Method for producing scleroglucan through sclerotium rolfsii fermentation based on dissolved oxygen regulation
By regulating the dissolved oxygen content in the fermentation broth of Cialis in stages, combined with the use of stirring paddles and mechanical gas rise, the problems of low yield and insufficient substrate utilization of Cialis in fermentation of Cialis in fermentation of Cialis in fermentation of Cialis in fermentation were solved. Achieve efficient and low-cost hard dextran production.
Patent Information
- Application Number
- CN202411624519.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-02-21
AI Technical Summary
In the prior art, the production of hard dextran by fermentation of Cialis microglycobacterium has problems such as long fermentation cycle, low yield and insufficient substrate utilization, and the traditional hot water extraction method has low extraction efficiency, high cost and long time.
By regulating the dissolved oxygen content in the fermentation broth in three stages, the initial stage of fermentation is reduced from 100% to 0-10%, the intermediate stage of fermentation is maintained at 0-10%, and the later stage of fermentation is controlled at 20-30%, combining stirring paddles and mechanical gas rise The use of the rotating speed and ventilation volume is adjusted to improve fermentation efficiency.
It significantly improves the yield of hard dextran and the viscosity of the fermentation broth, reduces production costs, and has more industrial application value.
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Figure CN119491029A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for producing scleroglucan by fermentation of Sclerotium truncatum based on dissolved oxygen regulation, and belongs to the technical field of microbial fermentation. Background Art
[0002] Fungal exopolysaccharides are an important component of the extracellular biofilm matrix of filamentous fungi and can diffuse into the fermentation culture medium. They have multiple physiological effects such as antioxidant, anti-inflammatory, analgesic, anti-tumor and hypoglycemic properties and are widely used in the healthcare industry. Scleroglucan is fermented by Sclerotium truncatum. Because its product polysaccharide is gel-like at low temperatures, it is also called sclerotium gel. As a new type of fungal polysaccharide, scleroglucan has good moisturizing ability, and its fermentation broth is thermally stable and salt-resistant. It is often added to cosmetics as a moisturizer and used as an oil-displacing agent in the field of oil extraction.
[0003] As a glucose polymer, scleroglucan is a β-glucan with three β-1,3-linked d-pyranose as repeating structural units, and each structural unit is connected to a glucose side chain by a single β-1,6 bond. At present, the industrial production method of scleroglucan is mainly based on microbial fermentation. Currently, Sclerotium uniformis is commonly used in industry to ferment and produce scleroglucan. The output of scleroglucan produced in each tank is about 20g / L, and it has defects such as long fermentation cycle, low yield and insufficient substrate utilization.
[0004] Sclerotium itself reproduces by producing sclerotia, so it is difficult to increase its production of scleroglucan by strain mutagenesis. In addition, Sclerotium has problems such as long fermentation cycle and insufficient utilization of substrate during the fermentation process. The downstream treatment process of scleroglucan mainly uses hot water extraction, which requires a large amount of anhydrous ethanol to precipitate the polysaccharide, and has the disadvantages of low extraction efficiency, high extraction cost and long extraction time. Therefore, it is necessary to improve the specific fermentation process to increase the industrial production of scleroglucan.
[0005] At present, many studies on fungal production of glucan have shown that the synthesis of polysaccharide polymers may be stimulated by dissolved oxygen (DO) limitation, and it is also mentioned that controlling dissolved oxygen will affect the metabolic process of oxalic acid, a byproduct of the synthesis of Sclerotium, but most of these studies only compare the dissolved oxygen regulation effect after the dissolved oxygen is reduced to a certain degree in the late fermentation period, and there are few studies on the influence of dissolved oxygen on Sclerotium at different stages in the early stage. Therefore, the present invention aims to provide a new fermentation dissolved oxygen regulation process to further increase the yield of scleroglucan. Summary of the invention
[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for producing scleroglucan by fermentation of Sclerotium uniformis based on dissolved oxygen regulation in view of the shortcomings of the prior art. The method combines a stirring paddle and a mechanical air lift to achieve regulation of dissolved oxygen in the fermentation liquid by changing the rotation speed and ventilation volume in three stages.
