Fermentation production method of mannase
Through mixed fermentation of Saccharomyces cerevisiae and Bacillus amylase, combined with the regulation of fermentation conditions, the problems of poor induction intensity and high cost in mannanase production are solved, and efficient and economical mannanase production is achieved.
Patent Information
- Application Number
- CN202510304444.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-05-06
AI Technical Summary
There are problems of poor induction intensity and high cost in the production process of existing mannanases, which affects the development of industrialization.
The mixed fermentation mode of Saccharomyces cerevisiae and Bacillus amylase is adopted to control fermentation conditions such as temperature, pH and ventilation, and regulate dissolved oxygen and feeding rate to promote spore germination and mannanase synthesis.
The enzyme activity of mannanase is improved, the production cost is reduced, the problem of poor induction intensity in the existing technology is overcome, and the development of industrialization is promoted.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a fermentation production method of mannanase. Background Art
[0002] When mannan is present in feed, it will increase the viscosity of chyme, and the ability of the intestine to mechanically mix the contents will be severely hindered. Mannan will mix with enzymes, substrates and other substances, reduce the activity of enzymes, affect the digestion and absorption of fat, reduce the absorption of water, and change the intestinal microbiome, intensify the competition between the host and bacteria for nutrients, increase the diarrhea rate of animals, thereby affecting the health of animals and inhibiting the growth of animals. In addition, it will also reduce the energy of feed. It not only acts as a nutrient diluent to directly affect the apparent digestible energy value of feed, but also reduces the rate of glucose absorption from the intestine and the metabolic process of carbohydrates by interfering with insulin secretion and insulin-like growth factor production. Mannan will over-stimulate the immune response in livestock and poultry in incompletely developed intestinal cells or under stressful environments, causing damage to growth performance, causing adverse immune responses, reduced food intake, slower growth, and an increase in the number of light-weight animals and a worsening of group uniformity.
[0003] Mannanase is a common growth promoter in feed, which can reduce the content of mannan in feed, thereby eliminating the adverse effects of mannan on livestock. However, the synthesis of mannanase has problems such as unsatisfactory induction cost (most mannan finished products are expensive) and induction strength (because mannan is easily mixed with enzymes, thus affecting its induction effect), which has great limitations and also affects the development of mannanase industrialization. Summary of the invention
[0004] The purpose of the invention is to overcome the problems of poor induction strength and high cost in the existing mannanase production process.
[0005] To this end, the present invention provides a fermentation production method of mannanase, comprising the following steps: simultaneously inoculating saccharomyces cerevisiae and bacillus amyloliquefaciens into a fermentation substrate for fermentation to obtain mannanase.
[0006] Specifically, based on the volume of the fermentation substrate, the inoculation amounts of saccharomyces cerevisiae and Bacillus amyloliquefaciens are 1-2% respectively.
[0007] Specifically, the initial fermentation temperature is 29-32° C., the initial ventilation volume is 0.5 vvm, the initial fermentation pH is controlled at 3.60-4.00, stirring and ventilation volume are controlled during the fermentation process, and feeding begins when the dissolved oxygen content rebounds.
[0008] Specifically, during the above-mentioned feeding process, the feeding speed is adjusted so that the dissolved oxygen content is controlled at 40-50%.
[0009] Specifically, the initial feeding rate is 40 mL / h. The feeding rate is adjusted to control the dissolved oxygen content at 40-50%. The maximum feeding rate is 145 mL / h.
[0010] Specifically, from 63 to 67 hours of fermentation, the pH setting value is increased by 0.2 per hour, the maximum pH value is 4.60-4.80, the fermentation temperature is increased to 36-38°C, and then the feeding is stopped. When the dissolved oxygen rebounds to more than 80% and the pH exceeds the setting value by more than 0.10, the feeding is started again.
[0011] Specifically, each liter of the feed contains 800-900 g / L of carbon source, and the remainder is water.
[0012] Specifically, each liter of the fermentation base material includes 24-30 g / L carbon source, 30-50 g / L nitrogen source, 0.2-0.4 g / L trace elements, 0.2 g / L defoaming agent, and the balance is water.
