Method for improving yield of saccharifying enzyme and aspergillus niger

Abstract

The invention relates to a microbial screening and culturing technology, and discloses a method for increasing the yield of saccharifying enzyme and aspergillus niger. The method comprises the following steps: inoculating saccharifying enzyme producing bacteria into a morphological regulation culture medium for culturing, so that bacterial colonies formed by culturing are fungus balls with the average diameter of less than or equal to 3500 microns; the form regulation culture medium contains a carbon source, a nitrogen source, a sodium salt, a magnesium salt and a calcium salt, and the nitrogen source is selected from at least one of soybean meal, casein and corn steep liquor. The preservation number of the aspergillus niger (Aspergillus niger) is GDMCC (China General Microbiological Culture Collection Center) NO: 63934. The method provided by the invention can effectively promote the production strain to synthesize the saccharifying enzyme and improve the saccharifying enzyme activity of the strain.

Description

Technical Field

[0001] The invention relates to microorganism screening and cultivation technology, in particular to a method for improving saccharifying enzyme production and Aspergillus niger. Background Art

[0002] The saccharifying enzyme system is named as starch α-1,4-glucan hydrolase (α-1,4-Glucanglucohydrolase), also known as glucoamylase (Glucoamylase), which has exoenzyme activity and can hydrolyze polysaccharides such as starch, glycogen, and dextrin from the end of non-reducing sugars to remove α-1,4-glucosidic bonds to obtain the final product β-D-glucose, and can also hydrolyze their α-1,6-glucosidic bonds to obtain glucose. Since the glucoamylase from Aspergillus niger was first purified in the 1970s, it has been widely used in industrial production such as starch sugar, food, medicine, and brewing. It is the second largest enzyme preparation in the world after protease, accounting for 25-33% of the global enzyme preparation trading market.

[0003] At present, the industrial production of saccharifying enzymes is mainly produced by fermentation of microorganisms of the genera Aspergillus and Rhizopu. Liquor is a traditional brewed food in my country. It is a beverage alcoholic beverage made from starchy raw materials such as sorghum as the main ingredients, and Daqu as a saccharification and fermentation agent, through solid-state fermentation and distillation. In the liquor industry, high-yield saccharifying enzyme strains play an extremely important role, which can improve the utilization rate of raw materials, save costs, and increase the yield of liquor. Aspergillus is a common filamentous fungus. Due to its strong protein secretion ability and good safety, it is widely used in the production of various enzyme preparations. It is an important saccharifying enzyme producing strain in liquor brewing.

[0004] However, the existing technology mainly improves the ability of Aspergillus niger to produce saccharifying enzymes from the perspective of genetic modification and metabolic regulation, but the effect of improving the saccharifying enzyme activity in Aspergillus niger is limited and needs further optimization. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of limited effect of improving the saccharifying enzyme activity in Aspergillus niger in the prior art, and to provide a method for increasing the yield of saccharifying enzyme and Aspergillus niger. The method can effectively promote the production bacteria to synthesize saccharifying enzyme and improve the saccharifying enzyme activity of the strain.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a method for increasing the production of saccharifying enzymes, which method comprises the following steps: inoculating saccharifying enzyme producing bacteria into a morphologically controlled culture medium for cultivation, so that the colonies formed by the culture are bacterial balls with an average diameter less than or equal to 3500 μm; the morphologically controlled culture medium contains a carbon source, a nitrogen source, a sodium salt, a magnesium salt and a calcium salt, wherein the nitrogen source is selected from at least one of soybean powder, casein and corn steep liquor.

[0007] Preferably, the average size of the bacterial balls is between 600-1500 μm.

[0008] Preferably, the nitrogen source is soybean flour and / or casein.

[0009] Preferably, the carbon source is selected from at least one of soluble starch, corn starch, sucrose, potato starch and β-cyclodextrin, more preferably corn starch and / or β-cyclodextrin.

