A fermentation method of Aspergillus niger and its application in controlling bacterial morphology and producing phytase

Abstract

The invention belongs to the field of biological fermentation technology, and in particular to a kind of fermentation method of aspergillus niger and its application in controlling thalline morphology and producing phytase.The present invention is in aspergillus niger fermentation process, and by controlling the reducing sugar content in the first stage fermentation liquid, it is 0.3g / 100g-0.45g / 100g to control thalline morphology, while by controlling the fermentation liquid reducing sugar content increasing rate of the second phase, it is 0.3g / 100g / 24h-0.6g / 100g / 24h to control the enzyme production rate and the highest enzyme production level of thalline.The present invention solves the problem that thalline morphology is difficult to control, enzyme production rate is unstable during aspergillus niger production phytase fermentation, while improving the fermentation level of aspergillus niger production phytase.The present invention has the characteristics of easy and simple to operate, low equipment requirements, obvious changes in thalline morphology, and obvious improvement in enzyme production level, and has broad development prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly 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 the class of phosphate monoester hydrolases and possesses a unique spatial structure. Its primary source is microbial phytase. Currently, phytase is widely used in feed. Phytase can degrade phytate-protein complexes, improving animal protein utilization, thereby reducing phytate chelation of trace elements and enhancing the nutritional value of feed. It can also reduce phosphorus content in feces, minimizing environmental pollution.

[0003] Many microorganisms in nature can produce phytase, such as bacteria, yeast, and filamentous fungi. Aspergillus species, such as Aspergillus niger, Aspergillus ficifolius, and Aspergillus oryzae, in particular, can produce highly active phytase. Compared to other sources, microbial phytase offers advantages such as a wider range of sources, shorter production cycles, and a wider pH tolerance range. Therefore, optimizing culture conditions and processes to further increase yields has become a hot topic in research on microbial fermentation-based phytase production.

[0004] Aspergillus niger belongs to the kingdom Fungi, subphylum Deuteromycotina, class Hypomycota, orders Hypomycota, family Hypomycota, genus Aspergillus. Its conidia, 15-20 microns in diameter and 1-3 mm long, extend from the stroma, with thick, smooth walls. A spherical capsule forms at the top, covered with a peduncle and a layer of peduncles, on which clusters of brownish-black, spherical conidia grow. The conidial heads are spherical, 700-800 microns in diameter, and brownish-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:

[0008] In one aspect, the present invention provides a fermentation method of Aspergillus niger, characterized in that it comprises the following steps:

[0009] S1. Seed liquid culture: Seed liquid is obtained by seed tank culture;

[0010] S2, first stage fermentation culture: the seed liquid is introduced into 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 maintain the reducing sugar content at 0.3g / 100g-0.45g / 100g for 10-15h to obtain the first stage fermentation liquid;

[0011] S3. Second stage fermentation culture: When the deformation ratio of the bacterial cells in the first stage fermentation broth is above 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 0.6 g / 100 g / 24 h until the bacterial cells autolyze and the fermentation is terminated, thereby obtaining the second stage fermentation broth.

[0012] 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; the conditions for the seed liquid culture are: pH 4.4-4.6, 25°C-34°C, and air volume 0.5-1.5vvm.

[0013] Preferably, the culture medium for the seed liquid culture in step S1 comprises, 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; and the conditions for the seed liquid culture are: pH 4.4-4.6, 30°C-34°C, and air volume 1-1.5vvm.

[0014] Preferably, the culture medium for the seed liquid culture in step S1 comprises, 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; and the conditions for the seed liquid culture are: pH 4.4-4.6, 34° C., and air volume 1.5 vvm.

[0015] 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 fermentation culture conditions are: pH 4.4-4.6, 25°C-34°C, and air volume 0.9-1.9vvm.

[0016] 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 fermentation culture conditions are: pH 4.4-4.6, 25°C-34°C, and air volume 1.5-1.9vvm.

