A strain with high production of thermostable acidic β-glucanase and its application

Abstract

The present invention relates to a green Trichoderma mutant strain with high beta-glucanase production, belonging to the field of microbial technology. The present invention provides a green Trichoderma (Trichoderma viride) UN-101 strain with high beta-glucanase production, with a deposit number of CGMCC NO.40414. The present invention also discloses an industrial production method for producing beta-glucanase using the strain, and the fermentation broth enzyme activity can reach more than 53000U / mL by seed culture and liquid deep fermentation with the strain, and the beta-glucanase product obtained by extraction has low finished product cost, high beta-glucanase enzyme activity, good stability and strong specificity. The invention is characterized in that the optimum reaction temperature is 45°C, and the enzyme activity can still be maintained at more than 85% when kept at 80°C for 2 hours, and the thermal stability is good; the optimum reaction pH is 3.0, and the enzyme can maintain more than 80% of its activity when treated in the pH range of 2.0-6.5 for 2 hours, and is an acidic glucanase; the invention has excellent resistance to hydrolysis by pepsin, trypsin and chymosin.

Description

Technical field:

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Trichoderma strain capable of producing high-temperature-resistant acidic beta-glucanase and a production method thereof. Background technology:

[0002] β-glucanase is an endoenzyme that specifically acts on the β-1,3 and β-1,4 glycosidic bonds in β-glucan molecules to produce oligosaccharides and glucose with 3-5 glucose units. β-glucan is a type of non-starch polysaccharide. As one of the components of the cell wall of cereal plants, it is particularly abundant in the endosperm cell walls of oats, wheat, barley, etc.

[0003] β-glucan is an anti-nutritional factor of wheat feed. Since β-glucan will wrap some nutrients, it reduces the chance of digestive enzymes in animals coming into contact with various nutrients in feed, thereby reducing the nutritional value of feed. At the same time, some β-glucans are also water-soluble, which can make feed very sticky in the animal intestines, bringing negative effects on animal growth and production. In addition, non-starch polysaccharides such as β-glucan can interact with the microbial zone in the latter part of the digestive tract, causing anaerobic fermentation, producing toxins, and inhibiting animal growth. Due to the anti-nutritional properties of β-glucan, the nutritional utilization of cereal raw materials in feed is limited. Therefore, adding β-glucanase to feed can effectively alleviate the negative effects of β-glucan, creating conditions for the large-scale use of cereal raw materials, especially barley, wheat, oats, etc. in feed.

[0004] β-glucanase is widely present in plants and microorganisms, but is lacking in humans and animals. It belongs to the class of hemicellulases. Cereal seeds themselves do not contain β-glucanase, but they can produce β-glucanase during their germination process to decompose β-glucan contained in the endosperm cell wall, thereby eliminating its resistance to the decomposition of other nutrients in the endosperm and ensuring the normal germination of seeds. In addition, β-glucanase can also be produced by microbial fermentation, mainly fungi (Aspergillus, Mucor, Trichoderma, etc.), bacteria (Bacillus) and rumen microorganisms. Microbial β-glucanase has become an important source of β-glucanase because of its high activity, wide source, low cost and convenient extraction.

[0005] At present, the research on the anti-nutritional effect of β-glucan has made great progress. A series of major achievements have been made in the use of β-glucanase to eliminate the anti-nutritional effect of β-glucan in cereal feed (especially wheat crops and their by-products) and improve the nutritional value of feed. Therefore, β-glucanase is a new feed additive with great development prospects, but there are also many problems that need further research.

[0006] Since feed pelleting, beer brewing and other processes all require high temperature treatment, and ordinary β-glucanase has poor heat resistance, it is necessary to develop β-glucanase with high temperature resistance so that it can be widely used in feed and beer brewing industries. In addition, the action environment of β-glucanase for feed is the gastrointestinal tract of animals. Since the stomach of monogastric animals is acidic, it is necessary to maintain high activity and stability in an acidic and neutral environment. Such enzymes have the longest action time in the gastrointestinal tract of animals and can exert their efficiency to the highest extent. This requires that the β-glucanase used in feed also needs to have high activity under acidic conditions.