[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0008] The invention discloses a method for producing scleroglucan by regulating Sclerotium truncatum based on dissolved oxygen. Sclerotium truncatum is used as a production strain to ferment and produce scleroglucan. Sclerotium truncatum is inoculated into a slant culture medium and a shaking flask for step-by-step expansion culture, and then inoculated into a mechanical airlift fermenter for fermentation. The whole fermentation cycle is 108 hours, which is divided into an early fermentation period, a middle fermentation period and a late fermentation period. The dissolved oxygen content in the fermentation liquid is controlled by controlling the stirring speed and the ventilation volume of the fermenter. In the early fermentation period, the dissolved oxygen is controlled to be reduced from 100% to 0-10%, the dissolved oxygen is controlled at 0-10% in the middle fermentation period, and the dissolved oxygen is controlled at 20-30% in the late fermentation period.
[0009] Wherein, the early fermentation period is 0 to 12 hours of the fermentation cycle.
[0010] The mid-fermentation period is 12 to 48 hours of the fermentation stage.
[0011] Wherein, the late fermentation period is 48 to 108 hours of the fermentation stage.
[0012] In the early stage of fermentation, the rotation speed is controlled at 100-200 rpm, the ventilation volume is controlled at 0.1-0.5 vvm, and the dissolved oxygen in the fermentation liquid is reduced to 0-10% in 10-12 hours. In the early stage of fermentation, the amount of cells of Sclerotium uniformis is taken as the target.
[0013] In the middle stage of fermentation, the rotation speed is controlled at 200-400 rpm, the ventilation volume is controlled at 0.5-1 vvm, and the dissolved oxygen in the fermentation liquid is continued to be maintained at 0-10%.
[0014] In the late fermentation stage, the rotation speed is controlled at 600-800 rpm, the ventilation volume is controlled at 2-3 vvm, and the dissolved oxygen in the fermentation liquid is continuously maintained at 20-30%.
[0015] Wherein, the deposit number of the Sclerotium uniformum is ATCC24965.
[0016] The DO of the fermentation broth is usually controlled to be above zero by increasing the impeller rotation speed of the agitator. The change in impeller speed will lead to a variety of effects, which control or maintain the target dissolved oxygen by changing the overall mixing, average shear stress, improving pH or temperature. Any or all of them may have a significant impact on the synthesis of biopolymers, so the present invention comprehensively evaluates the effects of the impeller rotation speed and the fermenter ventilation to obtain a better production method.
[0017] During the fermentation of scleroglucan, the lower dissolved oxygen in the early stage of fermentation can promote the growth of the fermentation biomass of Sclerotium uniformis and inhibit the formation of oxalic acid byproducts, but at the same time, insufficient mass transfer will also greatly inhibit the production of scleroglucan. Therefore, the stirring speed in the early stage of fermentation should not be higher than 200rpm. In the late stage of fermentation, the bacterial growth is sufficient and the viscosity of the fermentation liquid increases. At this time, the too low ventilation volume and stirring paddle speed cannot meet the needs of Sclerotium metabolic synthesis of scleroglucan. Therefore, the ventilation volume of the fermentation liquid and the stirring paddle speed can be increased to improve the mass transfer effect.
[0018] The fermentation method of the present invention has the logarithmic growth phases of Sclerotium in the early and middle stages of fermentation, and promotes the growth of Sclerotium by maintaining low dissolved oxygen; and improves the mass transfer efficiency by enhancing shear force and ventilation volume in the late stage of fermentation, so that the yield of scleroglucan in the fermentation process is increased.