[0017] Preferably, the culture medium for the fermentation culture in steps S2 and S3 comprises, 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; and the fermentation culture conditions are: pH 4.5, 34° C., and air volume 1.9 vvm.

[0018] 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 maintain the reducing sugar content at 0.3g / 100g-0.4g / 100g for 11-12 hours.

[0019] 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.

[0020] 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.

[0021] Specifically, 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 0.5 g / 100 g / 24 h.

[0022] Preferably, 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.

[0023] Preferably, in some embodiments, when the bacterial cell deformation ratio in the first stage fermentation broth in step S3 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.

[0024] Preferably, in some embodiments, when the bacterial cell deformation ratio in the first stage fermentation broth in step S3 is 50%, glucose is added to increase the reducing sugar content in the fermentation broth at a rate of 0.4 g / 100 g / 24 h.

[0025] In another aspect, the present invention provides use of the aforementioned fermentation method in controlling the morphology of Aspergillus niger.

[0026] In another aspect, the present invention provides the use of the aforementioned fermentation method in producing phytase.

[0027] The beneficial effects of the present invention are:

[0028] The present invention solves the problems of difficult control of bacterial morphology and unstable enzyme production rate during the fermentation process of Aspergillus niger producing phytase, while also improving the fermentation level of Aspergillus niger producing phytase. By controlling bacterial morphology to influence the enzyme production capacity of the bacterial body, the average phytase activity of the liquid fermentation supernatant of the strain is 79227 U / mL. The present invention is also characterized by simple operation, low equipment requirements, significant changes in bacterial morphology, and significantly improved enzyme production, and has broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The microbial deformation ratios of Examples 1 to 2 and Comparative Examples 1 to 3 were obtained by microscopic examination.

[0030] Figure 2 The relative enzyme activities of the phytases of Examples 3 to 6 and Comparative Examples 5 to 7 are shown. DETAILED DESCRIPTION

[0031] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0032] Basic Example

[0033] The enzyme activity determination method of phytase of the present invention is as follows:

[0034] 1. Preparation method of fermentation broth supernatant

[0035] According to the aseptic fermentation procedure, take an appropriate amount of fermentation broth and centrifuge it at 5000 rpm for 10 minutes. The supernatant is obtained, which is the supernatant of the phytase fermentation broth. The enzyme activity of the supernatant is tested according to the test requirements.

[0036] 2. Determination method

[0037] (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 per minute from a 5.0 mmol / L sodium phytate solution, which is one phytase activity unit, expressed as U.

[0038] (2) Principle of enzyme activity determination: Phytase hydrolyzes the substrate sodium phytate under certain temperature and pH conditions to produce orthophosphate and inositol derivatives. In acidic solution, it can form a yellow complex with ammonium vanadium molybdate, which can be measured colorimetrically at a wavelength of 415nm.

[0039] (3) Reagents and solutions:

[0040] Potassium dihydrogen phosphate (KH2PO4): reference substance.

[0041] Acetate buffer 1: c(CH3COONa) = 0.25 mol / L. Weigh 20.52 g of anhydrous sodium acetate into a 1000 mL beaker, add 900 mL of water, and stir to dissolve. Adjust the pH to 5.50 ± 0.01 with glacial acetic acid. Transfer to a 1000 mL volumetric flask and dilute to the mark with distilled water. Store at room temperature for 2 months.

[0042] Acetate buffer-2: c(CH3COONa) = 0.25 mol / L. Weigh 20.52 g of anhydrous sodium acetate, 0.5 g of Triton X-100, and 0.5 g of bovine serum albumin (BSA) into a 1000 mL beaker. Add 900 mL of water and stir to dissolve. Adjust the pH to 5.50 ± 0.01 with glacial acetic acid. Transfer to a 1000 mL volumetric flask and dilute to the mark with distilled water. Store at room temperature for 2 months.