[0007] Since β-glucanase itself is a protein, it may be degraded by pepsin and trypsin secreted in the gastrointestinal tract of animals and lose its activity. This requires that the β-glucanase used in feed must have a certain resistance to the proteases secreted in the gastrointestinal tract of animals. Therefore, the development of β-glucanase that is resistant to high temperature and acid and has a certain resistance to proteases is of great significance in the application of feed industry. Summary of the invention:

[0008] In order to solve the above technical problems, the present invention provides a strain with high production of thermostable acidic β-glucanase, which is obtained by UV and NTG composite mutagenesis of the green Trichoderma strain MHU-1 preserved in the laboratory, specifically green Trichoderma (Trichoderma viride) UN-101, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 8, 2022 (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with a deposit number of CGMCC NO.40414.

[0009] The present invention also provides the use of Trichoderma viride UN-101 in the production of β-glucanase, and in particular provides a liquid microbial fermentation production method of β-glucanase with high fermentation enzyme activity, high extraction yield and low manufacturing cost. The purpose of the present invention can be achieved by the following measures:

[0010] The method for producing β-glucanase by liquid fermentation is as follows:

[0011] Fermentation tank fermentation process conditions: inoculation amount 1.5-2.5%, tank pressure 0.05-0.08MPa, culture temperature 29-31℃, rotation speed 200-800r / min, start feeding when pH drops to the lowest and then rises to 6.5-7.0, control pH 6.0-6.3, culture to 115-125h, release the tank when enzyme activity increases slowly and the bacteria begin to partially autolyze;

[0012] Further, the feeding method is as follows: the initial feeding amount is 200 g / h, the feeding amount is reduced when the pH is lower than 6.0, and the feeding amount is increased when the pH is higher than 6.3, and the feeding amount is adjusted according to the pH situation;

[0013] Furthermore, the fermentation tank culture medium is composed of: 2.5-3.5% corn flour, 1.5-2.5% corn steep liquor, 3.0-4.0% barley flour, 2.5-3.0% soybean cake powder, 2-2.5% ammonium nitrate, 1-1.5% potassium dihydrogen phosphate, 0.3-0.5% magnesium sulfate, and the rest is water, pH 6.0-6.3 ("%" indicates mass volume percentage).

[0014] Furthermore, the fermentation tank sterilization process conditions are: 121°C-124°C, 0.11-0.12MPa, sterilization for 30min;

[0015] Further, the feed medium composition is as follows: 10-15% barley flour, 3.5-4% corn steep liquor, 1.5-2.0% magnesium sulfate, 2-2.5% potassium dihydrogen phosphate, and the rest is water, pH 6.0-6.3 ("%" indicates mass volume percentage);

[0016] At the end of fermentation, the β-glucanase activity of the fermentation broth was 53000-55000U / mL;

[0017] Further, the extraction and purification method of β-glucanase is as follows:

[0018] 40-45% of water and 0.25-0.35% of calcium chloride are added according to the volume of the fermentation liquid, and 2.5-3.5% of perlite filter aid is added according to the volume of the fermentation liquid to perform plate and frame filtration; the clarified filtered enzyme liquid is ultrafiltered and concentrated using an ultrafiltration membrane with a pore size of 20KDa; a stabilizer (2-3% trehalose and 1.5-2% glycerol are added according to the volume of the concentrated liquid) and a preservative (0.15-0.2% potassium sorbate and 0.15-0.25% sodium pyrosulfite) are added to the concentrated liquid, and then 5-8% diatomaceous earth is added according to the volume of the concentrated liquid to perform filtration and sterilization, so as to obtain a finished β-glucanase enzyme preparation ("%" represents the mass volume ratio).