[0019] Beneficial effects:
[0020] (1) As a facultative anaerobic bacterium, Sclerotium uniformis uses low dissolved oxygen in the early stage of fermentation to promote bacterial growth. In the later stage of fermentation, the viscosity of the fermentation liquid is too high. By increasing the ventilation volume and the speed of the agitator to enhance the mass transfer efficiency of the fermentation liquid, the yield of scleroglucan and the viscosity of the fermentation liquid are significantly improved. The high-viscosity fermentation liquid has better temperature and salt resistance.
[0021] (2) At present, the fermentation production of scleroglucan in the world mainly uses sucrose as the substrate for fermentation, consuming 150g / L of sucrose while only producing 26g / L of scleroglucan. However, when the method provided by the invention is used for fermentation, the dissolved oxygen in the fermentation liquid is regulated during the fermentation process. With 85g / L of glucose as the substrate, the scleroglucan output is 18.5g / L after fermentation in a 5L fermenter for 108h. When fermented in a 20L fermenter for 72h, the glucan output can reach 54-62g / L, and the fermentation process only consumes 85g / L of glucose. Compared with the common production processes at home and abroad, the cost of the invention is significantly reduced, and it has a wider industrial application. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.
[0023] Figure 1 The data graphs are of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth of Example 1.
[0024] Figure 2 The data graphs are of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the three groups of fermentation broths in Comparative Example 2; from left to right are Group 1, Group 2 and Group 3.
[0025] Figure 3 It is the CO2 content and O2 content in the tail gas of the fermentation broth of Group 1 and Group 3 in Comparative Example 2, as well as the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during fermentation.
[0026] Figure 4 The data graphs are of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the three groups of fermentation broths in Comparative Example 3; from left to right are Group 1, Group 2 and Group 3.
[0027] Figure 5 It is the CO2 content and O2 content in the tail gas of the fermentation broth of Group 1 and Group 3 in Comparative Example 3, as well as the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during fermentation.
[0028] Figure 6 The data graphs are of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth of Comparative Example 4.
[0029] Figure 7 The data graph is of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth of Comparative Example 5.
[0030] Figure 8 The CO2 content and O2 content in the tail gas of the fermentation broth of Example 1, Comparative Examples 4 and 5, as well as the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during fermentation; the red line in the figure represents Example 1, the blue line represents Comparative Example 4, and the black line represents Comparative Example 5. DETAILED DESCRIPTION
[0031] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.
[0032] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.
[0033] The culture medium and its components used in the following examples are as follows:
[0034] PDA solid culture medium: potato 200g / L; glucose 20g / L; agar 20g / L.
[0035] Seed liquid culture medium: glucose 30.0g / L; KH2PO4 10g / L; (NH4)2SO4 3.0g / L; yeast extract 1.0g / L; KCl 0.5g / L; MgSO4: 0.25g / L.
[0036] Fermentation medium: glucose 85.0 g / L; KH2PO4 1.0 g / L; (NH4)2SO4 3.0 g / L; yeast extract powder 1.0 g / L; KCl 0.5 g / L; citric acid 1.0 g / L; MgSO4 0.5 g / L.
[0037] All the above culture media were sterilized at 115°C for 20 min.
[0038] The deposit number of Sclerotium truncatum used in the examples is ATCC24965, which was purchased from Wuhan Huizao Biotechnology Co., Ltd. with the item number: HZB303567.
[0039] The method for measuring the scleroglucan yield and the biomass of the uniform microsclerotium in the embodiment is as follows: the fermentation broth sample is mixed with 80°C hot water in a ratio of 1:3, stirred in an oil bath at 60°C for 30min, and centrifuged at 12000r / s and 25°C for 10min. The supernatant is precipitated with ethanol overnight at a ratio of 1:2, the precipitated polysaccharide is collected by centrifugation, evaporated to constant weight at 105°C in a water evaporator, and weighed to obtain the mass of scleroglucan; the precipitate is dried to constant weight in a vacuum drying oven at 60°C, and weighed on an electronic balance to obtain the dry cell weight. Scleroglucan yield = scleroglucan mass / fermentation broth volume; uniform microsclerotium biomass = cell dry weight / fermentation broth volume.