[0043] Substrate solution: c(C6H6O 24 P6Na 12 ) = 7.5mmol / L, weigh 0.6929g sodium phytate (C6H6O 24 P6Na 12 , relative molecular mass 923.8, purity 95%), accurate to 0.1 mg, placed in a 100 mL beaker, dissolved with about 80 mL of acetic acid buffer, adjusted to pH 5.50 ± 0.01 with glacial acetic acid, transferred to a 100 mL volumetric flask, and made up to the mark with acetic acid buffer, prepared before use (final concentration in the actual reaction solution 5.0 mmol / L).

[0044] Nitric acid solution: an aqueous solution of nitric acid and water in a volume ratio of 1:2.

[0045] Ammonium molybdate solution: 100g / L, weigh 10g of ammonium molybdate [(NH4)6Mo7O 24 4H2O] is dissolved in water in a 50mL beaker. If necessary, it can be slightly heated. Then, it is transferred to a 100mL volumetric flask, 1.0mL of ammonia water (25%) is added, and the volume is made up to the mark with water.

[0046] Ammonium metavanadate solution: 2.35g / L. Weigh 0.235g of ammonium metavanadate (NH₄VO₃) into a 50mL beaker, add 2mL of nitric acid solution and a small amount of water, and grind with a glass rod to dissolve. Transfer to a 100mL brown volumetric flask and dilute to the mark with water. Store in the dark for one week.

[0047] Enzyme hydrolysis termination and color development solution: Pipette 2 parts (volume) nitric acid solution, 1 part (volume) ammonium molybdate solution, and 1 part (volume) ammonium metavanadate solution. Mix before use. Prepare immediately.

[0048] (4) Instruments and equipment

[0049] Analytical balance: sensitivity 0.1 mg;

[0050] Constant temperature water bath: 37℃±0.1℃;

[0051] Spectrophotometer: with 10mm cuvette, capable of measuring absorbance at 415nm;

[0052] Magnetic stirrer;

[0053] vortex mixer;

[0054] Acidity meter: pH accurate to 0.01;

[0055] Centrifuge: speed is above 4000r / min;

[0056] Ultrasonic dissolver;

[0057] Gyrotron oscillator.

[0058] (5) Drawing of standard curve

[0059] Accurately weigh 0.6804 g of standard potassium dihydrogen phosphate (KDP) dried to constant weight at 105°C into a 100 mL volumetric flask. Dissolve it in acetic acid buffer and bring the volume to 50.0 mmol / L. Dilute it with acetic acid buffer at specific ratios to different concentrations, and react with the sample to be tested. Use the inorganic phosphorus content as the abscissa and the absorbance as the ordinate to develop a linear regression equation: Y = (26.892 * A - 0.3378) / 30. Here, Y is the amount of inorganic phosphorus calculated from the linear regression equation based on the actual absorbance of the sample solution (in micromoles (μmol)); A is the measured absorbance.

[0060] (6) Preparation of enzyme solution to be tested

[0061] Weigh two phytase samples to the nearest 0.0001 g and place them in a 100 mL volumetric flask. Add acetate buffer, shake well, and bring to volume. Place a magnetic bar and stir on a magnetic stirrer at high speed for 30 minutes. Alternatively, sonicate the solution in an ultrasonic dissolver for 15 minutes, then shake on an orbital oscillator for 30 minutes.

[0062] (7) Determination of enzyme activity

[0063] Take a 25 mL test tube and operate according to the following reaction sequence, starting with the addition of substrate solution. The time interval for adding to each test tube should be consistent. Hydrolyze in a constant temperature water bath at 37℃ for 30 minutes.

[0064] The reaction steps, reagents and solution amounts are shown in Table 1:

[0065] Table 1 Reaction steps and reagent and solution amounts

[0066]

[0067] After the reaction, the sample was allowed to stand at room temperature for 10 minutes. If turbidity was present, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was zeroed using the blank of the standard curve. The absorbance of the sample blank (A0) and the sample solution (A1) was measured at a wavelength of 415 nm using a spectrophotometer. A1-A0 was the measured absorbance. The phytase activity was calculated using a linear regression equation.