[0013] Specifically, the carbon source includes carbohydrate hydrolyzate; the nitrogen source includes one or more of soybean meal, corn steep liquor, peptone, soybean cake powder, ammonium sulfate, and ammonium chloride; and the trace elements include one or more of ferrous sulfate, cobalt chloride, manganese sulfate, zinc sulfate, cupric chloride, and magnesium bromide.
[0014] Specifically, the method for preparing the above-mentioned carbohydrate hydrolyzate is: add water and phosphoric acid to one or more of lactose, sucrose, glucose, starch, glycerol, and microcrystalline cellulose, mix them evenly, and react to obtain the corresponding carbohydrate hydrolyzate.
[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0016] The fermentation production method of the mannanase provided by the present invention uses a mixed fermentation mode of saccharomyces cerevisiae and bacillus amyloliquefaciens to produce mannanase. During the fermentation process, saccharomyces cerevisiae can utilize reducing sugar metabolism to produce ethanol in large quantities, while for bacteria without cell walls, the damage of ethanol to cells can only be reduced by forming spores. With the growth and metabolism of saccharomyces cerevisiae, ethanol gradually accumulates in the fermentation system, resulting in more and more bacillus amyloliquefaciens forming spores. When the amount of saccharomyces cerevisiae is large, the growth of saccharomyces cerevisiae is slowed down by starvation and then feeding, and the temperature and pH are adjusted to artificially advance the spore germination. As the temperature increases, the ethanol volatilization is accelerated, which accelerates the decline of saccharomyces cerevisiae, and its cell wall (main component is mannan) is broken and metabolites are released from the cell, the fermentation system is rich in nutrients, and spores begin to germinate. Bacillus amyloliquefaciens will secrete mannanase to decompose the cell wall of saccharomyces cerevisiae and produce reducing sugars to provide energy for its own growth. DETAILED DESCRIPTION
[0017] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.
[0018] The present invention provides a fermentation production method of mannanase, comprising the following steps:
[0019] S1. Preparation of Saccharomyces cerevisiae and Bacillus amyloliquefaciens seed solution
[0020] 1.1 Saccharomyces cerevisiae seed culture
[0021] (1) Saccharomyces cerevisiae is inoculated onto a solid MEA medium and cultured at 28° C. for 2-3 days. When the bacterial balls cover the entire surface of the solid MEA medium, mature solid Saccharomyces cerevisiae seeds can be obtained.
[0022] (2) Scrape the brewer's yeast from the solid MEA medium with an inoculation loop and inoculate it into the solid MEA medium. Cultivate it at 28°C and 200 rpm for 2-3 days to obtain a mature brewer's yeast seed liquid.
[0023] 1.2 Seed culture of Bacillus amyloliquefaciens subspecies
[0024] (1) Inoculate Bacillus amyloliquefaciens onto solid NA medium and culture at 37° C. for 1-2 days to obtain mature solid seeds.
[0025] (2) Scrape the Bacillus amyloliquefaciens from the solid NA medium with an inoculation loop and inoculate it into the liquid NA medium. Cultivate it at 37° C. and 150 rpm for 2-3 days to obtain a mature seed liquid of the Bacillus amyloliquefaciens subspecies.
[0026] S2. Prepare fermentation base
[0027] Each liter of the fermentation base includes 24-30g / L carbon source, 30-50g / L nitrogen source, 0.2-0.4g / L trace elements, 0.2g / L defoamer, and the balance is water. That is, 24-30g carbon source, 30-50g nitrogen source, 0.2-0.4g trace elements, 0.2g defoamer are added with water to 1L.
[0028] Preferably, the carbon source includes carbohydrate hydrolyzate; the nitrogen source includes one or more of soybean meal, corn steep liquor, peptone, soybean cake powder, ammonium sulfate, and ammonium chloride; and the trace elements include one or more of ferrous sulfate, cobalt chloride, manganese sulfate, zinc sulfate, cupric chloride, and magnesium bromide.
[0029] The preparation method of the carbohydrate hydrolyzate is as follows: one or more of lactose, sucrose, glucose, starch, glycerol, and microcrystalline cellulose are dissolved in water at a solid-liquid ratio of 4:1 (heating is possible if necessary), 80% phosphoric acid is added at a ratio of 0.25 mL / kg, mixed evenly, reacted at 0.1 MPa for 40-50 minutes, and the volume is fixed to a solid mass concentration of 75%, thereby obtaining the corresponding carbohydrate hydrolyzate.
