Fermentation method of aspergillus niger and application of aspergillus niger in controlling thallus morphology and producing phytase

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation method of aspergillus niger and application of the aspergillus niger to control of thallus morphology and production of phytase. In the aspergillus niger fermentation process, the thallus form is controlled by controlling the reducing sugar content in the fermentation liquor at the first stage to be 0.3 g / 100g to 0.45 g / 100g, and meanwhile, the enzyme production rate and the highest enzyme production level of the thallus are controlled by controlling the reducing sugar content increase rate of the fermentation liquor at the second stage to be 0.3 g / 100g / 24h to 0.6 g / 100g / 24h. According to the method, the problems that the thallus form is difficult to control and the enzyme production rate is unstable in the fermentation process of producing the phytase by the aspergillus niger are solved, and meanwhile, the fermentation level of producing the phytase by the aspergillus niger is improved. The method disclosed by the invention has the characteristics of simplicity and convenience in operation, low equipment requirement, obvious thallus form change and obvious enzyme production level improvement, and has a wide development prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a fermentation method of Aspergillus niger and application of the method in controlling bacterial morphology and producing phytase. Background Art

[0002] Phytase is a general term for a class of enzymes that catalyze the hydrolysis of phytic acid and its salts into inositol and phosphate (salt). It belongs to phosphate monoester hydrolase and has a special spatial structure. Its main source is microbial phytase. At present, phytase is widely used in feed. Phytase can degrade phytate protein complexes and improve the utilization of protein by animals, thereby reducing the chelation of trace elements by phytate and improving the nutritional value of feed. On the other hand, it can reduce the phosphorus content in feces and reduce environmental pollution.

[0003] There are many microorganisms in nature that can produce phytase, such as bacteria, yeast and filamentous fungi, especially Aspergillus microorganisms, such as Aspergillus niger, Aspergillus fig, Aspergillus oryzae, etc., which can produce phytase with high activity. Compared with phytase from other sources, phytase from microorganisms has the advantages of wide sources, short production cycle and wide pH tolerance range. Therefore, optimizing culture conditions and processes and further improving yields have become hot topics in the research of phytase production by microbial fermentation.

[0004] Aspergillus niger belongs to the kingdom Fungi, subphylum Ascomycota, class Hypomycetes, orders Hypomycetes, family Hypophysosporaceae, genus Aspergillus. Its conidia extend from the matrix, with a diameter of 15-20 microns, a length of 1-3 mm, and thick and smooth walls. A spherical top capsule is formed on the top, which is fully covered with a layer of peduncles and a layer of peduncles, on which clusters of brown-black spherical conidia grow. The conidial head is spherical, 700-800 microns in diameter, and brown-black in color.

[0005] At present, Aspergillus niger is one of the main phytase producing bacteria. By optimizing the culture conditions and processes of Aspergillus niger and changing the bacterial morphology, the yield can be increased and the production cost can be reduced, which is of great significance for the large-scale fermentation production of cheap phytase preparations. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a method for controlling the bacterial morphology by controlling the reducing sugar content in the fermentation liquid during the fermentation process of Aspergillus niger, thereby improving the enzyme production capacity of Aspergillus niger liquid fermentation to produce phytase and significantly improving the fermentation level.

[0007] The technical solution of the present invention is: In one aspect, the present invention provides a fermentation method of Aspergillus niger, characterized in that it comprises the following steps: S1. Seed liquid culture: seed liquid is obtained by seed tank culture; S2, first stage fermentation culture: the seed liquid is connected to the fermentation tank for fermentation culture, and when the sugar content of the fermentation liquid drops to 0.3g / 100g-0.45g / 100g, glucose is added to keep the reducing sugar content at 0.3g / 100g-0.45g / 100g, and the mixture is maintained for 10-15h to obtain the first stage fermentation liquid; S3, second stage fermentation culture: when the proportion of bacterial deformation in the first stage fermentation liquid is more than 50%, glucose is added to increase the reducing sugar content in the fermentation liquid at a rate of 0.3g / 100g / 24h-0.6g / 100g / 24h, until the bacterial autolysis ends the fermentation, and the second stage fermentation liquid is obtained.