[0019] The β-glucanase prepared by the present invention has the following enzymatic properties:

[0020] (1) The optimum reaction temperature is 45°C. When kept at 80°C for 2 h, the enzyme activity can still be maintained at more than 85%, indicating good thermal stability.

[0021] (2) The optimum reaction pH is 3.0. After treatment at pH 2.0-6.5 for 2 h, the relative enzyme activity still remains above 80%.

[0022] (3) It has the ability to resist hydrolysis by pepsin, trypsin and chymosin. After being treated with the above three proteases for 2 hours, the relative enzyme activity can still be maintained above 90%.

[0023] Beneficial effects:

[0024] The Trichoderma and production method provided by the present invention have outstanding substantive characteristics and significant progress compared with the prior art, and can produce the following positive effects:

[0025] 1. The present invention first provides a mutant strain of Trichoderma viride with high β-glucanase production. The β-glucanase fermentation activity level produced by the strain in industrial fermentation can reach 53000-55000 U / mL or more, which greatly reduces the production cost, is conducive to the large-scale application of β-glucanase, and saves energy and reduces emissions.

[0026] 2. The β-glucanase has the characteristics of high temperature resistance, acid resistance, good pH stability and thermal stability, and protease resistance. The optimum reaction temperature is 45°C. After being kept at 80°C for 2 hours, the enzyme activity can still be maintained at more than 85%, and the thermal stability is good; the optimum reaction pH is 3.0. After being treated in the pH range of 2.0-6.5 for 2 hours, the enzyme can maintain more than 80% of its activity. It is an acidic glucanase with high activity and stability in acidic and neutral environments, so its enzyme activity will not be reduced under various pH environments throughout the animal digestive tract; at the same time, the results of the in vitro protease hydrolysis experiment show that the β-glucanase obtained by the present invention has excellent resistance to hydrolysis by pepsin, trypsin and chymotrypsin. After being treated with the above three proteases for 2 hours, the relative enzyme activity can still be maintained at more than 90%, so the β-glucanase in the present invention is easier to play a role in the stomach and intestines of animals and poultry than the wild type.

[0027] In addition, since the β-glucanase has good heat resistance, it is suitable for heat treatment in the process of preparing feed additives. Therefore, the thermostable acidic β-glucanase of the present invention will show its great potential in feed applications.

[0028] 3. The present invention provides a production method and production strain of β-glucanase liquid biological fermentation with higher fermentation activity, higher extraction yield and lower manufacturing cost. Description of the drawings:

[0029] Figure 1 Optimum reaction temperature curve;

[0030] Figure 2 Thermal stability curve;

[0031] Figure 3 Optimal reaction pH curve;

[0032] Figure 4 pH stability curve graph;

[0033] Figure 5 Pepsin digestion resistance curve;

[0034] Figure 6 Resistance curve to trypsin digestion;

[0035] Figure 7 Plot of resistance to chymotrypsin digestion. Specific implementation method:

[0036] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0037] Example 1 Mutation breeding of strains

[0038] 1. NTG mutagenesis

[0039] Take a fresh culture slant of wild-type green Trichoderma MHU-1, wash the cells with sterile saline, transfer to a triangular flask filled with glass beads, place it on a shaker for 30 minutes to disperse the cells, filter with sterile absorbent cotton, collect single spores, and adjust the single spore concentration to 10 with saline. 6 -10 7 / mL, and add NTG mother solution to make the final concentration of 0.5g / L. Then react at 30℃ for 30min, after appropriate dilution, take 100μL and spread it on the screening plate, culture at 30℃ for about 4 days, pick the single colony with larger transparent circle on the screening plate and inoculate it into the slant culture medium for culture, after the slant grows well, use a sterile iron shovel to take a piece and inoculate it into the seed culture medium for culture, after the seeds grow well, inoculate it into the fermentation shake bottle. A high-yield strain NG12-220 with β-glucanase activity increased by 1.8 times and good genetic stability was screened, and this strain was used as the starting strain for the next step of UV mutagenesis.