[0030] Each liter of the feed includes 800-900 g / L of carbon source, and the balance is water, that is, 800-900 g of carbon source is added with water to make up to 1 L. The carbon source preferably includes carbohydrate hydrolyzate.
[0031] After the base material and supplementary material are prepared according to the above formula, sterilize at 115°C, 0.065-0.075MPa for 30 minutes. After sterilization, cool to 30±2°C, and maintain positive pressure in the fermenter during this period.
[0032] S3. Vaccination
[0033] After the fermentation base is sterilized, the mature seed liquid is inoculated into the fermentation base in the presence of an external flame. The inoculation amount of brewer's yeast and Bacillus amyloliquefaciens is 1-2% respectively based on the volume of the fermentation base.
[0034] S4. Fermentation
[0035] The initial fermentation temperature is 29-32°C, the initial ventilation volume is 0.5vvm, the initial fermentation pH is controlled at 3.60-4.00, stirring and ventilation volume are controlled during the fermentation process, and feeding begins after the dissolved oxygen content rebounds. During the feeding process, the feeding speed is adjusted, the initial feeding speed is 40mL / h, and the feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The maximum feeding speed is 145mL / h, and the dissolved oxygen content is controlled at 40-50%.
[0036] From 63 to 67 hours of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.60-4.80, and the fermentation temperature was increased to 36-38°C. Feeding was then stopped. When the dissolved oxygen rebounded to above 80% and the online pH exceeded the set value, i.e. the pH was increased to a maximum value of above 0.10, feeding was started again. After the fermentation was completed, the mannanase activity was measured.
[0037] The effect of the fermentation production method of mannanase of the present invention is studied through specific examples below.
[0038] Embodiment 1:
[0039] This embodiment provides a fermentation production method of mannanase, comprising the following steps:
[0040] S1. Preparation of Saccharomyces cerevisiae and Bacillus amyloliquefaciens seed solution
[0041] 1.1 Saccharomyces cerevisiae seed culture
[0042] (1) Saccharomyces cerevisiae CICC 32883 was inoculated onto a solid MEA medium and cultured at 28° C. for 2 days. When the bacterial balls covered the entire surface of the solid MEA medium, mature solid Saccharomyces cerevisiae seeds were obtained.
[0043] (2) Scrape three rings of Saccharomyces cerevisiae from the solid MEA medium with an inoculation loop and inoculate into the solid MEA medium. Cultivate at 28°C and 200 rpm for 3 days to obtain mature Saccharomyces cerevisiae seed liquid.
[0044] 1.2 Seed culture of Bacillus amyloliquefaciens subspecies
[0045] (1) Inoculate Bacillus amyloliquefaciens CGMCC15838 onto solid NA medium and culture at 37°C for 2 days to obtain mature solid seeds.
[0046] (2) Scrape three rings of Bacillus amyloliquefaciens from the solid NA medium with an inoculation loop and inoculate them into liquid NA medium. Cultivate them at 37°C and 150 rpm for 2 days to obtain mature Bacillus amyloliquefaciens subspecies seed liquid.
[0047] S2. Prepare fermentation base material and supplementary material
[0048] The fermentation base material includes 405g of carbon source, 600g of nitrogen source, 4.5g of trace elements, 3g of defoaming agent, and water is added to make up to 15L.
[0049] The carbon source is glucose hydrolyzate, and the preparation method is as follows: dissolve 20 kg of glucose in 5 L of water, add 6.25 mL of 80% phosphoric acid, mix well, react at 0.1 MPa for 45 minutes, add water to make the volume to 26.7 kg, and obtain the corresponding carbohydrate hydrolyzate.
[0050] The nitrogen source includes ammonium sulfate and peptone in a mass ratio of 1:1.
[0051] Trace elements include ferrous sulfate, cobalt chloride, manganese sulfate and zinc sulfate in a mass ratio of 1:9:10:10.
[0052] The feed consisted of 17 kg of glucose hydrolysate, made up to 20 L with water.