[0008] Specifically, the culture medium for the seed liquid culture in step S1 includes, by mass volume ratio, 3-5% corn steep liquor, 1-3% ammonium sulfate, 0.5-2.5% potassium dihydrogen phosphate, 0.02-0.1% calcium carbonate, 0.5-2% yeast extract, 4-6% glucose and 0.01-0.05% polyether defoamer, with a natural pH value; the conditions for the seed liquid culture are: pH 4.4-4.6, 25°C-34°C, and air volume 0.5-1.5vvm.

[0009] Preferably, the culture medium for the seed liquid culture in step S1 includes, by mass volume ratio, 4-4.5% corn steep liquor, 1.5-2% ammonium sulfate, 1-2% potassium dihydrogen phosphate, 0.04-0.06% calcium carbonate, 1-1.5% yeast extract, 5-5.5% glucose and 0.02-0.03% polyether defoamer, with a natural pH value; the conditions for the seed liquid culture are: pH 4.4-4.6, 30°C-34°C, and air volume 1-1.5vvm.

[0010] Preferably, the culture medium for the seed liquid culture in step S1 includes, by mass volume ratio, 4.0% corn steep liquor, 2% ammonium sulfate, 2% potassium dihydrogen phosphate, 0.05% calcium carbonate, 1% yeast extract, 5% glucose and 0.02% polyether defoamer, with a natural pH; the conditions for the seed liquid culture are: pH 4.4-4.6, 34°C, and air volume 1.5vvm.

[0011] Specifically, the culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 3-5% corn steep liquor, 2-5% soybean meal powder, 1-3% ammonium sulfate, 0.5-2.5% potassium dihydrogen phosphate, 0.005-0.05% calcium chloride, 0.1-0.5% yeast peptone, 0.1-0.5% yeast extract powder, 0.1-0.5% citric acid, 0.05-0.1% magnesium sulfate, 0.01-0.2% manganese sulfate, 0.5-3% glucose and 0.01-0.1% polyether defoamer, and the pH of the fermentation liquid is natural; the conditions for the fermentation culture are: pH 4.4-4.6, 25°C-34°C, and air volume 0.9-1.9vvm.

[0012] The culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 3.5-4% corn steep liquor, 3-4% soybean meal powder, 1.5-2% ammonium sulfate, 2-2.5% potassium dihydrogen phosphate, 0.01-0.02% calcium chloride, 0.2-0.4% yeast peptone, 0.2-0.4% yeast extract powder, 0.2-0.4% citric acid, 0.08-0.1% magnesium sulfate, 0.08-0.1% manganese sulfate, 1-2% glucose and 0.01-0.1% polyether defoamer, and the pH of the fermentation liquid is natural; the conditions for the fermentation culture are: pH 4.4-4.6, 25°C-34°C, and air volume 1.5-1.9vvm.

[0013] Preferably, the culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 4.0% corn steep liquor, 3.6% soybean meal powder, 2% ammonium sulfate, 2% potassium dihydrogen phosphate, 0.01% calcium chloride, 0.3% yeast peptone, 0.3% yeast extract powder, 0.3% citric acid, 0.1% magnesium sulfate, 0.1% manganese sulfate, 1% glucose and 0.05% polyether defoamer, and the pH is natural; the conditions for the fermentation culture are: pH 4.5, 34°C, and air volume 1.9vvm.

[0014] Specifically, in step S2, the sugar content of the fermentation liquid is reduced to 0.3g / 100g-0.4g / 100g, and glucose is added to keep the reducing sugar content at 0.3g / 100g-0.4g / 100g for 11-12h.

[0015] Preferably, in some embodiments, the sugar content of the fermentation broth in step S2 is reduced to 0.3 g / 100 g, and glucose is added to maintain the reducing sugar content at 0.3 g / 100 g for 12 hours.

[0016] Preferably, in some embodiments, the sugar content of the fermentation broth in step S2 is reduced to 0.4 g / 100 g, and glucose is added to maintain the reducing sugar content at 0.4 g / 100 g for 12 hours.

[0017] Specifically, in step S3, when the bacterial deformation ratio in the first stage fermentation broth is 50%, the increasing rate of reducing sugar content in the fermentation broth is increased by 0.3 g / 100 g / 24 h-0.5 g / 100 g / 24 h by adding glucose.