[0010] Preferably, the carbon source content in the morphology control medium is 8-12 g / L, the nitrogen source content is 4-6 g / L, the sodium salt content is 0.1-0.5 g / L, the magnesium salt content is 0.2-0.8 g / L, and the calcium salt content is 0.1-0.3 g / L.

[0011] Preferably, the weight ratio of carbon source to nitrogen source in the morphology regulation medium is 2.5-3:1.

[0012] Preferably, the sodium salt is disodium hydrogen phosphate and / or sodium dihydrogen phosphate, preferably disodium hydrogen phosphate; the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, preferably magnesium sulfate; the calcium salt is selected from at least one of sodium chloride, sodium sulfate and sodium nitrate, preferably sodium chloride.

[0013] Preferably, the culture conditions include: temperature of 25-35°C, rotation speed of 150-200 rpm, and time of 5-7 days.

[0014] Preferably, the inoculum size in the morphology control medium is 4×10 4 -8×10 4 Pieces / mL.

[0015] Preferably, the saccharifying enzyme producing bacteria are obtained by separation and purification through a solid screening medium, wherein the solid screening medium contains peptone, soluble starch, potassium dihydrogen phosphate, magnesium sulfate, agar, bengal fuchsia and chloramphenicol.

[0016] Preferably, the solid screening medium contains 4-6 g / L of peptone, 8-10 g / L of soluble starch, 0.5-1.5 g / L of potassium dihydrogen phosphate, 0.3-0.8 g / L of magnesium sulfate, 12-20 g / L of agar, 0.02-0.05 g / L of red bengal, and 0.05-0.2 g / L of chloramphenicol.

[0017] Preferably, the separation and purification is carried out at a temperature of 25-35°C and the number of times is two or more times.

[0018] Preferably, the saccharifying enzyme producing bacteria is Aspergillus niger.

[0019] Preferably, the deposit number of the Aspergillus niger is GDMCC NO:63934.

[0020] The second aspect of the present invention provides an Aspergillus niger, wherein the deposit number of the Aspergillus niger is GDMCC NO:63934.

[0021] Through the above technical scheme, the method for increasing the yield of saccharifying enzyme provided by the present invention controls the average diameter of the fungus balls formed after the saccharifying enzyme producing bacteria are cultured under a specific nitrogen source, so that the mycelium of the saccharifying enzyme producing bacteria is more uniform and full, and the mycelium has better activity, thereby effectively promoting the production bacteria to synthesize saccharifying enzyme, increasing the saccharifying enzyme yield and the enzyme activity of saccharifying enzyme after strain culture; this method is particularly suitable for regulating the enzyme activity of Aspergillus niger provided by the present invention, and is applied to the production of enzyme preparations by the liquid method. The activity of the strain to produce saccharifying enzyme can be observed with the naked eye, so as to quickly take countermeasures and avoid losses, and has broad application prospects.

[0022] Furthermore, the Aspergillus niger saccharifying enzyme producing bacteria provided by the present invention are obtained by separation and purification through a modified solid screening medium, and glucose is replaced with soluble starch in the solid screening medium, which can effectively speed up the screening of the target strain and save time and experimental costs.

[0023] Biological Deposit

[0024] The strain provided by the present invention is Aspergillus niger, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center (abbreviated as GDMCC) on October 26, 2023, with a collection number of GDMCC No: 63934. The address of the collection center is: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1It is a standard curve diagram of glucose concentration and absorbance when the DNS method is used to determine the activity of saccharifying enzyme in the present invention;

[0026] Figure 2 is a diagram of the colony morphology in the fermentation broth when the nitrogen source is peptone in Example 2;

[0027] Figure 3 This is a diagram of the colony morphology in the fermentation liquid when the nitrogen source is soybean powder in Example 2;

[0028] Figure 4 is a diagram of the colony morphology in the fermentation broth when the nitrogen source is casein in Example 2;

[0029] Figure 5 is a diagram of the colony morphology in the fermentation broth when the nitrogen source is ammonium sulfate in Example 2;

[0030] Figure 6 is a diagram of the colony morphology in the fermentation liquid when the nitrogen source is corn steep liquor in Example 2;