[0053] Prepare the base material according to the above formula, place it in a fermentation tank, and sterilize it at 115℃, 0.065-0.075MPa for 30min. After sterilization, cool it to 28℃, and maintain positive pressure in the fermentation tank.
[0054] S3. Vaccination
[0055] In the presence of an external flame, the cultured mature seed liquid is inoculated into the fermentation base material. Based on the mass of the fermentation base material, the inoculation amount of brewer's yeast and Bacillus amyloliquefaciens is 2% each.
[0056] S4. Fermentation
[0057] The initial fermentation temperature is 29°C, the initial fermentation pH is controlled at 3.70, the fermentation process is stirred and the ventilation is controlled, when the dissolved oxygen in the fermentation tank is lower than 50%, the wind and speed are increased alternately, and the speed is increased by 12% of the maximum speed each time, and the upper limit of the stirring speed is 90% of the maximum stirring speed of the fermentation tank. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0058] After the stirring speed and ventilation volume are increased to the maximum, the dissolved oxygen content in the fermentation tank drops to the minimum and rebounds to more than 50%, and then the feeding begins. The initial feeding speed is 40mL / h. The feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The feeding speed is increased by 15mL / h each time, and the maximum feeding speed is 145mL / h. If the dissolved oxygen content is higher than 50% when the maximum feeding speed is reached, the stirring speed and ventilation volume can be appropriately reduced to keep the dissolved oxygen content no more than 50%.
[0059] From the 65th hour of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.80, and the fermentation temperature was increased to 38°C. Feeding was then stopped. When the dissolved oxygen rebounded to more than 80% and the online pH exceeded the set value by more than 0.10, feeding was started again. At this time, the initial feeding rate was 40 mL / h, and the feeding rate was adjusted by controlling the dissolved oxygen level at around 30%. The maximum feeding rate was 100 mL / h, and the fermentation cycle was 180 h. After the fermentation, the mannanase activity was measured. The results are shown in Table 1.
[0060] Embodiment 2:
[0061] This embodiment provides a fermentation production method of mannanase, comprising the following steps:
[0062] S1. Preparation of Saccharomyces cerevisiae and Bacillus amyloliquefaciens seed solution
[0063] 1.1 Saccharomyces cerevisiae seed culture
[0064] (1) Saccharomyces cerevisiae CGMCC 4747 was inoculated onto a solid MEA medium and cultured at 28° C. for 2 days. When the bacterial balls covered the entire surface of the solid MEA medium, mature solid Saccharomyces cerevisiae seeds were obtained.
[0065] (2) Scrape three rings of Saccharomyces cerevisiae from the solid MEA medium with an inoculation loop and inoculate into the solid MEA medium. Cultivate at 28°C and 200 rpm for 3 days to obtain mature Saccharomyces cerevisiae seed liquid.
[0066] 1.2 Seed culture of Bacillus amyloliquefaciens subspecies
[0067] (1) Inoculate Bacillus amyloliquefaciens CGMCC15838 onto solid NA medium and culture at 37°C for 2 days to obtain mature solid seeds.
[0068] (2) Scrape three rings of Bacillus amyloliquefaciens from the solid NA medium with an inoculation loop and inoculate them into liquid NA medium. Cultivate them at 37°C and 150 rpm for 2 days to obtain mature Bacillus amyloliquefaciens subspecies seed liquid.
[0069] S2. Prepare fermentation base material and supplementary material
[0070] The fermentation base material includes 405g of carbon source, 600g of nitrogen source, 4.5g of trace elements, 3g of defoaming agent, and water is added to make up to 15L.
[0071] The carbon source is glucose hydrolyzate, and the preparation method is as follows: dissolve 20 kg of glucose in 5 L of water, add 6.25 mL of 80% phosphoric acid, mix well, react at 0.1 MPa for 45 minutes, add water to make the volume to 26.7 kg, and obtain the corresponding carbohydrate hydrolyzate.
[0072] The nitrogen source includes ammonium sulfate and peptone in a mass ratio of 1:1.
[0073] Trace elements include ferrous sulfate, cobalt chloride, manganese sulfate and zinc sulfate in a mass ratio of 1:9:10:10.
[0074] The feed consisted of 17 kg of glucose hydrolysate, made up to 20 L with water.