[0018] Preferably, in step S3, when the bacterial deformation ratio in the first stage fermentation broth is 50%, glucose is added to increase the reducing sugar content in the fermentation broth at a rate of 0.3 g / 100 g / 24 h to 0.4 g / 100 g / 24 h.

[0019] Preferably, in some embodiments, when the bacterial deformation ratio in the first stage fermentation broth in step S3 is 50%, the rate of increase of the reducing sugar content in the fermentation broth is increased by 0.3 g / 100 g / 24 h by adding glucose.

[0020] Preferably, in some embodiments, when the bacterial deformation ratio in the first stage fermentation broth in step S3 is 50%, the rate of increase of the reducing sugar content in the fermentation broth is increased at a level of 0.4 g / 100 g / 24 h by adding glucose.

[0021] In yet another aspect, the present invention provides application of the aforementioned fermentation method in controlling the morphology of Aspergillus niger.

[0022] In another aspect, the present invention provides application of the aforementioned fermentation method in producing phytase.

[0023] The beneficial effects of the present invention are: The invention solves the problem that the cell morphology is difficult to control and the enzyme production rate is unstable during the fermentation process of Aspergillus niger producing phytase, and at the same time improves the fermentation level of Aspergillus niger producing phytase, and affects the enzyme production capacity of the cell by controlling the cell morphology, so that the average phytase activity of the liquid fermentation supernatant of the strain is 79227U / mL. At the same time, the invention has the characteristics of simple operation, low equipment requirements, obvious cell morphology changes, and obvious improvement of enzyme production level, and has broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the microscopic bacterial deformation ratio of Example 1-Example 2 and Comparative Example 1-Comparative Example 3.

[0025] Figure 2 It is the relative enzyme activity of the phytase of Example 3 to Example 6 and Comparative Example 5 to Comparative Example 7. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below by way of examples. The following examples are only a part of the present invention and are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0027] Basic Example The enzyme activity determination method of phytase of the present invention is as follows: 1. Preparation method of fermentation broth supernatant According to the aseptic fermentation operation, take an appropriate amount of fermentation liquid, centrifuge the fermentation liquid at 5000rpm for 10 minutes, and take the supernatant, which is the supernatant of the phytase fermentation liquid. The enzyme activity of the supernatant is tested according to the test requirements.

[0028] 2. Determination method (1) Definition of enzyme activity unit: Under the conditions of temperature of 37℃ and pH of 5.50, 1 μmol of inorganic phosphorus is released from 5.0 mmol / L sodium phytate solution per minute, which is one phytase activity unit, expressed as U.

[0029] (2) Principle of enzyme activity determination: Under certain temperature and pH conditions, phytase hydrolyzes the substrate sodium phytate to generate orthophosphate and inositol derivatives. In an acidic solution, it can generate a yellow complex with ammonium vanadium molybdate, which can be measured colorimetrically at a wavelength of 415nm.

[0030] (3) Reagents and solutions: Potassium dihydrogen phosphate (KH 2 PO 4 ): benchmark object.

[0031] Acetate buffer-1: c(CH 3 COONa) = 0.25mol / L, weigh 20.52g of anhydrous sodium acetate in a 1000mL beaker, add 900mL of water and stir to dissolve, adjust the pH to 5.50±0.01 with glacial acetic acid, transfer to a 1000mL volumetric flask, and dilute to the mark with distilled water. It is valid within 2 months when stored at room temperature.

[0032] Acetate buffer-2: c(CH 3 COONa) = 0.25mol / L, weigh 20.52g anhydrous sodium acetate, 0.5g Triton X-100, 0.5g bovine serum albumin (BSA) in a 1000mL beaker, add 900mL water and stir to dissolve, adjust the pH to 5.50±0.01 with glacial acetic acid, transfer to a 1000mL volumetric flask, and dilute to the mark with distilled water. It is valid within 2 months when stored at room temperature.

[0033] Substrate solution: c (C 6 H 6 O 24 P 6 Na 12 ) = 7.5mmol / L, weigh 0.6929g sodium phytate (C 6 H 6 O 24 P 6 Na 12, relative molecular mass is 923.8, purity is 95%), accurate to 0.1 mg, placed in a 100mL beaker, dissolved with about 80mL acetic acid buffer, adjusted pH to 5.50±0.01 with glacial acetic acid, transferred to a 100mL volumetric flask, and made up to the mark with acetic acid buffer, prepared before use (the final concentration in the actual reaction solution is 5.0mmol / L).