[0068] (8) Calculation of enzyme activity

[0069] The phytase activity in the sample is expressed as X, and the unit is enzyme activity unit per milliliter (U / mL), and is calculated according to the formula:

[0070]

[0071] Where:

[0072] X——phytase activity in the sample, the unit is enzyme activity unit per milliliter (U / mL);

[0073] 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);

[0074] t——enzymatic reaction time, in minutes (min);

[0075] n——dilution multiple of the sample;

[0076] m——the amount of the sample, in milliliters (mL).

[0077] Example 1

[0078] This example illustrates a method for controlling the bacterial morphology during Aspergillus niger fermentation by controlling the reducing sugar content in the fermentation broth.

[0079] The strain in this example is Aspergillus niger, a filamentous fungus commonly used in the company's industrial production. The strain was purchased from the China Industrial Microbiological Culture Collection Center in March 2015, and the collection number is: Aspergillus niger CICC 40700.

[0080] 1.1 Preparation of culture medium and sugar supplement solution:

[0081] (1) The mass volume ratio of the seed tank culture medium is as follows: 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 broth has a natural pH and is sterilized at 121-123°C for 30 min.

[0082] (2) The fermentation medium was composed of the following by weight and volume ratio: corn steep liquor 4.0%, soybean meal 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%, and polyether defoamer 0.05%. The fermentation broth was maintained at a natural pH and sterilized at 121-123°C for 35 min.

[0083] (3) Sugar rehydration solution is a glucose solution with a reducing sugar content of 50 g / 100 g (all reducing sugar values ​​are calculated based on glucose content). Sterilize at 118°C for 40 min.

[0084] 1.2 Culture conditions and methods:

[0085] (a) Seed tank culture: The seed liquid after shake flask fermentation was inoculated into the seed tank culture medium at a ratio of 5% v / v. The culture was carried out at an initial pH of 4.4-4.6, 34°C, an air volume of 1.5 vvm, and a rotation speed of 200-700 rpm for 46 h.

[0086] (b) Fermenter culture: Seed liquid fermented in the seed tank was added to the fermenter culture medium at a ratio of 5% v / v of the inoculum volume. During the culture, the pH of the fermentation liquid was automatically controlled at 4.5 using ammonia, the temperature was 34°C, and the air volume was 0.9-1.9 vvm. The rotation speed was 200-700 rpm. The dissolved oxygen level was required to be ≥ 20% during the culture. Sugar supplementation was not performed during the initial fermentation. Fermentation was carried out for approximately 14 hours. After the reducing sugar content in the fermentation liquid dropped to 0.3g / 100g, sugar supplementation was added to the fermenter midstream. The reducing sugar content of the fermentation liquid was monitored every 4 hours and the sugar feed flow rate was adjusted to maintain the reducing sugar content of the fermentation liquid at 0.3g / 100g.

[0087] Example 2

[0088] The difference from Example 1 is step (b): after the reducing sugar content in the fermentation broth drops to 0.45 g / 100 g, sugar replenishment is added to the fermenter to maintain the reducing sugar content in the fermentation broth at 0.45 g / 100 g.

[0089] Comparative Example 1

[0090] 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 fermenter to maintain the reducing sugar content in the fermentation broth at 0.15 g / 100 g.

[0091] Comparative Example 2

[0092] 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 fermenter to maintain the reducing sugar content in the fermentation broth at 0.6 g / 100 g.

[0093] Comparative Example 3

[0094] 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 fermenter to maintain the reducing sugar content in the fermentation broth at 0.75 g / 100 g.

[0095] Comparative Example 4

[0096] 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 fermenter to maintain the reducing sugar content in the fermentation broth at 1 g / 100 g.