[0075] Prepare the base material according to the above formula, place it in a fermentation tank, and sterilize it at 115℃, 0.065-0.075MPa for 30min. After sterilization, cool it to 28℃, and maintain positive pressure in the fermentation tank.
[0076] S3. Vaccination
[0077] In the presence of an external flame, the cultured mature seed liquid is inoculated into the fermentation base material. Based on the mass of the fermentation base material, the inoculation amount of brewer's yeast and Bacillus amyloliquefaciens is 2% each.
[0078] S4. Fermentation
[0079] The initial fermentation temperature is 29°C, the initial fermentation pH is controlled at 3.70, the fermentation process is stirred and the ventilation is controlled, when the dissolved oxygen in the fermentation tank is lower than 50%, the wind and speed are increased alternately, and the speed is increased by 12% of the maximum speed each time, and the upper limit of the stirring speed is 90% of the maximum stirring speed of the fermentation tank. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0080] After the stirring speed and ventilation volume are increased to the maximum, the dissolved oxygen content in the fermentation tank drops to the minimum and rebounds to more than 50%, and then the feeding begins. The initial feeding speed is 40mL / h. The feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The feeding speed is increased by 15mL / h each time, and the maximum feeding speed is 145mL / h. If the dissolved oxygen content is higher than 50% when the maximum feeding speed is reached, the stirring speed and ventilation volume can be appropriately reduced to keep the dissolved oxygen content no more than 50%.
[0081] From the 65th hour of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.80, and the fermentation temperature was increased to 38°C. Feeding was then stopped. When the dissolved oxygen rebounded to more than 80% and the online pH exceeded the set value by more than 0.10, feeding was started again. At this time, the initial feeding rate was 40 mL / h, and the feeding rate was adjusted by controlling the dissolved oxygen level at around 30%. The maximum feeding rate was 100 mL / h, and the fermentation cycle was 180 h. After the fermentation, the mannanase activity was measured. The results are shown in Table 1.
[0082] Embodiment 3:
[0083] This embodiment provides a fermentation production method of mannanase, comprising the following steps:
[0084] S1. Preparation of Saccharomyces cerevisiae and Bacillus amyloliquefaciens seed solution
[0085] 1.1 Saccharomyces cerevisiae seed culture
[0086] (1) Saccharomyces cerevisiae CICC30024 was inoculated onto a solid MEA medium and cultured at 28° C. for 2 days. When the bacterial balls covered the entire surface of the solid MEA medium, mature solid Saccharomyces cerevisiae seeds were obtained.
[0087] (2) Scrape three rings of Saccharomyces cerevisiae from the solid MEA medium with an inoculation loop and inoculate into the solid MEA medium. Cultivate at 28°C and 200 rpm for 3 days to obtain mature Saccharomyces cerevisiae seed liquid.
[0088] 1.2 Seed culture of Bacillus amyloliquefaciens subspecies
[0089] (1) Inoculate Bacillus amyloliquefaciens CGMCC15838 onto solid NA medium and culture at 37°C for 2 days to obtain mature solid seeds.
[0090] (2) Scrape three rings of Bacillus amyloliquefaciens from the solid NA medium with an inoculation loop and inoculate them into liquid NA medium. Cultivate them at 37°C and 150 rpm for 2 days to obtain mature Bacillus amyloliquefaciens subspecies seed liquid.
[0091] S2. Prepare fermentation base material and supplementary material
[0092] The fermentation base material includes 405g of carbon source, 600g of nitrogen source, 4.5g of trace elements, 3g of defoaming agent, and water is added to make up to 15L.
[0093] The carbon source is glucose hydrolyzate, and the preparation method is as follows: dissolve 20 kg of glucose in 5 L of water, add 6.25 mL of 80% phosphoric acid, mix well, react at 0.1 MPa for 45 minutes, add water to make the volume to 26.7 kg, and obtain the corresponding carbohydrate hydrolyzate.
[0094] The nitrogen source includes ammonium sulfate and peptone in a mass ratio of 1:1.
[0095] The trace elements include ferrous sulfate, cobalt chloride, manganese sulfate and zinc sulfate in a mass ratio of 1:9:10:10.
[0096] The feed consisted of 17 kg of glucose hydrolysate, made up to 20 L with water.