[0034] Nitric acid solution: an aqueous solution with a volume ratio of nitric acid to water of 1:2.

[0035] Ammonium molybdate solution: 100 g / L, weigh 10 g of ammonium molybdate [(NH 4 )6Mo 7 O 24 ·4H 2 O] Dissolve in water in a 50mL beaker, heat slightly if necessary, transfer to a 100mL volumetric flask, add 1.0mL of ammonia water (25%), and dilute to the mark with water.

[0036] Ammonium metavanadate solution: 2.35 g / L, weigh 0.235 g ammonium metavanadate (NH 4 VO 3 ) in a 50mL beaker, add 2mL nitric acid solution and a small amount of water, grind with a glass rod to dissolve, then transfer to a 100mL brown volumetric flask and dilute to the mark with water. It is valid for one week when stored in a dark environment.

[0037] Enzyme hydrolysis termination and color development solution: pipette 2 parts (volume) of nitric acid solution, 1 part (volume) of ammonium molybdate solution, and 1 part (volume) of ammonium metavanadate solution. Mix them before use and prepare them immediately.

[0038] (4) Instruments and equipment Analytical balance: sensitivity 0.1mg; Constant temperature water bath: 37℃±0.1℃; Spectrophotometer: with 10mm cuvette, can measure absorbance at 415nm; Magnetic stirrer; Vortex mixer; Acidity meter: pH accurate to 0.01; Centrifuge: speed is above 4000r / min; Ultrasonic dissolver; Gyrotron oscillator.

[0039] (5) Drawing of standard curve Accurately weigh 0.6804g of standard potassium dihydrogen phosphate dried to constant weight at 105℃ into a 100mL volumetric flask, dissolve it with acetic acid buffer, and dilute to the mark with a concentration of 50.0mmol / L. Dilute it with acetic acid buffer in a certain proportion to different concentrations, and react with the sample to be tested for determination. With the amount of inorganic phosphorus as the horizontal axis and the absorbance value as the vertical axis, list the linear regression equation (Y=(26.892*A-0.3378) / 30), where Y is the amount of inorganic phosphorus calculated by the linear regression equation based on the absorbance value of the actual sample solution, in micromoles (μmol); A is the measured absorbance value.

[0040] (6) Preparation of enzyme solution to be tested Weigh two portions of phytase samples to an accuracy of 0.0001 g, place in a 100 mL volumetric flask, add acetic acid buffer, shake well and dilute to scale. Place a magnetic bar and stir at high speed on a magnetic stirrer for 30 minutes. Alternatively, ultrasonically dissolve in an ultrasonic dissolver for 15 minutes, and then place in a gyroscopic oscillator for 30 minutes.

[0041] (7) Determination of enzyme activity Take a 25mL test tube and operate according to the following reaction sequence, starting with adding the substrate solution. The time interval for adding to each test tube should be consistent, and hydrolyze at 37℃ in a constant temperature water bath for 30 minutes.

[0042] The reaction steps and the amounts of reagents and solutions are shown in Table 1: Table 1 Reaction steps and reagent and solution dosage

[0043] The sample after reaction was allowed to stand at room temperature for 10 minutes. If turbidity appeared, it was centrifuged at 4000r / min for 10 minutes. The supernatant was adjusted to zero with the blank of the standard curve. The absorbance of the sample blank (A0) and the sample solution (A1) was measured at a wavelength of 415nm on a spectrophotometer. A1-A0 was the measured absorbance. The activity of phytase was calculated using a linear regression equation.

[0044] (8) Calculation of enzyme activity The phytase activity in the sample is represented by X, and the unit is enzyme activity unit per milliliter (U / mL), and is calculated according to the formula:

[0045] Where: X——phytase activity in the test sample, in units of enzyme activity per milliliter (U / mL); y——the amount of inorganic phosphorus calculated by the linear regression equation based on the absorbance value of the actual sample solution, in micromoles (μmol); t——enzymatic reaction time, in minutes (min); n——dilution multiple of the sample; m——the amount of sample, in milliliters (mL).

