Method for producing scleroglucan by immobilized continuous fermentation of sclerotium rolfsii
Fixed neat Rhizome micropores by micropore stirring paddles, solving the problem of decreased activity and low yield caused by accumulation of oxalic acid during continuous fermentation of Rhizome micropores, and achieving more efficient hard dextran production.
Patent Information
- Application Number
- CN202411663435.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-03-11
AI Technical Summary
In the prior art, when Rhizophobia continuously fermentes and produces hard dextran, the activity of bacteria decreases, due to the accumulation of harmful by-products such as oxalic acid, the fermentation cycle is long and the yield is low.
The micropore stirring paddle is used to fix the neat Rhizome microbial bacteria, and the mass transfer and immobilization of the fermentation broth are enhanced through the screw tape and micropore structure, achieving continuous fermentation.
Through immobilization treatment, the strain activity is maintained, the fermentation cycle is shortened, the productivity of hard dextran is improved, and the cost of product isolation and purification is reduced.
Smart Images
Figure CN119592643A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biological fermentation, and in particular relates to a method for producing scleroglucan by immobilized continuous fermentation of Sclerotium truncatum. Background Art
[0002] Sclerotium, also known as sclerotium gum, is a natural microbial polysaccharide produced by filamentous fungi of the genus Sclerotium. It has excellent antioxidant, analgesic, antitumor activity and thermal stability, and is widely used in medicine, food, and oil extraction. As a glucose polymer, sclerotium is a β-glucan composed of 3 β-1,3-linked d-pyranose as a repeating structural unit, and each structural unit is connected by a single β-1,6 bond to a glucose side chain. As an extracellular polysaccharide produced by fungi, sclerotium requires a low pH in the fermentation environment, which makes Sclerotium have the advantage of stable growth and low contamination in the early stage of fermentation. However, with the accumulation of harmful products such as oxalic acid during the fermentation process, the production of sclerotium in the late fermentation period is limited. In addition, sclerotium has a longer fermentation cycle than other fungal polysaccharides (one fermentation cycle is 96h), and in the pretreatment before the formal start of fermentation, strain activation requires 4 days and shake flask culture requires 3 days.
[0003] Fermentation in industry can be mostly divided into batch fermentation, fed-batch fermentation and continuous fermentation, among which batch fermentation has the characteristic of not being easy to be contaminated. When Sclerotium is continuously fermented to produce scleroglucan, due to the accumulation of toxic byproducts such as oxalic acid, the activity of the bacteria cannot be maintained for a long time. However, it is precisely because the pH of the environment in which Sclerotium ferments to produce scleroglucan is low that it is not easy to be contaminated during the fermentation process. However, due to the long preparation time for fermentation, the yield is low. The present invention is committed to maintaining the activity of the strain by fixing the sclerotium bacteria with a microporous stirring paddle for continuous fermentation, so as to shorten the fermentation cycle of the sclerotium bacteria and improve productivity. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a method for producing scleroglucan by immobilizing and continuously fermenting Sclerotium uniformis in view of the deficiencies of the prior art. The method uses the blades of a microporous stirring paddle to immobilize Sclerotium uniformis and continuously ferment to produce scleroglucan. During the fermentation process, the spiral ribbon and microporous structure of the blade enhance the mass transfer of the fermentation liquid and the immobilization effect of Sclerotium uniformis.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0006] A method for producing scleroglucan by immobilized continuous fermentation of Sclerotium truncatum comprises the following steps:
[0007] (1) Installing a microporous stirring paddle in a bioreactor;
[0008] (2) injecting fermentation medium into the bioreactor, inoculating the seed liquid of the Sclerotium truncatum into the fermentation medium, and performing the first batch of fermentation. As the fermentation proceeds, a large number of Sclerotium truncatum cells are fixed to the micropores of the microporous stirring paddle through hyphae, thereby achieving the immobilization of the Sclerotium truncatum;
[0009] (3) After the fermentation of the first batch is completed, the fermentation liquid obtained by the fermentation flows out of the bioreactor, the fermentation medium is added to the bioreactor, and the next batch of fermentation is continued using the uniform microsclerotium fixed on the microporous stirring paddle;
[0010] (4) After the fermentation is completed, step (3) is repeated to carry out continuous batch fermentation of immobilized Sclerotium truncatum to produce scleroglucan.
[0011] Wherein, nano-micropores are distributed on the microporous stirring paddle.
[0012] Wherein, the diameter of the nanopores is 100nm to 1000nm.
[0013] Wherein, the distribution density of the nano-micropores on the microporous stirring paddle is 30-80%.
[0014] Among them, the specific structural composition, structural parameters and manufacturing method of the microporous stirring paddle are disclosed in patent application number 2023112578685.