[0031] Figure 7 is a diagram of the state of mycelium in the fermentation broth when the nitrogen source is peptone in Example 2;

[0032] Figure 8 is a diagram of the state of mycelium in the fermentation broth when the nitrogen source is casein in Example 2;

[0033] Fig. 9 This is a graph of the saccharifying enzyme activity of the crude enzyme solution under different nitrogen sources in Example 2. DETAILED DESCRIPTION

[0034] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] The first aspect of the present invention provides a method for increasing the yield of saccharifying enzymes, the method comprising the following steps: inoculating saccharifying enzyme producing bacteria into a morphology-controlled culture medium for culturing, so that the colonies formed by the culture are bacterial balls with an average diameter less than or equal to 3500 μm; the morphology-controlled culture medium contains a carbon source, a nitrogen source, a sodium salt, a magnesium salt and a calcium salt, wherein the nitrogen source is selected from at least one of soybean powder, casein and corn steep liquor.

[0036] The method for increasing the yield of saccharifying enzyme provided by the present invention controls the average diameter of the fungus balls formed after the saccharifying enzyme producing bacteria are cultured under a specific nitrogen source, so that the mycelium of the saccharifying enzyme producing bacteria is more uniform and full, and the mycelium has better activity, thereby effectively promoting the production bacteria to synthesize saccharifying enzyme, increasing the yield of saccharifying enzyme and the enzyme activity of saccharifying enzyme after the strain is cultured. The method can observe the activity of the strain to produce saccharifying enzyme by the naked eye, so as to quickly take countermeasures and avoid losses, and has broad application prospects.

[0037] In the present invention, the bacterial morphology is observed and photographed by a scanning electron microscope (SEM); the diameter of the bacterial ball is measured by a microscope ruler, and the average diameter of the bacterial balls is obtained by randomly selecting 20 bacterial balls to measure the diameters and taking the average value.

[0038] In the present invention, the average diameter of the bacterial colony ball formed by the culture can be specifically 100 μm, 300 μm, 600 μm, 900 μm, 1200 μm, 1500 μm, 1800 μm, 2100 μm, 2400 μm, 2700 μm, 3000 μm, 3300 μm, 3500 μm, or any value between the above two values; preferably, the average diameter of the bacterial ball is 600-1500 μm, and more preferably 600-900 μm. The inventors found that under the influence of the regulation of the nitrogen source, the enzyme activity is positively correlated with the decrease in the average diameter of the bacterial ball (i.e., the volume of the bacterial ball). The smaller the average diameter, the higher the enzyme activity of the saccharifying enzyme production bacteria after cultivation.

[0039] According to the present invention, when the colony of the saccharifying enzyme producing bacteria is spherical, the large relative specific surface area is more conducive to the mycelium absorbing nutrients and metabolism, and the saccharifying enzyme output effect is better than the output effect when the colony is strip-shaped. Preferably, the nitrogen source is soybean powder and / or casein, and more preferably casein. The inventors found that under this preferred embodiment, it is more conducive to the cultivation of saccharifying enzyme producing bacteria to form bacterial balls with small average diameter and high enzyme activity.

[0040] According to the present invention, the type of carbon source in the morphological regulation culture medium can also regulate the culture morphology of the saccharifying enzyme producing bacteria to promote the synthesis of saccharifying enzymes and improve the enzymatic activity of saccharifying enzymes in the bacterial pellets. Preferably, the carbon source is selected from at least one of soluble starch, corn starch, sucrose, potato starch and β-cyclodextrin, more preferably corn starch and / or soluble starch, and further preferably corn starch.