[0046] Example 1 This example illustrates a method for controlling the morphology of Aspergillus niger by controlling the reducing sugar content in the fermentation broth during fermentation.

[0047] The strain of this example is a filamentous fungus commonly used in the company's industrial production - Aspergillus niger. The strain was purchased from the China Industrial Microbiological Culture Collection Administration Center in March 2015, and the collection number is: Aspergillus niger CICC 40700.

[0048] 1.1 Preparation of culture medium and sugar supplement solution: (1) The mass volume ratio of the seed tank culture medium is: corn steep liquor 4.0%, ammonium sulfate 2%, potassium dihydrogen phosphate 2%, calcium carbonate 0.05%, yeast extract 1%, glucose 5%, polyether defoamer 0.02%. The fermentation liquid has a natural pH and is sterilized at 121-123℃ for 30 minutes.

[0049] (2) The mass volume ratio of the fermentation tank culture medium is as follows: corn steep liquor 4.0%, soybean meal powder 3.6%, ammonium sulfate 2%, potassium dihydrogen phosphate 2%, calcium chloride 0.01%, yeast peptone 0.3%, yeast extract powder 0.3%, citric acid 0.3%, magnesium sulfate 0.1%, manganese sulfate 0.1%, glucose 1%, polyether defoamer 0.05%. The fermentation liquid pH is natural and sterilized at 121-123℃ for 35min.

[0050] (3) Sugar supplement solution is a glucose solution with a reducing sugar content of 50 g / 100 g (all reducing sugar values ​​are measured in terms of glucose content). Sterilize at 118°C for 40 min.

[0051] 1.2 Culture conditions and methods: (a) Seed tank culture: The seed liquid after shake flask fermentation was inoculated into the seed tank culture medium at an inoculation rate of 5% v / v, and cultured for 46 h at an initial pH of 4.4-4.6, 34°C, air volume of 1.5 vvm, and rotation speed of 200-700 rpm.

[0052] (b) Fermentation tank culture: The seed liquid fermented in the seed tank is added to the fermentation tank culture medium at a ratio of 5% v / v of the inoculation amount. During the culture process, the pH of the fermentation liquid is automatically controlled at 4.5, 34°C, and the air volume is 0.9-1.9 vvm using ammonia water. The rotation speed is 200-700 rpm, and the dissolved oxygen is required to be ≥ 20% during the culture process. No sugar supplementation is performed during the culture process. The fermentation culture is carried out for about 14 hours. After the reduced sugar content in the fermentation liquid drops to 0.3g / 100g, sugar supplementation is added to the fermentation tank midstream. The reducing sugar content in the fermentation liquid is detected every 4 hours and the sugar supplement flow rate of the feed pump is adjusted to maintain the reducing sugar content in the fermentation liquid at 0.3g / 100g.

[0053] Example 2 The difference from Example 1 is step (b): after the detected reducing sugar content in the fermentation broth drops to 0.45 g / 100 g, sugar replenishment is added to the fermentation tank to maintain the reducing sugar content in the fermentation broth at 0.45 g / 100 g.

[0054] Comparative Example 1 The difference from Example 1 is step (b): after the reducing sugar content in the fermentation broth drops to 0.15 g / 100 g, sugar replenishment is added to the fermentation tank to maintain the reducing sugar content in the fermentation broth at 0.15 g / 100 g.

[0055] Comparative Example 2 The difference from Example 1 is step (b): after the reducing sugar content in the fermentation broth drops to 0.6 g / 100 g, sugar replenishment is added to the fermentation tank to maintain the reducing sugar content in the fermentation broth at 0.6 g / 100 g.

[0056] Comparative Example 3 The difference from Example 1 is step (b): after the reducing sugar content in the fermentation broth drops to 0.75 g / 100 g, sugar replenishment is added to the fermentation tank to maintain the reducing sugar content in the fermentation broth at 0.75 g / 100 g.

[0057] Comparative Example 4 The difference from Example 1 is step (b): after the reducing sugar content in the fermentation broth drops to 1 g / 100 g, sugar replenishment is added to the fermentation tank to maintain the reducing sugar content in the fermentation broth at 1 g / 100 g.