[0015] Preferably, the microporous stirring paddle is manufactured by a 3D printing process to obtain finer micropores, which can effectively improve the mixing efficiency of the fermentation liquid.
[0016] Preferably, taking a 5L bioreactor as an example, the specific structure and size design of the microporous stirring paddle are as follows:
[0017] The stirring shaft of the bioreactor is 30 cm long, the hub diameter is 12 mm, the height is 15 mm, the thickness is 2 mm, the spacing between the upper and lower hubs is 100 mm, the side of the hub contains three M5 threaded openings, the horizontal blades and the spiral blades are both set to 3 pieces, the spiral blades of the ribbon stirring paddle are 9 mm wide, the twist is 90°, the spiral blades of the ribbon stirring paddle are integrally formed with the two horizontal blades corresponding to the upper and lower horizontal stirring paddles, and are circumferentially installed on the hub at an interval of 120°. The blades of the ribbon stirring paddle and the horizontal stirring paddle are distributed with 800 nm micropores with a density of 80%.
[0018] Preferably, the microporous stirring paddle used in the present invention can also be formed by 3D printing using nylon material.
[0019] The microporous stirring paddle described in the present invention is a 3D printed microporous stirring paddle specialized for high-viscosity pseudoplastic fermentation fluids. During the fermentation of sclerotium gum, the paddle can fix a large number of fungal hyphae between the spiral ribbons of the paddle and can be used for continuous fermentation. Figure 1 The three views and the actual picture of the microporous stirring paddle used in the present invention; among them, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is the actual picture. Figure 2 This is a schematic diagram of the blade of the microporous stirring paddle used in the present invention; wherein, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is a schematic diagram of the micropores on the blade.
[0020] Wherein, the deposit number of the Sclerotium uniformum is ATCC24965.
[0021] Wherein, the inoculation amount of the Sclerotium uniformis seed solution is 10-13% of the volume of the fermentation medium.
[0022] Wherein, during the fermentation, the temperature is 28-30° C., the rotation speed of the microporous stirring paddle is 400-600 rpm, the ventilation volume of the fermentation tank is 1-2 vvm, and the fermentation time is 96-108 h.
[0023] Beneficial effects:
[0024] (1) The microporous stirring paddle used in the present invention fixes and adsorbs the uniform microorganisms of Sclerotium through the microporous structure on the ribbon stirring paddle, and screens out microorganisms with strong fermentation activity at the end of fermentation for continuous fermentation, thereby reducing the waste of microorganisms during fermentation, shortening the fermentation cycle of uniform microorganisms, and improving the productivity of scleroglucan.
[0025] (2) Compared with the existing batch fermentation method for producing scleroglucan, the continuous batch fermentation method can produce scleroglucan while avoiding the excessive growth of Sclerotium biomass in the fermentation broth and the excessive viscosity of the fermentation broth, thereby reducing the cost of product separation and purification.
[0026] (3) The method of the present invention is used to produce scleroglucan by fixed continuous batch fermentation. The yield of scleroglucan in the second batch of fermentation can still reach 13 g / L.
[0027] (4) The method of the present invention is used to carry out immobilized continuous fermentation of Sclerotium uniformis. Under immobilized batch fermentation, the blades used in the present invention have a larger contact area with the fungus and the fermentation liquid than traditional blades, and have a stronger ability to promote gas-liquid mass transfer in the highly viscous environment of the fungal fermentation liquid, which is beneficial to the fermentation of Sclerotium uniformis. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] 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.
[0029] Figure 1 The three views and the real picture of the microporous stirring paddle used in the present invention; wherein, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is the real picture;
[0030] The reference numerals represent: 1-hub; 2-horizontal stirring paddle; 3-ribbon stirring paddle; 4-micro-nano pores; 5-threaded hole.
[0031] Figure 2 This is a schematic diagram of the blade of the microporous stirring paddle used in the present invention; wherein, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is a schematic diagram of the micropores on the blade.
[0032] Figure 3 The graph shows the changes in scleroglucan yield, residual sugar, viscosity of the fermentation broth, and DCW in the fermentation broth during the first batch of fermentation using a microporous stirring paddle in Example 1.
[0033] Figure 4 This is a graph showing the changes in scleroglucan yield, residual sugar, fermentation broth viscosity, and DCW in the fermentation broth during the second batch of fermentation using a microporous stirring paddle in Example 2.
[0034] Figure 5 These are fermentation liquid state diagrams at 0h, 24h, 36h and 72h of the second batch fermentation during the immobilized continuous batch fermentation in Example 2; wherein, Figure A is for fermentation 0h, Figure B is for fermentation 24h, Figure C is for fermentation 36h, and Figure D is for fermentation 72h.