[0041] In the present invention, the morphology control medium is a liquid culture medium. In order to further make the saccharifying enzyme producing bacteria culture form bacterial balls with small average diameter and high enzyme activity, preferably, the content of carbon source in the morphology control medium is 8-12g / L, specifically 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, or any value between the above two values; the content of nitrogen source is 4-6g / L, specifically 4g / L, 4.5g / L, 5g / L, 5.5g / L, 6g / L, or any value between the above two values; the content of sodium salt is 0.1-0.5g / L, Specifically, it can be 4g / L, 4.5g / L, 5 / L, 5.5g / L, 6g / L, or any value between the above two values; the content of magnesium salt is 0.2-0.8g / L, specifically 0.2g / L, 0.3g / L, 0.4 / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, or any value between the above two values; the content of calcium salt is 0.1-0.3g / L, specifically 0.1g / L, 0.15g / L, 0.2 / L, 0.25g / L, 0.3g / L, or any value between the above two values.

[0042] In the present invention, in order to further make the saccharifying enzyme producing bacteria culture form bacterial balls with small average diameter and high enzyme activity, preferably, the weight ratio of the carbon source to the nitrogen source in the morphology control medium is 2.5-3:1, specifically 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, or any value between the above two values.

[0043] In the present invention, the sodium salt, magnesium salt and calcium salt can be conventional salt compounds that can be applied to the growth of saccharifying enzyme production bacteria. Preferably, the sodium salt is disodium hydrogen phosphate and / or sodium dihydrogen phosphate, more preferably disodium hydrogen phosphate; the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, more preferably magnesium sulfate; the calcium salt is selected from at least one of sodium chloride, sodium sulfate and sodium nitrate, more preferably sodium chloride.

[0044] According to the present invention, the culture conditions can be selected according to the growth environment required by the saccharifying enzyme production bacteria. Preferably, the culture conditions include: a temperature of 25-35°C, specifically 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the above two values; a rotation speed of 150-200rpm, specifically 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the above two values; a time of 5-7 days, specifically 5 days, 5.5 days, 6 days, 6.5 days, 7 days, or any value between the above two values.

[0045] In the present invention, in order to further make the saccharifying enzyme production bacteria culture form bacterial balls with small average diameter and high enzyme activity, preferably, the inoculation amount in the morphology control medium is 4×10 4 -8×10 4 For example, when the saccharifying enzyme producing bacteria is a filamentous fungus, the saccharifying enzyme producing bacteria can be subjected to solid seed culture, the spores obtained by solid seed culture can be scraped and dispersed, counted with a hemocytometer and diluted to a spore count of 10 6 -10 7 / mL to obtain a bacterial suspension, which was then inoculated into a morphology control medium.

[0046] According to the present invention, the saccharifying enzyme production bacteria can be separated and screened from wine koji (collected from Luzhou Laojiao Co., Ltd.) by conventional methods. Preferably, the saccharifying enzyme production bacteria is separated and purified by solid screening medium, and the solid screening medium contains peptone, soluble starch, potassium dihydrogen phosphate, magnesium sulfate, agar, tiger red and chloramphenicol. By replacing the glucose in the solid screening medium with soluble starch, the saccharifying enzyme production bacteria are obtained by separation and purification as an improved solid screening medium, which can effectively speed up the screening speed of the target strain, save time and experimental cost, and further verify that the saccharifying enzyme activity after its cultivation can be adjusted by regulating and changing the colony morphology.

[0047] According to the present invention, preferably, the content of peptone in the solid screening medium is 4-6g / L, specifically 4g / L, 4.5g / L, 5g / L, 5.5g / L, 6g / L, or any value between the above two values; the content of soluble starch is 8-10g / L, specifically 8g / L, 8.5g / L, 9g / L, 9.5g / L, 10g / L, or any value between the above two values; the content of potassium dihydrogen phosphate is 0.5-1.5g / L, specifically 0.5g / L, 0.7g / L, 0.9g / L, 1.1g / L, 1.3g / L, 1.5g / L, or any value between the above two values; the content of magnesium sulfate is 0.3-0.8g / L, specifically 0.3 g / L, 0.4 / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, or any value between the above two values; the content of agar is 12-20g / L, specifically 12g / L, 14g / L, 16g / L, 18g / L, 20g / L, or any value between the above two values; the content of bengal is 0.02-0.05g / L, specifically 0.02g / L, 0.03g / L, 0.04g / L, 0.05g / L, or any value between the above two values; the content of chloramphenicol is 0.05-0.2g / L, specifically 0.05g / L, 0.1g / L, 0.15g / L, 0.2g / L, or any value between the above two values.