[0058] Bacterial morphology results: Comparison of bacterial morphology: The bacterial morphology under fermentation culture with the reducing sugar content in the fermentation broth at 1g / 100g was used as the control. The results are as follows Figure 1As shown, for the fermentation batches in which the reducing sugar content in the fermentation broth was maintained at 0.15g / 100g and 0.3g / 100g, the proportion of deformed bacteria in microscopic examination after 50 hours of cultivation was significantly higher than that in the other batches. Since the bacteria in the batches in which the reducing sugar content in the fermentation broth was maintained at 0.15g / 100g were prone to produce pigments in the later stage of fermentation and the supernatant turned black, the condition of maintaining the reducing sugar content in the fermentation broth at 0.3g / 100g was selected for the subsequent fermentation experiments.

[0059] The method for calculating the bacterial deformation ratio is: the calculation formula for the bacterial deformation ratio under microscopic examination for 50 hours = the bacterial deformation ratio observed under microscopic examination in the experimental groups (Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3) for 50 hours - the bacterial deformation ratio observed under microscopic examination in the control group (Comparative Example 4) for 50 hours.

[0060] Example 3 This example shows that the enzyme production capacity of Aspergillus niger in liquid fermentation for producing phytase can be improved by controlling the bacterial morphology, thereby significantly improving the fermentation level.

[0061] 3.1 Preparation of culture medium and sugar supplement solution: (1) The mass volume ratio of the seed tank culture medium is: corn steep liquor 4.0%, ammonium sulfate 2%, potassium dihydrogen phosphate 2%, calcium carbonate 0.05%, yeast extract 1%, glucose 5%, polyether defoamer 0.02%. The fermentation liquid has a natural pH and is sterilized at 121-123℃ for 30 minutes.

[0062] (2) The mass volume ratio of the fermentation tank culture medium is as follows: corn steep liquor 4.0%, soybean meal powder 3.6%, ammonium sulfate 2%, potassium dihydrogen phosphate 2%, calcium chloride 0.01%, yeast peptone 0.3%, yeast extract powder 0.3%, citric acid 0.3%, magnesium sulfate 0.1%, manganese sulfate 0.1%, glucose 1%, polyether defoamer 0.05%. The fermentation liquid pH is natural and sterilized at 121-123℃ for 35min.

[0063] (3) Sugar rehydration solution is a glucose solution with a reducing sugar content of 50%. Sterilize at 118℃ for 40 minutes.

[0064] 3.2 Culture conditions and methods: (a) Seed tank culture process: The seed liquid after shake flask fermentation was inoculated into the seed tank culture medium at an inoculation rate of 5% v / v, and cultured for 46 h at an initial pH of 4.4-4.6, 34°C, air volume of 1.5 vvm, and rotation speed of 200-700 rpm.

[0065] (b) Fermentation tank culture process: The seed liquid fermented in the seed tank is added to the fermentation tank culture medium at a ratio of 5% of the inoculation amount. During the culture process, the pH of the fermentation liquid is automatically controlled at 4.5 using ammonia water, 34°C, and the air volume is 0.9-1.9vvm. The rotation speed is 200-700rpm, and the dissolved oxygen is required to be ≥20% during the culture process.

[0066] (c) Sugar supplementation process during the cultivation process: Step 1: After the fermentation tank is inoculated, sugar is not added first. After the reducing sugar content in the fermentation liquid drops to 0.3g / 100g, glucose replenishment with a reducing sugar content of 50% is added to the fermentation tank, and the reducing sugar content in the fermentation liquid is maintained at 0.3g / 100g-0.4g / 100g and maintained for 12h.

[0067] Step 2: When the proportion of microbial deformation under microscopic examination reaches 50%, the sugar supplement flow rate is gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.3 g / 100 g / 24 h. The fermentation is terminated when the enzyme activity increases slowly and the microbial autolysis is serious. The culture period is 160 h. The enzyme activity of phytase in the supernatant of the fermentation broth of 6 batches (i.e., 6 parallel experiments) of the 50 L fermenter is detected with reference to the basic embodiment.

[0068] Example 4 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.4 g / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Others were the same as in Example 3.

[0069] Example 5 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.5 g / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Others were the same as in Example 3.

[0070] Example 6 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.6 g / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Others were the same as in Example 3.

[0071] Comparative Example 5 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.7 g / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Others were the same as in Example 3.

[0072] Comparative Example 6 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.8 g / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Other conditions were the same as those in Example 3.