[0040] Measurement of residual sugar in the examples: A biosensor analyzer (purchased from Hillman Technology Co., Ltd., model: S-10) was used to measure the residual sugar content of glucose at different fermentation stages.
[0041] The viscosity of the fermentation broth was measured using an NDJ-8J viscometer (purchased from Shanghai Fangrui Technology Co., Ltd.).
[0042] A portable fermentation process exhaust gas analyzer (purchased from Shanghai Baiyuan Biotechnology Co., Ltd., model: BY-001) was used to measure the CO2 and O2 contents in the fermentation exhaust gas as well as the respiratory metabolic parameters CO2 release rate (CER) and oxygen uptake rate (OUR).
[0043] The 5L fermenter used in the examples and comparative examples was purchased from Debier Biotechnology (Shanghai) Co., Ltd., model number T&J-Intelli-Ferm A5L.
[0044] Example 1
[0045] (1) Slant culture of Sclerotium sclerotiorum strains
[0046] The sclerotia of Sclerotium uniformis were inoculated onto PDA solid medium (potato dextrose agar medium) plates and cultured at 30°C for 3-4 days until the plates were covered with white hyphae and a small amount of white sclerotia, and a large number of mature Sclerotium uniformis were obtained for shake flask culture of seed liquid.
[0047] (2) Shake flask culture of seed solution of Sclerotium sclerotiorum
[0048] Scrape the mature mycelium of Sclerotium uniformis into the seed liquid culture medium, and culture the seed liquid on a shaker. The culture temperature is 30°C, the culture time is 72h, and the shaker speed is 220rpm. At the end of the culture, the seed liquid is in the form of white porridge, and the biomass of Sclerotium uniformis in the seed liquid is 9-12g / L.
[0049] (3) Production of scleroglucan by fermentation of Sclerotium sclerotium
[0050] The seed liquid was inoculated with 12% inoculation into a 5L fermentation tank (purchased from Shanghai Dibil Biotechnology Co., Ltd.) containing 3L fermentation medium for fermentation. The stirring paddle speed in the early stage of fermentation (0-12h) was 200rpm, and the fermentation tank ventilation was 0.1vvm. The DO in the fermentation liquid was monitored and it was found that the dissolved oxygen gradually decreased from 100% to 10% in 8-12h; the stirring paddle speed in the middle stage of fermentation (12-48h) was 400rpm, and the fermentation tank ventilation was 0.5vvm, maintaining the dissolved oxygen in the fermentation liquid at about 10%; the stirring paddle speed in the late stage of fermentation (48-108h) was 600rpm, and the fermentation tank ventilation was 1vvm. During the fermentation, the temperature was controlled at 30°C, and there was no need to adjust the pH of the fermentation liquid.
[0051] Figure 1The data of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth of this example are shown in FIG. Figure 1 It can be seen that the scleroglucan yield is 18.5 g / L and the glucose utilization rate is 64.7%.
[0052] Comparative Example 2
[0053] (1) Slant culture of Sclerotium sclerotiorum strains
[0054] The sclerotia of Sclerotium uniformis were inoculated onto PDA solid culture medium plates and cultured at 30°C for 3-4 days until the plates were covered with white hyphae and a small amount of white sclerotia, and a large number of mature Sclerotium uniformis were obtained for shake flask culture of seed liquid.
[0055] (2) Shake flask culture of seed solution of Sclerotium sclerotiorum
[0056] Scrape the mature mycelium of Sclerotium uniformis into the seed liquid culture medium, and culture the seed liquid on a shaker. The culture temperature is 30°C, the culture time is 72h, and the shaker speed is 220rpm. At the end of the culture, the seed liquid is in the form of white porridge, and the biomass of Sclerotium uniformis in the seed liquid is 9-12g / L.