[0048] According to the present invention, preferably, the separation and purification temperature is 25-35°C, specifically 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the above two values; the number of times is more than two times.

[0049] Exemplarily, the process of separating and purifying the saccharifying enzyme producing bacteria includes: taking the crushed koji sample and placing it in a conical flask with physiological saline and sterile glass beads (sterilized and dried in advance), shaking it at a temperature of 25-35°C and a speed of 150-200rpm for 15-25min, taking it out and placing it in an ultra-clean workbench, taking the bacterial suspension and placing it in a sterile centrifuge tube filled with sterile water, and performing a 10-fold gradient dilution to dilute it to 10 -6 , take 10 -2 to 10 -6 The gradient bacterial suspension is spread on a red Bengal culture medium and cultured at a constant temperature of 25-35°C; strains with differences in morphology, color, etc. are selected and inoculated on the above-mentioned improved solid screening culture medium, and separation and purification are carried out at least twice. The purified strains are subjected to liquid fermentation, and the enzyme activity of the saccharifying enzyme is determined by the DNS method, and the strain with the best enzyme activity is selected as the saccharifying enzyme production bacteria.

[0050] According to the present invention, preferably, the saccharifying enzyme producing bacteria is Aspergillus niger. More preferably, the deposit number of Aspergillus niger is GDMCC NO:63934.

[0051] The second aspect of the present invention provides an Aspergillus niger, and the Aspergillus niger (Aspergillus niger) has a deposit number of GDMCC NO: 63934. The Aspergillus niger strain can change its colony morphology by the method of the present invention to adjust its enzyme activity, and is applied to the production of enzyme preparations by a liquid method. The activity of the enzyme produced by the strain can be observed by the naked eye, so that countermeasures can be quickly taken to avoid losses, and the strain has broad application prospects.

[0052] The present invention will be described in detail below through examples.

[0053] In the following examples, soluble starch was purchased from Aladdin Reagent (Shanghai) Co., Ltd., product model / specification is AR, 500g,

[0054] Corn starch was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The product model / specification is reagent grade, 1 kg,

[0055] Sucrose was purchased from Aladdin Reagent (Shanghai) Co., Ltd., product model / specification is AR, 250g,

[0056] Potato starch was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The product model / specification is powder, 250g,

[0057] β-Cyclodextrin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The product model / specification is 98% biotechnology grade, 250g,

[0058] Peptone was purchased from Nanjing Houxin Biotechnology Co., Ltd. The product model / specification is biotechnology grade, 500g,

[0059] Soybean powder was purchased from Beijing Hongrun Baoshun Technology Co., Ltd. The product specification was reagent grade 250g.

[0060] Casein was purchased from Nanjing Kaijia Biotechnology Co., Ltd., with a product specification of 100g.

[0061] Ammonium sulfate was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., with a product specification of 500 g of reagent grade.

[0062] Corn syrup was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., with a product specification of 500 g;

[0063] The remaining raw materials and reagents are conventional commercially available products.

[0064] In the following examples, the formula of red Bengal medium is: peptone 5g / L, glucose 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, agar 15g / L, red Bengal 0.035g / L, chloramphenicol 0.1g / L, and the final pH is 7.2±0.2;

[0065] The formula of the liquid culture medium is: trypsin 10 g / L, corn starch 5 g / L, disodium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, and calcium chloride 0.2 g / L.

