yield peak were recorded.
[0063] The time at the peak of ergosterol production, wet weight, dry weight, ergosterol content, and ergosterol production of Examples 2 to 16 and Comparative Example 1 are shown in Table 4 below.
[0064] Table 4. Ergosterol test results of Examples 2 to 16 and Comparative Example 1 In conjunction with the above table, it can be seen from the comparison of Example 1 and Comparative Example 1 that the ergosterol content (dry) and ergosterol yield of Example 1 are significantly better than those of Comparative Example 1, that is, the amount of ergosterol that can be produced per unit volume of culture fluid in Example 1 is greater than that of Comparative Example 1, and the ergosterol content in the output yeast cell membrane is greater than that of Comparative Example 1. Therefore, the present application combines physical ultraviolet mutagenesis, pure culture optimization, and amplified culture to exert a synergistic effect, broaden the practical application significance of induction, and indeed increase the high ergosterol content of yeast and the reproduction rate and survival rate of high ergosterol content yeast, thereby obtaining a high ergosterol yeast raw material.
[0065] Comparing Examples 1 to 4, the four strains used different screening culture medium components in the mutation screening and used sterile culture fluids with matching components in the amplification cultivation. The strains screened in Examples 1 to 4 expressed different biological characteristics in terms of the metabolic production rate of ergosterol and the cell membrane ergosterol content. From the test results, it can be seen that the ergosterol content (dry) and ergosterol yield of Example 1 are both better than those of Examples 2 to 4. The present application optimizes the screening culture medium components, adjusts the carbon and nitrogen source ratios, and adds specific trace elements to give yeast with high ergosterol content in the cell membrane a growth advantage, thereby enhancing the screening effect. The resulting yeast raw material has a higher ergosterol content and yield. Therefore, the screening culture medium in the present application is preferably: 5wt% glucose, 1.5wt% corn steep liquor, 0.1wt% KH2PO4, 0.2wt% K2HPO4, 0.0014wt% FeSO4·7H2O, 0.0012wt% MgSO4·5H2O, and 0.0018wt% CuSO4·5H2O.
[0066] Comparing Example 1 with Examples 5 to 6, it can be seen that the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Examples 5 to 6; combined with Example 1 and Example 7, the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Example 7. Therefore, the seed cultivation conditions in this application are preferably pH 4.5 to 5.5 and maintaining a dissolved oxygen value ≥ 20%. The strain community obtained by seed cultivation in the time-amplification cultivation is more stable in the biological characteristics of high ergosterol content in the cell membrane during the reproduction process, and the yeast raw material finally obtained has a higher ergosterol content and yield.
[0067] Comparing Example 1 with Examples 8 to 9, it can be seen that the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Examples 8 to 9; in combination with Example 1 and Example 10, the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Example 10. Therefore, the seed cultivation conditions in the present application are preferably pH 4.5 to 5.5 and maintaining a dissolved oxygen value ≥ 15%, so that the yeast reproduction speed in the fermentation culture process of the present application is fast, the biological characteristics of high ergosterol content in the cell membrane are iteratively stable, and the ergosterol yield of the obtained yeast raw material is higher.
[0068] Comparing Example 1 with Examples 11 to 12, it can be seen that the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Examples 11 to 12. Therefore, during the fermentation and cultivation process, the culture medium is supplemented with sugar to control the sugar content of the culture medium, and the following control is preferably performed: sugar content in the initial growth period ≤1 g / L; sugar content in the logarithmic growth period 1 to 2 g / L; sugar content in the stable period 1±0.1 g / L; sugar supplementation is stopped during the decline period, so that the yeast propagates quickly during the fermentation and cultivation process of the present application, rapidly propagates in the logarithmic growth period, and maintains efficient metabolic output of ergosterol in the stable period, and the obtained yeast raw material has a higher ergosterol content and yield.
[0069] Comparing Example 1 with Examples 13 to 14, it can be seen that the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Examples 13 to 14. In the early stage of fermentation, as the microorganisms accumulate, glucose consumption increases, and the pH changes rapidly during the logarithmic growth period of yeast accumulation. Controlling the pH within the range of 4.5 to 5.0 is not only beneficial to the accumulation of yeast, but also to the metabolic production of ergosterol. The resulting yeast raw material has a higher ergosterol yield. Therefore, during the initial growth phase and logarithmic growth phase of the fermentation cultivation process, the culture medium pH is adjusted to 4.5 to 5.0; during the stable period and the decay period, the culture medium pH is preferably adjusted to 5.0 to 5.5.
[0070] Combined with Examples 15 to 16, the ergosterol content (dry) and ergosterol yield of Example 1 are better than those of Examples 15 to 16. This is because ammonia water is used to adjust the pH in the early stage. Ammonia water is easily metabolized, which can reduce the effect of electrolyte balance inside and outside the cell membrane on ergosterol metabolism, thereby increasing the content and yield of ergosterol in the yeast raw material. Therefore, it is better to use ammonia water to adjust the pH during the initial growth phase and the logarithmic growth phase during the fermentation cultivation process, and to use NaOH solution to adjust the pH during the stable phase and the decay phase.