[0040] 2. UV mutagenesis

[0041] Take a fresh culture slant of the high-yield strain NG12-220 obtained by NTG mutagenesis, wash the cells with sterile saline, transfer to a triangular flask filled with glass beads, place it on a shaker for 30 minutes to disperse the cells, filter with sterile absorbent cotton, collect single spores, and adjust the single spore concentration to 10 with saline. 6 -10 7 / mL. Pipette 1mL of the single spore suspension prepared by the above method into a sterile culture dish, place the culture dish vertically at 30cm under a UV lamp, and irradiate for 0-5min under magnetic stirring. After dilution and coating, place it in a 30℃ incubator away from light for 4 days. After the culture is completed, observe and count the single colonies grown. The irradiation time with a lethality of about 80% was selected as the optimal mutagenic dose. Through ultraviolet mutagenesis, a high-yield strain UN-101 with an enzyme activity 2.2 times higher than that of MHU-1 was finally obtained. The β-glucanase produced by this high-yield strain has better high temperature resistance, acid resistance, good pH stability and thermal stability, and protease resistance than the starting strain.

[0042] Screening plate culture medium: 0.2% barley β-glucan, 0.5% yeast powder, 0.1% potassium nitrate, 0.2% sodium dihydrogen phosphate, 0.5% magnesium sulfate, 0.3% Congo red, 2% agar, and the rest is water, pH 6.5.

[0043] Slant medium: 20% potato, 2% sucrose, 2% agar, the rest water, natural pH.

[0044] Seed culture medium: sucrose 3%, sodium nitrate 0.2%, magnesium sulfate 0.5%, potassium dihydrogen phosphate 0.3%, potassium chloride 0.05%, pH 6.5.

[0045] Fermentation shake flask medium: 2% barley flour; 2% corn flour, 1.6% corn steep liquor, 0.6% ammonium nitrate, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate, 0.5% calcium carbonate, and the rest is water, pH 6.2.

[0046] 3. Stability test of β-glucanase high-producing strain UN-101

[0047] Take a piece of the cultured β-glucanase high-yielding strain UN-101 fresh slant with a sterile iron shovel and inoculate it into the seed culture medium, and culture it at 30℃, 200r / min for 36h. Inoculate the cultured seed solution into the fermentation shake flask at a 2% inoculation rate, and culture it at 30℃, 200r / min for 120h, and measure the β-glucanase activity. The results of the shake flask after 10 consecutive passages of the strain are shown in Table 1:

[0048] Table 1. Stability test results of strain UN-101

[0049] Shake flask enzyme activity (U / mL) Relative enzyme activity (%) F1 12679 100 F2 13766 109 F3 11790 93 F4 12356 99 F5 13288 97 F6 12606 105 F7 11988 99 F8 12603 95 F9 13410 106 F10 12597 99

[0050] The mutant strain was subcultured for 10 generations. The experimental results shown in Table 1 show that the mutant strain has good genetic stability.

[0051] Example 2 β-glucanase activity determination method

[0052] (1) Definition of β-glucanase activity unit of the present invention

[0053] The amount of enzyme required to decompose β-glucan to produce 1 μmol of reducing sugar per minute at pH 5.0 and 40°C is defined as 1 unit of enzyme activity.

[0054] (2) Principle of β-glucanase activity assay

[0055] The activity of the β-glucanase of the present invention is detected by the DNS method, and 3,5-dinitrosalicylic acid solution is reduced to a brown-red amino compound after being heated with a reducing sugar solution. Within a certain range, the amount of reducing sugar and the depth of the color of the brown-red substance are in direct proportion, so it can be used for colorimetric determination.