[0073] Comparative Example 7 The difference from Example 3 is step 2: When the proportion of microbial deformation under microscopy reached 50%, the sugar supplementation flow rate was gradually increased to increase the reducing sugar content in the fermentation broth at a rate of 0.9 / 100 g / 24 h, until the enzyme activity increased slowly and the microbial autolysis was serious, the fermentation was terminated, and the culture period was 160 h. The enzyme activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example. Other conditions were the same as those in Example 3.

[0074] Comparative Example 8 The difference from Example 3 is step 1: After the fermentation tank was inoculated, sugar supplementation was not performed first. After the reducing sugar content in the fermentation liquid dropped to 0.75 g / 100 g, a sugar supplement solution with a reducing sugar content of 50% was added to the fermentation tank, and the reducing sugar content in the fermentation liquid was maintained at 0.75 g for 12 h. Other conditions were the same as those in Example 3.

[0075] The enzymatic activity of phytase in the supernatant of 6 batches of fermentation broth in a 50 L fermenter was detected with reference to the basic example.

[0076] The results of phytase enzyme activity test are shown in Table 2: Table 2 Phytase enzyme activity test results

[0077] Figure 2 The relative enzyme activities of the phytases of Examples 3 to 6 and Comparative Examples 5 to 7 are shown, and the average enzyme activity of the supernatant fermented by the method of Example 3 is taken as 100% enzyme activity as a control.

[0078] The above results show that: (1) Example 3 shows that sugar supplementation starts and the reducing sugar content in the fermentation broth is controlled to maintain a concentration of 0.3g / 100g-0.4g / 100g and maintained for 12h, so that the bacteria are fully deformed. By adjusting the sugar supplementation flow rate, the reducing sugar content in the fermentation broth increases at a rate of 0.3g / 100g / 24h. After 160h of fermentation, the enzyme activity of the supernatant is the highest, and the average enzyme activity of 6 batches reaches 79227U / mL. This is the most suitable sugar supplementation process for enzyme production of this strain.

[0079] (2) Examples 4 to 6 show that: sugar supplementation is started and the reducing sugar content in the fermentation broth is controlled to be maintained at a concentration of 0.3g / 100g-0.4g / 100g and maintained for 12 hours, so that the bacterial cells are fully deformed. By adjusting the sugar supplementation flow rate, the reducing sugar content in the fermentation broth is gradually increased at a rate of 0.3g / 100g / 24h-0.6g / 100g / 24h. After 160 hours of fermentation, the enzyme activity of the supernatant gradually decreases, proving that the sugar supplementation process of Example 3 is the optimal sugar supplementation process. However, its enzyme activity is still higher than the fermentation level under the traditional process, so the sugar supplementation process of Examples 4 to 6 is within the claims of the present invention.

[0080] (3) Comparative Examples 5 to 7 show that: sugar supplementation was started and the reducing sugar content in the fermentation broth was controlled to be maintained at a concentration of 0.3g / 100g-0.4g / 100g and maintained for 12 hours, so that the bacterial cells were fully deformed. By adjusting the sugar supplementation flow rate, the reducing sugar content in the fermentation broth was gradually increased at a rate of 0.7g / 100g / 24h-0.9g / 100g / 24h. After 160 hours of fermentation, the enzyme activity of the supernatant decreased significantly, and its fermentation enzyme activity was lower than the fermentation level of the traditional process. This further proves that the sugar supplementation process of Example 3 is the optimal process.

[0081] (4) The experiment of comparative example 8 shows that if the reducing sugar content in the fermentation broth is not controlled and maintained for a period of time at the beginning of sugar supplementation, the bacteria cannot be fully deformed. Even if the reducing sugar content in the fermentation broth is increased according to the optimal growth rate of 0.3g / 100g / 24h by adjusting the sugar supplementation flow rate in the later stage, the enzyme activity is 28522U / mL after 160h of fermentation operation. It is far lower than the fermentation level of sugar control at the beginning of sugar supplementation. Therefore, it is proved that controlling the reducing sugar content in the fermentation broth to maintain a concentration of 0.3g / 100g-0.4g / 100g and maintaining it for 12h at the beginning of sugar supplementation, so that the bacteria are fully deformed, is an important condition for the bacteria to produce high levels of enzymes in the later fermentation culture.