[0097] Bacterial morphology results:

[0098] Comparison of bacterial morphology: The bacterial morphology under fermentation culture with the reducing sugar content of the fermentation broth at 1g / 100g was used as the control. The results are as follows: Figure 1 As shown, the fermentation batches with the reducing sugar content in the fermentation broth maintained at 0.15g / 100g and 0.3g / 100g, the proportion of bacterial deformation detected by microscopic examination after 50h of cultivation was significantly higher than that of the other batches. Since the bacteria in the batches with the reducing sugar content in the fermentation broth maintained at 0.15g / 100g are prone to produce pigments in the later stage of fermentation and the supernatant turns black, the subsequent fermentation experiments were carried out under the condition of maintaining the reducing sugar content in the fermentation broth at 0.3g / 100g.

[0099] The method for calculating the bacterial deformation ratio is: the calculation formula of the bacterial deformation ratio under microscopic examination for 50 hours = the bacterial deformation ratio observed under microscopic examination for 50 hours in the experimental groups (Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3) - the bacterial deformation ratio observed under microscopic examination for 50 hours in the control group (Comparative Example 4).

[0100] Example 3

[0101] 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.

[0102] 3.1 Preparation of culture medium and sugar supplement solution:

[0103] (1) The mass volume ratio of the seed tank culture medium is as follows: 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 broth has a natural pH and is sterilized at 121-123°C for 30 min.

[0104] (2) The fermentation medium was composed of the following by weight and volume ratio: corn steep liquor 4.0%, soybean meal 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%, and polyether defoamer 0.05%. The fermentation broth was maintained at a natural pH and sterilized at 121-123°C for 35 min.

[0105] (3) Sugar rehydration solution is a glucose solution with a reducing sugar content of 50%. Sterilize at 118°C for 40 minutes.

[0106] 3.2 Culture conditions and methods:

[0107] (a) Seed tank culture process: The seed liquid after shake flask fermentation was inoculated into the seed tank culture medium at an inoculum ratio 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.

[0108] (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 inoculum volume. During the culture process, the pH of the fermentation liquid is automatically controlled at 4.5 using ammonia water, the temperature is 34°C, the air volume is 0.9-1.9 vvm, and the rotation speed is 200-700 rpm. The dissolved oxygen content during the culture process must be ≥ 20%.

[0109] (c) Sugar supplementation process during the cultivation process:

[0110] 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 with a reducing sugar content of 50% is added to the fermentation tank to make up the reducing sugar content in the fermentation liquid, and the reducing sugar content in the fermentation liquid is maintained at 0.3g / 100g-0.4g / 100g and maintained for 12h.

[0111] Step 2: When the proportion of microbial deformation observed under microscopic examination reached 50%, the sugar feed flow rate was 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 was terminated when the enzyme activity increased slowly and microbial autolysis was severe. The culture period was 160 h. The phytase activity in the supernatant of the fermentation broth from six batches (i.e., six parallel experiments) of fermentation broth in a 50 L fermentor was measured according to the basic example.

[0112] Example 4

[0113] The difference from Example 3 is step 2:

[0114] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.4 g / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0115] Example 5

[0116] The difference from Example 3 is step 2:

[0117] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.5 g / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0118] Example 6

[0119] The difference from Example 3 is step 2:

[0120] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.6 g / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0121] Comparative Example 5

[0122] The difference from Example 3 is step 2:

[0123] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.7 g / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0124] Comparative Example 6

[0125] The difference from Example 3 is step 2:

[0126] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.8 g / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0127] Comparative Example 7

[0128] The difference from Example 3 is step 2:

[0129] When the percentage of microbial deformation observed under microscopy reached 50%, the sugar addition flow rate was gradually increased until the reducing sugar content in the fermentation broth increased at a rate of 0.9 / 100 g / 24 h. Fermentation was terminated when enzyme activity slowed to a crawl and microbial autolysis became severe. The culture period was 160 h, and the phytase activity in the supernatant of six batches of fermentation broth from a 50 L fermentor was measured according to the basic example. Other conditions were the same as in Example 3.

[0130] Comparative Example 8

[0131] The difference from Example 3 is step 1:

[0132] After the fermenter was inoculated, sugar supplementation was not performed. After the reducing sugar content in the fermentation broth dropped to 0.75 g / 100 g, a sugar supplement solution with a reduced sugar content of 50% was added to the fermenter, and the reducing sugar content in the fermentation broth was maintained at 0.75 g for 12 hours. Other conditions were the same as in Example 3.