[0071] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as the modifications are within the scope of protection required by the present invention, they will be protected by patent law.
Claims
1. A method for induced growth and fermentation culture of high ergosterol content yeast, characterized in that: Including purification culture, ultraviolet light mutagenesis, mutation screening, and amplification culture; The ultraviolet light mutagenesis step is as follows: Pour the suspension containing the purified bacteria into a sterilized culture dish and spread it evenly; Place the culture dish 30-40 cm below the UV lamp and stir it to ensure that the bacteria in the suspension are evenly exposed to the UV light. Take out a culture dish every 10 seconds and mark it, and place the sample irradiated for 10 to 60 seconds in the incubator in the dark; The samples irradiated for 10-60 seconds were diluted and inoculated into sterilized PAD agar medium, and cultured at 28-30°C for 48-72 hours until colonies grew to obtain the mutagenic strains. The mutation screening steps are as follows: Prepare screening culture medium, sterilize it, and cool it for use; The induced strains irradiated for 10 to 60 seconds were selected and inoculated into the screening culture medium respectively. The strains were placed in a shaker at 28 to 30°C and a rotation speed of 160 to 220 r / min for 18 to 24 hours. Preliminary screening was carried out based on the bacterial growth rate, cumulative total amount, and metabolic production capacity of high ergosterol to obtain dominant strains with high growth rate, high ergosterol content, and high ergosterol production.
2. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 1, wherein: The screening medium composition is glucose 5wt%, corn steep liquor 1.5wt%, KH2PO4 0.1wt%, K2HPO4 0.2wt%, FeSO4•7H2O 0.0014wt%, MgSO4•5H2O 0.0012wt%, CuSO4•5H2O 0.0018wt%, and the rest is water.
3. The method for induced growth and fermentation of high-ergosterol yeast according to claim 1, wherein: The amplification culture is followed by seed cultivation and fermentation cultivation. The seed cultivation steps are as follows: The dominant strain selected from the mutation screening was selected as the strain, inoculated into PAD agar medium, and cultured at a temperature of 28-30°C for 48-72 hours; Select induced bacterial strains cultured on PAD agar medium at different UV irradiation time periods, select single colonies and inoculate into sterilized YPD liquid medium, culture at 28-30°C and shaking at 160-200 rpm for 18 hours to obtain seed bacterial solution; Add 60-70% of the volume of sterile culture solution to the seed culture tank. The sterile culture solution consists of 5wt% glucose, 1.5wt% corn steep liquor, 0.1wt% KH2PO4, 0.2wt% K2HPO4, 0.0014wt% FeSO4•7H2O, 0.0012wt% MgSO4•5H2O, 0.0012wt% ZnSO4•5H2O, 0.0018wt% CuSO4•5H2O, and the rest is water. The seed culture solution is inoculated according to 2wt% of the sterilized culture solution in the seed culture tank, the culture temperature is 30-33°C, the pH is 4.5-5.5, the pressure is 0.04-0.05MPa, the dissolved oxygen value is maintained at ≥20%, and the culture time is 18-24h to obtain the seed culture solution.
4. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 3, wherein: The fermentation culture steps are as follows: The culture medium in the fermentation culture tank is inoculated with seed culture medium, the inoculation amount of the seed culture medium is 10-15wt% of the culture medium, the culture temperature is 30-35°C, the pH is 4.5-5.5, the pressure is 0.04-0.05MPa, and the culture time is 28-30h.
5. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 4, wherein: During fermentation culture, the dissolved oxygen value of the culture medium in the fermentation culture tank is ≥15%.
6. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 4, wherein: The fermentation process is divided into initial growth phase, logarithmic growth phase, stable phase and decay phase according to the yeast growth situation; During the fermentation process, the culture medium is supplemented with sugar to control the sugar content of the culture medium. Sugar content in the initial growth period ≤1g / L; Sugar content during the logarithmic growth period is 1~2g / L; Sugar content during the stable period: 1±0.1g / L; Stop supplementing sugar during the decline period.
7. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 4, wherein: Fermentation cultivation is divided into initial growth phase, logarithmic growth phase, stable phase and decay phase according to the yeast growth during the fermentation process; During the initial growth phase and logarithmic growth phase, the pH of the culture medium was adjusted to 4.5-5.0; During the stable and decay phases, the pH of the culture medium was adjusted to 5.0~5.
5.
8. The method for induced growth and fermentation culture of high-ergosterol yeast according to claim 7, wherein: Ammonia solution was used to adjust the pH during the initial growth phase and logarithmic growth phase, and NaOH solution was used to adjust the pH during the stable phase and decay phase.