[0057] (3) Production of scleroglucan by fermentation of Sclerotium sclerotium
[0058] The seed liquid was inoculated with 12% inoculum into a 5L fermentation tank (purchased from Dibil Bioengineering Shanghai Co., Ltd.) containing 3L fermentation medium, and the fermentation experiment was carried out in three groups. The stirring paddle speed of the first group during the entire fermentation period (108h) was 200rpm, and the fermentation tank ventilation was 1vvm. The stirring paddle speed of the second group during the entire fermentation period (108h) was 400rpm, and the fermentation tank ventilation was 1vvm. The stirring paddle speed of the third group during the entire fermentation period (108h) was 600rpm, and the fermentation tank ventilation was 1vvm. During the fermentation of the three groups of fermenters, the temperature was controlled at 30°C, and there was no need to regulate the pH of the fermentation liquid.
[0059] Figure 2 The data graphs are the scleroglucan yield, residual sugar, viscosity of the fermentation broth, and DCW of the three fermentation experiments in this comparative example, from left to right they are Group 1, Group 2, and Group 3. Figure 2 It can be seen that the scleroglucan production of group 1, group 2 and group 3 were 10.5 g / L, 14.5 g / L and 11.5 g / L, respectively, and the glucose utilization rates were 45.88%, 64.71% and 55.29%, respectively. Figure 3 is the CO2 content and O2 content in the tail gas of the fermentation broth of Group 1 and Group 3 in this example, as well as the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during the fermentation process, from Figure 3 It can be seen that in the first 36h of the fermentation process of Group 1 (200rpm), the CO2 and CER in the tail gas were much higher than those of Group 3 (600rpm), and the biomass in the fermentation broth of Group 1 was 25g / L, which was much higher than 14g / L of Group 3 ( Figure 2 ), which proves that low ventilation conditions in the early stage of fermentation are conducive to promoting the growth of the strain. After 36h of fermentation, the CER of Group 1 and Group 3 remained stable, indicating that the amount of carbon source used by the two was similar, and the carbon source of Group 1 (200rpm) was mostly used for strain growth (reaching 25g / L, Figure 1 ), while in group 3 (600 rpm), the pH decreased significantly ( Figure 2 ), indicating that although the production of scleroglucan increased in the later period, the carbon source was obviously more utilized in the production of organic acids.
[0060] Comparative Example 3
[0061] (1) Slant culture of Sclerotium sclerotiorum strains
[0062] The sclerotia of Sclerotium uniformis were inoculated onto PDA solid culture medium plates and cultured at 30°C for 3-4 days until the plates were covered with white hyphae and a small amount of white sclerotia, and a large number of mature Sclerotium uniformis were obtained for shake flask culture of seed liquid.
[0063] (2) Shake flask culture of seed solution of Sclerotium uniformis
[0064] Scrape the mature mycelium of Sclerotium uniformis into the seed liquid culture medium, and culture the seed liquid on a shaker. The culture temperature is 30°C, the culture time is 72h, and the shaker speed is 220rpm. At the end of the culture, the seed liquid is in the form of white porridge, and the biomass of Sclerotium uniformis in the seed liquid is 9-12g / L.
[0065] (3) Production of scleroglucan by fermentation of Sclerotium sclerotium
[0066] The seed liquid was inoculated with 12% inoculum into a 5L fermentation tank (purchased from Dibil Bioengineering Shanghai Co., Ltd.) containing 3L fermentation medium, and the fermentation experiment was carried out in three groups. The stirring paddle speed of the first group during the entire fermentation period (108h) was 400rpm, and the fermentation tank ventilation was 0.5vvm, the stirring paddle speed of the second group during the entire fermentation period was 400rpm, and the fermentation tank ventilation was 1vvm, and the stirring paddle speed of the third group during the entire fermentation period was 400rpm, and the fermentation tank ventilation was 2vvm. During the fermentation of the three groups of fermenters, the temperature was controlled at 30°C, and there was no need to regulate the pH of the fermentation liquid.