[0056] (3) Drawing of glucose standard curve

[0057] Take 2.0, 4.0, 6.0, 8.0, 10.0 mL of 1.0% standard glucose solution and dilute to 100 mL respectively, and prepare standard solutions with concentrations of 200, 400, 600, 800, and 1000 μg / mL. Take 1 mL of standard solution and distilled water of different concentrations respectively, and place them in 5 stoppered colorimetric tubes. Then, add 3 mL of DNS reagent to each tube, shake well, and place in a 100℃ water bath for accurate reflection for 5 minutes. After taking out, immediately place in an ice water bath to cool to room temperature, adjust to zero with a blank tube (blank is replaced by distilled water instead of glucose standard solution), and then compare colors at 540 nm to measure OD 540 Value, in terms of absorbance OD 540 Draw a standard curve with the concentration of the corresponding standard solution as the horizontal axis and the concentration of the corresponding standard solution as the vertical axis.

[0058] (4) β-glucanase assay method

[0059] Sample determination: dilute the enzyme solution with pH 5.0 acetic acid-sodium acetate buffer to an appropriate multiple, take 1.0 mL of the enzyme solution preheated at 40°C, add 1.0 mL of 1% β-glucan solution preheated at 40°C, mix well, react at 40°C for 10 min, then add 3.0 mL of DNS to terminate the reaction, shake well, incubate at 100°C in a water bath for 5 min, cool to room temperature, and measure OD 540 .

[0060] Preparation of blank: Take 1.0 mL of diluted enzyme solution, add 3 mL of DNS first, and then add 1.0 mL of 1.0% β-glucan solution.

[0061] Enzyme activity unit (U / mL) = (R×N) / (180×t)

[0062] Where R is the concentration (μg / mL) corresponding to the standard glucose solution obtained from the standard curve through the OD value; N is the dilution factor of the enzyme solution to be tested; t is the reaction time (min), and 180 is the molecular weight of glucose.

[0063] Example 3 Production of β-glucanase by liquid fermentation and its extraction

[0064] 1. Seed cultivation

[0065] The mass volume percentage composition of the culture medium is as follows:

[0066] (1) Plate separation medium: 0.2% barley β-glucan, 0.5% yeast powder, 0.1% potassium nitrate, 0.2% sodium dihydrogen phosphate, 0.5% magnesium sulfate, 0.3% Congo red, 2% agar, and the rest water, pH 6.5.

[0067] (2) Slant culture medium: 20% potato, 2% sucrose, 2% agar, and the rest water, natural pH.

[0068] (3) Seed culture medium: 3% sucrose, 1.5% fine bran, 0.2% sodium nitrate, 0.5% magnesium sulfate, 0.3% potassium dihydrogen phosphate, 0.05% potassium chloride, pH 6.5.

[0069] (4) Fermentation shake flask fermentation medium: 2% barley flour; 2% corn flour, 1.6% corn steep liquor, 0.6% ammonium nitrate, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate, 0.5% calcium carbonate, and the remainder water, pH 6.2.

[0070] (5) Culture conditions

[0071] Separation plate: culture at 30°C for 4 days;

[0072] Slant: culture at 30°C for 7 days;

[0073] Liquid seeds: Take a piece of bacterial lawn from the cultivated slope, inoculate it into the seed culture medium, and culture it at 30℃ for 36h with a shaking speed of 200r / min;

[0074] Fermentation shake flask: Take the cultured seed liquid and inoculate it into the fermentation shake flask medium at an inoculation rate of 2%, culture at 30°C for 120 hours, and the shaking speed is 200r / min.

[0075] 2. Seed tank expansion culture

[0076] The mass volume percentage composition of the culture medium is as follows:

[0077] (1) Seed tank culture medium: 3.5% corn starch, 2% yeast powder, 3% soybean meal powder, 3% dipotassium hydrogen phosphate, 0.5% calcium chloride, 2.3% ammonium sulfate, 0.3% magnesium sulfate, and the rest water, pH 6.5;

[0078] (2) Seed tank sterilization process conditions: 121°C, 0.12 MPa, sterilization for 30 min;

[0079] (3) Seed tank culture process conditions

[0080] Tank pressure 0.05MPa, culture temperature 30℃, stirring speed 200r / min, pH control 6.5;

[0081] (4) Seed tank transplanting conditions: The bacteria are darkly stained, robust, and free of foreign bacteria.