[0097] Prepare the base material according to the above formula, place it in a fermentation tank, and sterilize it at 115℃, 0.065-0.075MPa for 30min. After sterilization, cool it to 28℃, and maintain positive pressure in the fermentation tank.
[0098] S3. Vaccination
[0099] In the presence of an external flame, the cultured mature seed liquid is inoculated into the fermentation base material. Based on the mass of the fermentation base material, the inoculation amount of brewer's yeast and Bacillus amyloliquefaciens is 2% each.
[0100] S4. Fermentation
[0101] The initial fermentation temperature is 29°C, the initial fermentation pH is controlled at 3.70, the fermentation process is stirred and the ventilation is controlled, when the dissolved oxygen in the fermentation tank is lower than 50%, the wind and speed are increased alternately, and the speed is increased by 12% of the maximum speed each time, and the upper limit of the stirring speed is 90% of the maximum stirring speed of the fermentation tank. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0102] After the stirring speed and ventilation volume are increased to the maximum, the dissolved oxygen content in the fermentation tank drops to the minimum and rebounds to more than 50%, and then the feeding begins. The initial feeding speed is 40mL / h. The feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The feeding speed is increased by 15mL / h each time, and the maximum feeding speed is 145mL / h. If the dissolved oxygen content is higher than 50% when the maximum feeding speed is reached, the stirring speed and ventilation volume can be appropriately reduced to keep the dissolved oxygen content no more than 50%.
[0103] From the 65th hour of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.80, and the fermentation temperature was increased to 38°C. Feeding was then stopped. When the dissolved oxygen rebounded to more than 80% and the online pH exceeded the set value by more than 0.10, feeding was started again. At this time, the initial feeding rate was 40 mL / h, and the feeding rate was adjusted by controlling the dissolved oxygen level at around 30%. The maximum feeding rate was 100 mL / h, and the fermentation cycle was 180 h. After the fermentation, the mannanase activity was measured. The results are shown in Table 1.
[0104] Comparative Example 1:
[0105] This comparative example provides a fermentation production method of mannanase, comprising the following steps:
[0106] S1. Preparation of Saccharomyces cerevisiae seed solution
[0107] (1) Saccharomyces cerevisiae CICC 32883 was inoculated onto a solid MEA medium and cultured at 28° C. for 2 days. When the bacterial balls covered the entire surface of the solid MEA medium, mature solid Saccharomyces cerevisiae seeds were obtained.
[0108] (2) Scrape three rings of Saccharomyces cerevisiae from the solid MEA medium with an inoculation loop and inoculate into the solid MEA medium. Cultivate at 28°C and 200 rpm for 3 days to obtain mature Saccharomyces cerevisiae seed liquid.
[0109] S2. Prepare fermentation base material and supplementary material
[0110] The fermentation base material includes 405g of carbon source, 600g of nitrogen source, 4.5g of trace elements, 3g of defoaming agent, and water is added to make up to 15L.
[0111] The carbon source is glucose hydrolyzate, and the preparation method is as follows: dissolve 20 kg of glucose in 5 L of water, add 6.25 mL of 80% phosphoric acid, mix well, react at 0.1 MPa for 45 minutes, add water to make the volume to 26.7 kg, and obtain the corresponding carbohydrate hydrolyzate.
[0112] The nitrogen source includes ammonium sulfate and peptone in a mass ratio of 1:1.
[0113] The trace elements include ferrous sulfate, cobalt chloride, manganese sulfate and zinc sulfate in a mass ratio of 1:9:10:10.
[0114] The feed consisted of 17 kg of glucose hydrolysate, made up to 20 L with water.
[0115] Prepare the base material according to the above formula, place it in a fermentation tank, and sterilize it at 115℃, 0.065-0.075MPa for 30min. After sterilization, cool it to 28℃, and maintain positive pressure in the fermentation tank.
[0116] S3. Vaccination
[0117] In the presence of an external flame, the cultured mature seed liquid is inoculated into the fermentation base material, and the inoculation amount of brewer's yeast is 2% based on the mass of the fermentation base material.