[0067] Figure 4 The data graphs are the scleroglucan yield, residual sugar, viscosity of the fermentation broth, and DCW of the three fermentation experiments in this comparative example, from left to right they are Group 1, Group 2, and Group 3. Figure 4 It can be seen that the scleroglucan production of group 1, group 2 and group 3 were 13.5 g / L, 15.5 g / L and 12.5 g / L, respectively, and the glucose utilization rates were 52.94%, 58.82% and 62.35%, respectively. Figure 5 is the CO2 content and O2 content in the tail gas of the fermentation broth of Group 1 and Group 3 in this example, as well as the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during the fermentation process, from Figure 5 It can be seen that when only the ventilation volume is increased, the oxygen uptake rate and CO2 release rate of Sclerotium are at a low level, indicating that only changing the ventilation volume cannot significantly improve the mass transfer and dissolved oxygen content of the fermentation liquid.
[0068] Comparative Example 4
[0069] The sclerotia of Sclerotium uniformis were inoculated onto PDA solid culture medium plates and cultured at 30°C for 3-4 days until the plates were covered with white hyphae and a small amount of white sclerotia, and a large number of mature Sclerotium uniformis were obtained for shake flask culture of seed liquid.
[0070] (2) Shake flask culture of seed solution of Sclerotium uniformis
[0071] Scrape the mature mycelium of Sclerotium uniformis into the seed liquid culture medium, and culture the seed liquid on a shaker. The culture temperature is 30°C, the culture time is 72h, and the shaker speed is 220rpm. At the end of the culture, the seed liquid is in the form of white porridge, and the biomass of Sclerotium uniformis in the seed liquid is 9-12g / L.
[0072] (3) Production of scleroglucan by fermentation of Sclerotium sclerotium
[0073] The seed liquid was inoculated with an inoculation rate of 12% in a 5L fermentation tank (purchased from Shanghai Dibil Biotechnology Co., Ltd.) containing 3L fermentation medium for fermentation. In the early stage of fermentation (0-48h), the speed of the stirring paddle was 600rpm, the ventilation volume of the fermentation tank was 1vvm, and the dissolved oxygen in the fermentation liquid was about 40%. In the subsequent late fermentation period (48-108h), the speed of the stirring paddle was adjusted to 400rpm, the ventilation volume of the fermentation tank was 0.5vvm, and the dissolved oxygen in the Sclerotium fermentation liquid dropped to 10% in 60h, and then the dissolved oxygen was maintained at about 10%. During the entire fermentation period, the temperature was controlled at 30°C, and there was no need to adjust the pH of the fermentation liquid.
[0074] Figure 6The data of scleroglucan yield, residual sugar, viscosity of fermentation broth and DCW in the fermentation broth of this example are shown in FIG. Figure 6 It can be seen that the scleroglucan yield is 14 g / L and the glucose utilization rate is 62.3%.
[0075] Comparative Example 5
[0076] (1) Slant culture of Sclerotium sclerotiorum strains
[0077] The sclerotia of Sclerotium uniformis were inoculated onto PDA solid culture medium plates and cultured at 30°C for 3-4 days until the plates were covered with white hyphae and a small amount of white sclerotia, and a large number of mature Sclerotium uniformis were obtained for shake flask culture of seed liquid.
[0078] (2) Shake flask culture of seed solution of Sclerotium uniformis
[0079] Scrape the mature mycelium of Sclerotium uniformis into the seed liquid culture medium, and culture the seed liquid on a shaker. The culture temperature is 30°C, the culture time is 72h, and the shaker speed is 220rpm. At the end of the culture, the seed liquid is in the form of white porridge, and the biomass of Sclerotium uniformis in the seed liquid is 9-12g / L.