[0066] In the following examples, the enzyme activity of the saccharifying enzyme was determined by the DNS method, and the specific process was as follows: 1 mL of the prepared crude enzyme solution was taken, diluted 2-10 times to be tested, and 5 mL of a soluble starch solution with a concentration of 2wt% and 4 mL of acetic acid-sodium acetate buffer solution with a pH of 4.6 were added to a 25 mL test tube, respectively, and the mixture was shaken in a 40°C water bath for 10 min; 1 mL of the crude enzyme solution to be tested was added to the test tube and shaken, reacted at 40°C for 10 min, and immediately stopped the reaction in a boiling water bath; 1 mL of the reaction solution was taken, and then 0.5 mL of water and 2 mL of DNS solution were added in sequence, and the mixture was boiled in a water bath for 5 min, immediately cooled with running water, and 9 mL of water was added and shaken, and the absorbance value was measured at 540 nm; wherein, the standard curve of glucose concentration y and absorbance x is y=0.9157x-0.034, see Figure 1 The enzyme activity unit U / mL of saccharifying enzyme is specifically defined as: the amount of glucose released per minute per milliliter of crude enzyme solution at 40°C and pH 4.6 (in μg).

[0067] Example 1

[0068] 1. Take 10g of the crushed koji sample (collected from Luzhou Laojiao Co., Ltd.), place it in a conical flask (250mL, the conical flask was sterilized and dried in advance) with 90mL of normal saline and 2 sterile glass beads, shake it in a shaker at 30℃ and 180rpm for 20min, take it out and place it in an ultra-clean workbench, take 1mL of the bacterial suspension and place it in a sterile centrifuge tube containing 9mL of sterile water, and perform a 10-fold gradient dilution to dilute it to 10 -6 , take 10 -2 to 10 -6 0.1 mL of the gradient bacterial suspension was spread on Bengal red agar medium and cultured in a constant temperature incubator at 30°C to obtain multiple strain points;

[0069] 2. Select strains with differences in morphology, color, etc., and inoculate them on improved solid screening culture medium (5 g / L peptone, 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 15 g / L agar, 0.035 g / L Bengal red, 0.1 g / L chloramphenicol), separate and purify them at least twice at 30°C, inoculate the purified strains into liquid culture medium, ferment them at 30°C and 180 rpm, determine the saccharifying enzyme activity of the fermentation broth by DNS method, and select strain Y1 corresponding to the best enzyme activity for identification.

[0070] Strain Y1 was sent to Qingke Biotechnology Co., Ltd. for 18s sequencing, and the whole genome of the strain was compared with the NCBI database. It was finally determined that strain Y1 was Aspergillus niger and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 26, 2023, with the preservation number GDMCC No: 63934.

[0071] Example 2

[0072] 2.1 Preparation of bacterial suspension: The strain Y1 obtained in Example 1 was inoculated into a slant medium (5 g / L peptone, 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 15 g / L agar, 0.1 g / L chloramphenicol), and activated at 30°C for 120 h. The activated strain slant was rinsed with physiological saline, spores were scraped off, and after being broken up, the spores were counted with a hemocytometer and diluted to a spore count of 10. 6 / mL to obtain a bacterial suspension.

[0073] 2.2 Enzyme fermentation: The bacterial suspension obtained in step 2.1 was inoculated into a fermentation shake flask medium (formula: 10 g / L soluble starch, 5 g / L peptone, 0.3 g / L disodium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride, sterilized at 121°C for 20 min, cooled to 30°C) at an inoculum size of 6% (v / v). After shaking, the culture was cultured at a constant temperature of 30°C and a rotation speed of 180 rpm for 6 days. After the culture was completed, the fermentation broth was centrifuged at 6000 rpm for 15 min, the supernatant was aspirated, and the precipitate was discarded to obtain a crude saccharifying enzyme solution.

[0074] The average diameter of the bacterial pellets in the fermentation broth was determined to be 975 μm. The activity of the saccharifying enzyme in the crude saccharifying enzyme solution was determined to be 728.040 U / mL using the DNS method.