[0082] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A fermentation method of Aspergillus niger, characterized in that: The following steps are involved: S1. Seed liquid culture: seed liquid is obtained by seed tank culture; S2, first stage fermentation culture: the seed liquid is connected to the fermentation tank for fermentation culture, and when the sugar content of the fermentation liquid drops to 0.3g / 100g-0.45g / 100g, glucose is added to keep the reducing sugar content at 0.3g / 100g-0.45g / 100g, and the mixture is maintained for 10-15h to obtain the first stage fermentation liquid; S3, second stage fermentation culture: when the proportion of bacterial deformation in the first stage fermentation liquid is more than 50%, glucose is added to increase the reducing sugar content in the fermentation liquid at a rate of 0.3g / 100g / 24h-0.6g / 100g / 24h, until the bacterial autolysis ends the fermentation, and the second stage fermentation liquid is obtained.

2. The fermentation method according to claim 1, characterized in that The culture medium for the seed liquid culture in step S1 includes, by mass volume ratio, 3-5% corn steep liquor, 1-3% ammonium sulfate, 0.5-2.5% potassium dihydrogen phosphate, 0.02-0.1% calcium carbonate, 0.5-2% yeast extract, 4-6% glucose and 0.01-0.05% polyether defoamer, with a natural pH value; the conditions for the seed liquid culture are: pH 4.4-4.6, 25°C-34°C, and air volume 0.5-1.5vvm.

3. The fermentation method according to claim 2, characterized in that The culture medium for the seed liquid culture in step S1 includes, by mass volume ratio, 4.0% corn steep liquor, 2% ammonium sulfate, 2% potassium dihydrogen phosphate, 0.05% calcium carbonate, 1% yeast extract, 5% glucose and 0.02% polyether defoamer, with a natural pH value; the conditions for the seed liquid culture are: pH 4.4-4.6, 34°C, and air volume 1.5vvm.

4. The fermentation method according to claim 3, characterized in that The culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 3-5% corn steep liquor, 2-5% soybean meal powder, 1-3% ammonium sulfate, 0.5-2.5% potassium dihydrogen phosphate, 0.005-0.05% calcium chloride, 0.1-0.5% yeast peptone, 0.1-0.5% yeast extract powder, 0.1-0.5% citric acid, 0.05-0.1% magnesium sulfate, 0.01-0.2% manganese sulfate, 0.5-3% glucose and 0.01-0.1% polyether defoamer, and the pH of the fermentation liquid is natural; the conditions for the fermentation culture are: pH 4.4-4.6, 25°C-34°C, and air volume 0.9-1.9vvm.

5. The fermentation method according to claim 4, characterized in that The culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 4.0% corn steep liquor, 3.6% soybean meal powder, 2% ammonium sulfate, 2% potassium dihydrogen phosphate, 0.01% calcium chloride, 0.3% yeast peptone, 0.3% yeast extract powder, 0.3% citric acid, 0.1% magnesium sulfate, 0.1% manganese sulfate, 1% glucose and 0.05% polyether defoamer, with a natural pH; the fermentation culture conditions are: pH 4.5, 34°C, and air volume 1.9vvm.

6. The fermentation method according to any one of claims 1 to 5, characterized in that: In step S2, the sugar content of the fermentation liquid is reduced to 0.3g / 100g-0.4g / 100g, and glucose is added to keep the reducing sugar content at 0.3g / 100g-0.4g / 100g for 11-12h.

7. The fermentation method according to claim 6, characterized in that In step S3, when the bacterial deformation ratio in the fermentation broth of the first stage is 50%, glucose is added to increase the reducing sugar content in the fermentation broth at a rate of 0.3 g / 100 g / 24 h to 0.5 g / 100 g / 24 h.

8. The fermentation method according to claim 7, characterized in that In step S3, when the bacterial deformation ratio in the fermentation broth of the first stage is 50%, glucose is added to increase the reducing sugar content in the fermentation broth at a rate of 0.3 g / 100 g / 24 h to 0.4 g / 100 g / 24 h.

9. Use of the fermentation method according to any one of claims 1 to 8 in controlling the morphology of Aspergillus niger.

10. Use of the fermentation method according to any one of claims 1 to 8 in producing phytase.

Images