[0133] The phytase activity in the supernatant of 6 batches of fermentation broth from a 50 L fermenter was detected with reference to the basic example.

[0134] The results of phytase activity test are shown in Table 2:

[0135] Table 2 Phytase enzyme activity test results

[0136]

[0137] Figure 2 The relative enzyme activities of the phytases of Examples 3 to 6 and Comparative Examples 5 to 7 are shown, with the average enzyme activity of the supernatant fermented by the method of Example 3 being 100% as the control.

[0138] The above results show that:

[0139] (1) Example 3 shows that sugar supplementation was started and the reducing sugar content in the fermentation broth was controlled to maintain a concentration of 0.3g / 100g-0.4g / 100g for 12 hours, under the premise that the bacterial cells were fully deformed. By adjusting the sugar supplementation flow rate, the reducing sugar content in the fermentation broth increased at a rate of 0.3g / 100g / 24h. After 160 hours of fermentation, the supernatant enzyme activity was the highest, and the average enzyme activity of 6 batches reached 79227U / mL. This is the most suitable sugar supplementation process for enzyme production of this strain.

[0140] (2) Examples 4 to 6 show that sugar supplementation was initiated and the reducing sugar content in the fermentation broth was controlled to maintain a concentration of 0.3g / 100g-0.4g / 100g 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 0.3g / 100g / 24h-0.6g / 100g / 24h. After 160 hours of fermentation, the enzyme activity in the supernatant gradually decreased, proving that the sugar supplementation process of Example 3 was the optimal sugar supplementation process. However, its enzyme activity was still higher than the fermentation level under the traditional process, so the sugar supplementation processes of Examples 4 to 6 are within the scope of the claims of the present invention.

[0141] (3) Comparative Examples 5 to 7 show that: sugar supplementation was started and the reducing sugar content in the fermentation broth was controlled to maintain a concentration of 0.3g / 100g-0.4g / 100g 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 an increase 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.

[0142] (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 at 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. This is far lower than the fermentation level when sugar is controlled 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 to allow the bacteria to fully deform is an important condition for the bacteria to produce high levels of enzymes in the later fermentation culture.

[0143] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A fermentation method for producing phytase by 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 introduced into the fermentation tank for fermentation culture. When the sugar content of the fermentation liquid drops to 0.3g / 100g-0.45g / 100g, glucose is added to maintain the reducing sugar content at 0.3g / 100g-0.4g / 100g for 12 hours to obtain the first stage fermentation liquid; S3, second stage fermentation culture: when the cell deformation ratio in the first stage fermentation broth reaches 50%, glucose is fed to increase the reducing sugar content in the fermentation broth at a rate of 0.3 g / 100 g / 24 h to 0.6 g / 100 g / 24 h until the cells autolyze and the fermentation is terminated, thereby obtaining the second stage fermentation broth; The culture medium for the seed solution culture in step S1 comprises, 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. The conditions for the seed solution culture are: pH 4.4-4.6, 25° C.-34° C., and air volume 0.5-1.5 vvm; 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 fermentation culture conditions are: pH 4.4-4.6, 25°C-34°C, and air volume 0.9-1.9vvm.

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, 4% 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.

3. The fermentation method according to claim 1, characterized in that The culture medium for the fermentation culture in steps S2 and S3 includes, by mass volume ratio, 4% 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 1.9 vvm.

4. The fermentation method according to any one of claims 1 to 3, 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 maintain the reducing sugar content at 0.3g / 100g-0.4g / 100g for 12 hours.

5. The fermentation method according to claim 4, 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.

6. The fermentation method according to claim 5, 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.

7. Use of the fermentation method according to any one of claims 1 to 6 in controlling the morphology of Aspergillus niger.

8. Use of the fermentation method according to any one of claims 1 to 6 in producing phytase.

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