[0075] 2.3 Regulation of nitrogen source form

[0076] The same process as in step 2.2 is used to obtain a crude saccharifying enzyme solution, except that the peptone in the fermentation shake bottle medium is replaced with soybean powder, casein, ammonium sulfate or corn steep liquor of equal concentration;

[0077] The colony morphology and mycelium state in each fermentation broth were observed and photographed under an optical microscope. The colony morphology under various nitrogen sources was shown in Figure 2. Figures 2 to 6 When peptone was used as nitrogen source, the state of mycelium was shown in Figure 7 , the state of mycelium when casein is used as nitrogen source is shown in Figure 8 ; Determine the average diameter of the bacterial balls in each fermentation broth, and use the DNS method to determine the saccharifying enzyme activity of the crude enzyme solution under different nitrogen sources. The results are shown in Fig. 9 and Table 1.

[0078] Table 1

[0079] Nitrogen source type Average diameter of bacterial pellets in fermentation broth, μm Saccharifying enzyme activity of crude enzyme solution, U / mL Soybean powder 910 1827.382 Casein 700 2559.062 Corn Syrup 3400 1259.510 Ammonium sulfate 5000 808.125 Peptone 975 728.040

[0080] Example 3

[0081] 3.1 Preparation of bacterial suspension: The strain Y1 obtained in Example 1 was inoculated into a slant medium (5 g / L peptone, 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 15 g / L agar, 0.1 g / L chloramphenicol), and activated at 25°C for 120 h. The activated strain slant was rinsed with physiological saline, spores were scraped off, and after being broken up, the spores were counted with a hemocytometer and diluted to a spore count of 10. 6 / mL to obtain a bacterial suspension.

[0082] 3.2 Enzyme fermentation: The bacterial suspension obtained in step 3.1 was inoculated into a fermentation shake bottle medium (formula: 12 g / L soluble starch, 6 g / L casein, 0.5 g / L disodium hydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride, sterilized at 121°C for 20 min, cooled to 30°C) at an inoculum amount of 8%, shaken well and cultured at a constant temperature of 25°C and 200 rpm for 7 days. After the culture was completed, the fermentation broth was centrifuged at 6000 rpm for 15 min, the supernatant was aspirated, and the precipitate was discarded to obtain a crude saccharifying enzyme solution.

[0083] The average diameter of the bacterial balls in the fermentation broth was determined to be 700 μm. The activity of the saccharifying enzyme in the crude saccharifying enzyme solution was determined to be 2530.426 U / mL using the DNS method.

[0084] 3.3 Regulation of carbon source morphology

[0085] The same process as in step 3.2 is used to obtain a crude saccharifying enzyme solution, except that the soluble starch in the fermentation shake bottle medium is replaced with corn starch, sucrose, potato starch or β-cyclodextrin of equal concentration;

[0086] The colony morphology and mycelium state in each fermentation broth were observed and photographed using an optical microscope; the average diameter of the bacterial balls in each fermentation broth was determined, and the saccharifying enzyme activity of the crude enzyme solution under different carbon sources was determined using the DNS method. The results are shown in Table 2.

[0087] Table 2

[0088] Carbon source type Average diameter of bacterial pellets in fermentation broth, μm Saccharifying enzyme activity of crude enzyme solution, U / mL Soluble starch 975 2368.040 Corn starch 700 2530.426 sucrose 1440 2093.564 Potato starch 1190 2279.003 β-Cyclodextrin 1445 2037.247

[0089] Example 4

[0090] 4.1 Preparation of bacterial suspension: The strain Y1 obtained in Example 1 was inoculated into a slant medium (5 g / L peptone, 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 15 g / L agar, 0.1 g / L chloramphenicol), and activated at 35°C for 120 h. The activated strain slant was rinsed with physiological saline, and the spores were scraped off and broken up. The spores were counted with a hemocytometer and diluted to a spore count of 10 6 / mL to obtain a bacterial suspension.

[0091] 4.2 Enzyme fermentation: The bacterial suspension obtained in step 4.1 was inoculated into a fermentation shake bottle medium (formula: 8 g / L corn starch, 4 g / L casein, 0.1 g / L disodium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium chloride, sterilized at 121°C for 20 min, cooled to 30°C) at an inoculum size of 4%. After shaking, the medium was cultured at a constant temperature of 35°C and a rotation speed of 150 rpm for 5 days. After the culture was completed, the fermentation liquid was centrifuged at 6000 rpm for 15 min, the supernatant was aspirated, and the precipitate was discarded to obtain a crude saccharifying enzyme solution.