[0118] S4. Fermentation
[0119] The initial fermentation temperature is 29°C, the initial fermentation pH is controlled at 3.70, the fermentation process is stirred and the ventilation is controlled, when the dissolved oxygen in the fermentation tank is lower than 50%, the wind and speed are increased alternately, and the speed is increased by 12% of the maximum speed each time, and the upper limit of the stirring speed is 90% of the maximum stirring speed of the fermentation tank. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0120] After the stirring speed and ventilation volume are increased to the maximum, the dissolved oxygen content in the fermentation tank drops to the minimum and rebounds to more than 50%, and then the feeding begins. The initial feeding speed is 40mL / h. The feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The feeding speed is increased by 15mL / h each time, and the maximum feeding speed is 145mL / h. If the dissolved oxygen content is higher than 50% when the maximum feeding speed is reached, the stirring speed and ventilation volume can be appropriately reduced to keep the dissolved oxygen content no more than 50%.
[0121] From the 65th hour of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.80, and the fermentation temperature was increased to 38°C. Feeding was then stopped. When the dissolved oxygen rebounded to more than 80% and the online pH exceeded the set value by more than 0.10, feeding was started again. At this time, the initial feeding rate was 40 mL / h, and the feeding rate was adjusted by controlling the dissolved oxygen level at around 30%. The maximum feeding rate was 100 mL / h, and the fermentation cycle was 180 h. After the fermentation, the mannanase activity was measured. The results are shown in Table 1.
[0122] Comparative Example 2:
[0123] This comparative example provides a fermentation production method of mannanase, comprising the following steps:
[0124] S1. Preparation of Bacillus amyloliquefaciens seed solution
[0125] (1) Inoculate Bacillus amyloliquefaciens CGMCC15838 onto solid NA medium and culture at 37°C for 2 days to obtain mature solid seeds.
[0126] (2) Scrape three rings of Bacillus amyloliquefaciens from the solid NA medium with an inoculation loop and inoculate them into liquid NA medium. Cultivate them at 37°C and 150 rpm for 2 days to obtain mature Bacillus amyloliquefaciens subspecies seed liquid.
[0127] S2. Prepare fermentation base material and supplementary material
[0128] The fermentation base material includes 405g of carbon source, 600g of nitrogen source, 4.5g of trace elements, 3g of defoaming agent, and water is added to make up to 15L.
[0129] The carbon source is glucose hydrolyzate, and the preparation method is as follows: dissolve 20 kg of glucose in 5 L of water, add 6.25 mL of 80% phosphoric acid, mix well, react at 0.1 MPa for 45 minutes, add water to make the volume to 26.7 kg, and obtain the corresponding carbohydrate hydrolyzate.
[0130] The nitrogen source includes ammonium sulfate and peptone in a mass ratio of 1:1.
[0131] The trace elements include ferrous sulfate, cobalt chloride, manganese sulfate and zinc sulfate in a mass ratio of 1:9:10:10.
[0132] The feed consisted of 17 kg of glucose hydrolysate, made up to 20 L with water.
[0133] Prepare the base material according to the above formula, place it in a fermentation tank, and sterilize it at 115℃, 0.065-0.075MPa for 30min. After sterilization, cool it to 28℃, and maintain positive pressure in the fermentation tank.
[0134] S3. Vaccination
[0135] In the presence of an external flame, the cultured mature seed liquid was inoculated into the fermentation base material, and the inoculation amount of Bacillus amyloliquefaciens was 2% based on the mass of the fermentation base material.
[0136] S4. Fermentation
[0137] The initial fermentation temperature is 29°C, the initial fermentation pH is controlled at 3.70, the fermentation process is stirred and the ventilation is controlled, when the dissolved oxygen in the fermentation tank is lower than 50%, the wind and speed are increased alternately, and the speed is increased by 12% of the maximum speed each time, and the upper limit of the stirring speed is 90% of the maximum stirring speed of the fermentation tank. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0138] After the stirring speed and ventilation volume are increased to the maximum, the dissolved oxygen content in the fermentation tank drops to the minimum and rebounds to more than 50%, and then the feeding begins. The initial feeding speed is 40mL / h. The feeding speed is adjusted to control the dissolved oxygen content at 40-50%. The feeding speed is increased by 15mL / h each time, and the maximum feeding speed is 145mL / h. If the dissolved oxygen content is higher than 50% when the maximum feeding speed is reached, the stirring speed and ventilation volume can be appropriately reduced to keep the dissolved oxygen content no more than 50%.