[0035] Figure 6 The graph is a change graph of scleroglucan yield, residual sugar, fermentation broth viscosity and DCW in the fermentation broth during the fermentation process using a common stirring paddle in Comparative Example 3.
[0036] Figure 7The graph is a graph showing the changes in scleroglucan yield, residual sugar, viscosity of the fermentation broth, and DCW in the fermentation broth during the fermentation process using a non-porous stirring paddle in Comparative Example 4.
[0037] Figure 8 These are the three views and the actual picture of the hole-free stirring paddle used in Comparative Example 4; among them, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is the actual picture. DETAILED DESCRIPTION
[0038] 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.
[0039] 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.
[0040] The fermenter used in the embodiments and comparative examples of the present invention was purchased from Dibier Biotechnology (Shanghai) Co., Ltd., model T&J-Intelli-Ferm A 5L. The fermenter was originally equipped with three ordinary stirring paddles (flat-blade disc turbine agitators), and the top stirring paddle in the fermenter was a serrated paddle, which was mainly used to handle the overflow bubbles in the early stage of fermentation.
[0041] The specific structure and parameters of the microporous stirring paddle used in the following examples are as follows:
[0042] The stirring shaft of the fermenter is 30cm long, the hub diameter is 12mm, the height is 15mm, the thickness is 2mm, the spacing between the upper and lower hubs is 100mm, the side of the hub contains three M5 threaded openings, the horizontal blades and the spiral blades are set to 3 pieces, the width of the spiral blades of the ribbon stirring paddle is 9mm, the twist is 90°, the spiral blades of the ribbon stirring paddle and the two horizontal blades corresponding to the upper and lower horizontal stirring paddles are integrally formed, and are circumferentially installed on the hub at an interval of 120°. The blades of the ribbon stirring paddle and the horizontal stirring paddle are distributed with 800nm micropores with a density of 80%. Nylon material is used for 3D printing.
[0043] The formulas of the solid PDA medium, seed liquid medium and fermentation medium used in the following examples are as follows:
[0044] PDA solid culture medium: potato 200g / L; glucose 20g / L; agar 20g / L.
[0045] Seed liquid culture medium: glucose 30.0g / L; KH2 PO 4 10g / L; (NH 4 ) 2 SO 4 3.0g / L; yeast extract 1.0g / L; KCl 0.5g / L; MgSO 4 :0.25g / L.
[0046] Fermentation medium: glucose 85.0g / L; KH 2 PO 4 1.0g / L; (NH 4 ) 2 SO 4 3.0g / L; Yeast extract powder 1.0g / L; KCl 0.5g / L; Citric acid 1.0g / L; MgSO 4 0.5g / L.
[0047] All the above culture media were sterilized at 115℃ for 20min before use.
[0048] 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.
[0049] 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.
[0050] The residual sugar content of glucose at different fermentation stages was determined using a biosensor analyzer (purchased from Hillman Technology Co., Ltd., model: S-10).
[0051] The viscosity of the fermentation broth was measured using an NDJ-8J viscometer (purchased from Shanghai Fangrui Technology Co., Ltd.).
[0052] Example 1
[0053] (1) Activation of Sclerotium sclerotium strains
[0054] The sclerotia of Sclerotium uniformis stored at -60°C were inoculated into PDA solid culture medium (potato dextrose agar medium) and cultured at 30°C for 3-4 days until the plate was 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 uniformis
[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 second and third common stirring paddles (flat blade disc turbine stirrer) in the 5L fermenter from top to bottom were replaced with the microporous stirring paddles, and the spacing between the stirring paddles was 60 mm. The seed liquid was inoculated with an inoculum of 12% in a 5L fermenter containing 3L fermentation medium for fermentation. During the entire fermentation process, there was no need to adjust the pH of the fermentation liquid, the stirring paddle speed was controlled to 400 rpm, the fermenter ventilation was 1 vvm, the temperature was 30°C, and the fermentation time was 108 h.
[0059] During the fermentation process, the production of scleroglucan, residual sugar, viscosity of the fermentation broth and DCW were monitored. Figure 3 The results showed that the yield of scleroglucan reached 17.5 g / L when the microporous stirring blade was used for fermentation, and the biomass of the whole Sclerotium was 22 g / L at the end of the fermentation.
[0060] Example 2
[0061] After the fermentation of Example 1 was completed, a large number of neat Sclerotium bodies were fixed on the microporous stirring paddle, 90% of the fermentation liquid in the fermenter was discharged, and then 3L of fresh fermentation medium was added to the fermenter, and the second batch of fermentation was continued using the microporous stirring paddle with neat Sclerotium fixed. The fermentation conditions during the fermentation process were the same as those in Example 1.