[0080] (3) Production of scleroglucan by fermentation of Sclerotium sclerotium
[0081] The seed liquid was inoculated at an inoculation rate of 12% in a 5L fermentation tank (purchased from Debier Biotechnology Shanghai Co., Ltd.) containing 3L fermentation medium for fermentation. At the beginning of fermentation, the stirring paddle speed was controlled to 400rpm and the fermentation tank ventilation volume was 1vvm. When the dissolved oxygen in the fermentation liquid was reduced to 20%, the stirring paddle speed was adjusted to 600rpm and the fermentation tank ventilation volume was 2vvm to maintain the dissolved oxygen in the fermentation liquid at 20%.
[0082] Figure 7 The data of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth of this example are shown in FIG. Figure 7 It can be seen that the scleroglucan yield is 15 g / L and the glucose utilization rate is 57.6%.
[0083] Figure 8 The CO2 content and O2 content in the fermentation liquid tail gas of Example 1, Comparative Examples 4 and 5, and the CO2 release rate (CER) and oxygen uptake rate (OUR) of Sclerotium during fermentation, the red line in the figure represents Example 1, the blue line represents Comparative Example 4, and the black line represents Comparative Example 5. Figure 8It can be seen that Example 1 optimizes the dissolved oxygen conditions in the fermentation broth by adjusting the rotation speed and ventilation volume before, during and after fermentation. The CER of the microsclerotium in the fermentation broth is much higher than that of the other two comparative examples, proving that optimizing the dissolved oxygen conditions greatly increases the growth intensity of the bacteria in the logarithmic growth phase and is more conducive to the formation of later products. The OUR during the entire fermentation period of Example 1 is at a relatively low level, proving that the fermentation production of scleroglucan by the neat microsclerotium strain is an anaerobic process as a whole. Comparing the scleroglucan yields of Example 1 and Comparative Examples 2 to 5, it can be concluded that the fermentation process is divided into the early, middle and late stages using the method of the present invention, and the dissolved oxygen of the fermentation broth is regulated in stages. The final scleroglucan yield is significantly higher than that of other comparative examples that do not regulate dissolved oxygen in stages.
[0084] The present invention provides a concept and method for producing scleroglucan by fermentation of Sclerotium sclerotium based on dissolved oxygen regulation. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.
Claims
1. A method for producing scleroglucan by fermentation of Sclerotium sclerotium based on dissolved oxygen regulation, characterized in that: Scleroglucan is produced by fermentation using Sclerotium uniformis as the production strain. The entire fermentation cycle is divided into early fermentation period, middle fermentation period and late fermentation period. The dissolved oxygen content in the fermentation liquid is controlled by controlling the stirring speed and ventilation volume of the fermentation tank. In the early fermentation period, the dissolved oxygen is controlled to be reduced from 100% to 0-10%, in the middle fermentation period, the dissolved oxygen is controlled at 0-10%, and in the late fermentation period, the dissolved oxygen is controlled at 20-30%.
2. The method according to claim 1, characterized in that The early fermentation period is greater than or equal to the 0th hour of the fermentation cycle and less than the 12th hour of the fermentation cycle.
3. The method according to claim 1, characterized in that The mid-fermentation period is greater than or equal to the 12th hour of the fermentation cycle and less than the 48th hour of the fermentation cycle.
4. The method according to claim 1, characterized in that The late fermentation period is greater than or equal to the 48th hour of the fermentation cycle and less than or equal to the 108th hour of the fermentation cycle.
5. The method according to claim 1, characterized in that In the early stage of fermentation, the rotation speed is controlled at 100-200 rpm and the ventilation volume is controlled at 0.1-0.5 vvm.
6. The method according to claim 1, characterized in that In the middle stage of fermentation, the rotation speed is controlled at 200-400 rpm and the ventilation volume is controlled at 0.5-1 vvm.
7. The method according to claim 1, characterized in that In the late fermentation stage, the rotation speed is controlled at 600-800 rpm and the ventilation volume is controlled at 2-3 vvm.