[0139] From the 65th hour of fermentation, the pH set value was increased by 0.2 per hour to a maximum of 4.80, and the fermentation temperature was increased to 38°C. Feeding was then stopped. When the dissolved oxygen rebounded to more than 80% and the online pH exceeded the set value by more than 0.10, feeding was started again. At this time, the initial feeding rate was 40 mL / h, and the feeding rate was adjusted by controlling the dissolved oxygen level at around 30%. The maximum feeding rate was 100 mL / h, and the fermentation cycle was 180 h. After the fermentation, the mannanase activity was measured. The results are shown in Table 1.
[0140] Table 1 Mannanase activity
[0141]
[0142]
[0143] As can be seen from the above table, in Comparative Example 1, Saccharomyces cerevisiae has almost no ability to synthesize mannanase, and in Comparative Example 2, because there is no mannan as an inducer, the enzyme activity produced by Bacillus amyloliquefaciens subsp. amyloliquefaciens is relatively low. However, in the mixed fermentation method provided by the present invention, because of the presence of mannan synthesized by Saccharomyces cerevisiae, mannan as an inducer can greatly improve the activity of mannanase synthesized by Bacillus amyloliquefaciens, and its enzyme activity is increased by 170% compared with single culture.
[0144] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.
Claims
1. A fermentation production method of mannanase, characterized in that: The following steps are involved: Saccharomyces cerevisiae and Bacillus amyloliquefaciens are inoculated into the fermentation base material simultaneously for fermentation to obtain mannanase.
2. The fermentation production method of mannanase according to claim 1, characterized in that: Based on the volume of the fermentation substrate, the inoculation amounts of saccharomyces cerevisiae and Bacillus amyloliquefaciens are 1-2% respectively.
3. The fermentation production method of mannanase according to claim 1, characterized in that: The initial fermentation temperature is 29-32°C, the initial ventilation volume is 0.5vvm, and the initial fermentation pH is controlled at 3.60-4.
00. During the fermentation process, the fermentation is stirred and the ventilation volume is controlled. When the dissolved oxygen content rebounds, feeding begins.
4. The fermentation production method of mannanase according to claim 3, characterized in that: During the feeding process, the feeding speed was adjusted to control the dissolved oxygen content at 40-50%.
5. The fermentation production method of mannanase according to claim 4, characterized in that: The initial feeding rate was 40 mL / h. The feeding rate was adjusted to control the dissolved oxygen content at 40-50%. The maximum feeding rate was 145 mL / h.
6. The fermentation production method of mannanase according to claim 3, characterized in that: From 63 to 67 hours of fermentation, the pH setting value is increased by 0.2 per hour, and the maximum pH value is 4.60-4.
80. At the same time, the fermentation temperature is increased to 36-38°C, and then feeding is stopped. When the dissolved oxygen rebounds to more than 80% and the pH exceeds the setting value by more than 0.10, feeding is started again.
7. The fermentation production method of mannanase according to claim 3, characterized in that: Each liter of the feed includes 800-900 g / L of carbon source, and the balance is water.
8. The fermentation production method of mannanase according to claim 1, characterized in that: Each liter of the fermentation base material includes 24-30 g / L of carbon source, 30-50 g / L of nitrogen source, 0.2-0.4 g / L of trace elements, 0.2 g / L of defoaming agent, and the balance is water.
9. The fermentation production method of mannanase according to claim 8, characterized in that: The carbon source includes carbohydrate hydrolyzate; the nitrogen source includes one or more of soybean meal, corn steep liquor, peptone, soybean cake powder, ammonium sulfate, and ammonium chloride; and the trace elements include one or more of ferrous sulfate, cobalt chloride, manganese sulfate, zinc sulfate, cupric chloride, and magnesium bromide.
10. The fermentation production method of mannanase according to claim 9, characterized in that: The method for preparing the carbohydrate hydrolyzate is as follows: adding water and phosphoric acid to one or more of lactose, sucrose, glucose, starch, glycerol and microcrystalline cellulose, mixing them evenly and reacting them to obtain the corresponding carbohydrate hydrolyzate.