[0062] During the fermentation process, the production of scleroglucan, residual sugar, viscosity of the fermentation broth and DCW were monitored. Figure 4The results showed that after 48 hours of fermentation, the yield of scleroglucan was maintained at 13 g / L, and the biomass of Sclerotium uniformis was 15.5 g / L at the end of fermentation. There was no bacterial contamination during the fermentation process, and the biomass of Sclerotium uniformis in the fermentation broth of the second batch of fermentation was less than that of the first batch (22 g / L). Therefore, the cost of extracting and separating scleroglucan from the fermentation broth can be reduced, and the difficulty of cleaning the stirring paddle is also reduced.
[0063] Figure 5 These are fermentation liquid state diagrams at 0h, 24h, 36h and 72h of the second batch fermentation during the immobilized continuous batch fermentation in this embodiment; wherein, Figure A is for fermentation 0h, Figure B is for fermentation 24h, Figure C is for fermentation 36h, and Figure D is for fermentation 72h.
[0064] Comparative Example 3
[0065] Fermentation was carried out using a common stirring paddle (flat-blade disc turbine stirrer) in a 5L fermenter. The neat Sclerotium seed liquid obtained by culture in Example 1 was inoculated at an inoculation rate of 12% into a 5L fermenter containing 3L fermentation medium for fermentation. The fermentation conditions during the fermentation process were the same as in Example 1.
[0066] During the fermentation process, the production of scleroglucan, residual sugar, viscosity of the fermentation broth and DCW were monitored. Figure 6 The results showed that the yield of scleroglucan reached 15.5 g / L when fermented with a common stirring paddle.
[0067] During the fermentation process of this comparative example, almost no bacteria attached and grew on the ordinary stirring paddle, so continuous batch fermentation could not be carried out.
[0068] Comparative Example 4
[0069] The second and third common stirring paddles (flat blade disc turbine agitators) from top to bottom in the 5L fermenter were replaced with non-porous stirring paddles with the same structure, parameters and manufacturing method as the microporous stirring paddles, and the spacing between the paddles was 60 mm. The neat Sclerotium seed liquid obtained by culture in Example 1 was inoculated with an inoculum amount of 12% in a 5L fermenter containing 3L fermentation medium for fermentation, and the fermentation conditions during the fermentation process were the same as those in Example 1.
[0070] During the fermentation process, the production of scleroglucan, residual sugar, viscosity of the fermentation broth and DCW were monitored. Figure 7The results showed that the yield of scleroglucan reached 16.5 g / L when the non-porous stirring paddle was used for fermentation.
[0071] During the fermentation process of this comparative example, almost no bacteria attached and grew on the non-porous stirring paddle, so continuous batch fermentation could not be carried out.
[0072] Figure 8 These are the three views and the actual picture of the hole-free stirring paddle used in this comparative example; among them, Figure A is the front view; Figure B is the top view; Figure C is the horizontal view; and Figure D is the actual picture.
[0073] The present invention provides a concept and method for producing scleroglucan by immobilized continuous fermentation of Sclerotium sclerotium. 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 protection scope 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 immobilized continuous fermentation of Sclerotium truncatum, characterized in that: The following steps are involved: (1) Installing a microporous stirring paddle in a bioreactor; (2) injecting a fermentation medium into the bioreactor, inoculating a seed solution of the Sclerotium truncatum into the fermentation medium, and performing a first batch of fermentation, wherein as the fermentation proceeds, the Sclerotium truncatum is fixedly grown on the microporous stirring paddle; (3) After the fermentation of the first batch is completed, the fermentation liquid obtained by the fermentation flows out of the bioreactor, the fermentation medium is added to the bioreactor, and the next batch of fermentation is continued using the uniform microsclerotium fixed on the microporous stirring paddle; (4) After the fermentation is completed, step (3) is repeated to carry out continuous batch fermentation of immobilized Sclerotium truncatum to produce scleroglucan.
2. The method according to claim 1, characterized in that: In step (1), nanopores are distributed on the microporous stirring paddle.
3. The method according to claim 2, characterized in that The diameter of the nanopores is 100nm to 1000nm.
4. The method according to claim 2, characterized in that: The distribution density of the nano-micropores on the microporous stirring paddle is 30-80%.
5. The method according to claim 1, characterized in that In step (1), the inoculation amount of the Sclerotium uniformis seed solution is 10-13% of the volume of the fermentation medium.
6. The method according to claim 1, characterized in that In step (1) and step (3), during the fermentation, the rotation speed of the microporous stirring paddle is 400-600 rpm, and the ventilation volume of the bioreactor is 1-2 vvm.
7. The method according to claim 1, characterized in that In step (1) and step (3), when the fermentation is carried out, the temperature of the fermentation liquid is 28-30° C. and the fermentation time is 96-108 hours.