[0092] The average diameter of the bacterial pellets in the fermentation broth was determined to be 730 μm. The activity of the saccharifying enzyme in the crude saccharifying enzyme solution was determined to be 2208.086 U / mL using the DNS method.

[0093] 4.3 Regulation of nitrogen source form

[0094] The same process as in step 4.2 was used to obtain a crude saccharifying enzyme solution, except that the contents of corn starch and casein in the fermentation shake bottle medium were respectively as shown in Table 3;

[0095] The colony morphology and mycelium state in each fermentation broth were observed and photographed under an optical microscope; the average diameter of the bacterial balls in each fermentation broth was determined, and the saccharifying enzyme activity of the crude enzyme solution under different nitrogen sources was determined by the DNS method. The results are shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for increasing the yield of saccharifying enzyme, characterized in that: The method comprises the following steps: inoculating saccharifying enzyme production bacteria into a morphology control medium for cultivation, so that the colonies formed by the cultivation are bacterial balls with an average diameter less than or equal to 3500 μm; The morphology regulation medium contains a carbon source, a nitrogen source, a sodium salt, a magnesium salt and a calcium salt, wherein the nitrogen source is selected from at least one of soybean powder, casein and corn steep liquor.

2. The method according to claim 1, characterized in that The average size of the bacterial balls is 600-1500 μm; Preferably, the nitrogen source is soybean flour and / or casein; Preferably, the carbon source is selected from at least one of soluble starch, corn starch, sucrose, potato starch and β-cyclodextrin, more preferably corn starch and / or soluble starch.

3. The method according to claim 1 or 2, characterized in that: The content of carbon source in the morphology control medium is 8-12 g / L, the content of nitrogen source is 4-6 g / L, the content of sodium salt is 0.1-0.5 g / L, the content of magnesium salt is 0.2-0.8 g / L, and the content of calcium salt is 0.1-0.3 g / L.

4. The method according to claim 3, characterized in that The weight ratio of the carbon source to the nitrogen source in the morphology regulation medium is 2.5-3:

1.

5. The method according to claim 1 or 2, characterized in that: The sodium salt is disodium hydrogen phosphate and / or sodium dihydrogen phosphate, preferably disodium hydrogen phosphate; the magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate, preferably magnesium sulfate; the calcium salt is selected from at least one of sodium chloride, sodium sulfate and sodium nitrate, preferably sodium chloride.

6. The method according to claim 1 or 2, characterized in that: The culture conditions include: temperature of 25-35°C, rotation speed of 150-200 rpm, and time of 5-7 days; Preferably, the inoculum size in the morphology control medium is 4×10 4 -8×10 4 Pieces / mL.

7. The method according to claim 1 or 2, characterized in that: The saccharifying enzyme producing bacteria are obtained by separation and purification through a solid screening medium, wherein the solid screening medium contains peptone, soluble starch, potassium dihydrogen phosphate, magnesium sulfate, agar, bengalensis and chloramphenicol; Preferably, the solid screening medium contains 4-6 g / L of peptone, 8-10 g / L of soluble starch, 0.5-1.5 g / L of potassium dihydrogen phosphate, 0.3-0.8 g / L of magnesium sulfate, 12-20 g / L of agar, 0.02-0.05 g / L of red bengal, and 0.05-0.2 g / L of chloramphenicol.

8. The method according to claim 7, characterized in that The separation and purification is performed at a temperature of 25-35° C. and the number of times is more than two times.

9. The method according to claim 8, characterized in that The saccharifying enzyme producing bacteria is Aspergillus niger; Preferably, the deposit number of the Aspergillus niger is GDMCC NO:63934.

10. An Aspergillus niger, characterized in that The deposit number of Aspergillus niger is GDMCC NO:63934.

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