[0082] 3. Liquid fermentation to produce β-glucanase

[0083] The mass volume percentage composition of the culture medium is as follows:

[0084] (1) Fermentation tank culture medium: 3.5% barley flour; 3% corn flour, 2.5% soybean meal, 1.6% corn steep liquor, 2.2% ammonium nitrate, 1.2% potassium dihydrogen phosphate, 0.3% magnesium sulfate, and the rest water, pH 6.2.

[0085] (2) Fermentation tank sterilization process conditions: 121°C, 0.12 MPa, sterilization for 30 min.

[0086] (3) Fermentation process conditions in the fermenter: inoculation volume 2%, tank pressure 0.06 MPa, culture temperature 30°C, rotation speed 600 r / min, feed was started when the pH dropped to the lowest and then rose to 6.8, and the pH was controlled at 6.0-6.3 (the initial feed amount was 200 g / h, the feed amount was reduced when the pH was lower than 6.0, and the feed amount was increased when the pH was higher than 6.3, and the feed amount was adjusted appropriately according to the pH situation), and the culture was released from the tank when the enzyme activity increased slowly and the bacteria began to partially autolyze after about 115-125 hours;

[0087] 4. Feed

[0088] (1) Feed medium: 10% barley flour, 3.5% corn steep liquor, 1.8% magnesium sulfate, 2.2% potassium dihydrogen phosphate, and the remainder water, pH 6.2.

[0089] (2) Feeding method: Start feeding when the pH drops to the lowest and then rises to 6.8, control the pH at 6.0-6.3, and the initial feeding amount is 200 g / h. Reduce the feeding amount when the pH is lower than 6.0, and increase the feeding amount when the pH is higher than 6.3. Adjust the feeding amount appropriately according to the pH situation.

[0090] 5. Place the can

[0091] After culturing for about 115-125 hours, the enzyme activity increases slowly and the bacteria begin to partially autolyze, then they can be released from the tank.

[0092] 6. Extraction and purification of β-glucanase

[0093] After the fermentation is completed, 40% water and 0.3% calcium chloride are added according to the volume of the fermentation liquid, and 3% perlite filter aid is added to perform plate and frame filtration; the clarified filtered enzyme liquid is ultrafiltered and concentrated using a 20KDa ultrafiltration membrane; stabilizers: 2.5% trehalose, 1.6% glycerol, preservatives: 0.15% potassium sorbate, 0.2% sodium pyrosulfite are added to the concentrate, and then 6% diatomaceous earth is used for filtration and sterilization to obtain a finished β-glucanase enzyme preparation (% is the mass volume ratio).

[0094] Fermentation was performed using the Trichoderma induced strain CGMCC NO.40414 and the culture method. Table 2 shows the fermentation cycle and enzyme activity of the fermentation broth for 6 batches of fermentation. The enzyme activity of the fermentation broth was 53000-55000 U / mL.

[0095] Table 2.50L small tank fermentation test results

[0096] batch Fermentation cycle (h) Fermentation enzyme activity (U / mL) 1 123 54506 2 120 53087 3 118 53565 4 125 54021 5 117 54231 6 115 53290

[0097] As can be seen from Table 2, the fermentation level of the mutant strain UN-101 was relatively stable, and the fermentation enzyme activity reached more than 53000U / mL.

[0098] Example 4 Optimum reaction temperature

[0099] The UN-101 fermentation supernatant obtained in batch 1 of Example 3 was taken as the β-glucanase sample, and the MHU-1 fermentation supernatant prepared in the same way was taken as the wild-type control sample. The method described in Example 2 was used to measure the enzyme activity at different temperatures of 30, 35, 40, 45, 50, 55, and 60°C at pH 5.0. The highest activity was taken as 100%, and the enzyme activities measured at other temperatures were compared with it to obtain the relative enzyme activity at that temperature. The results are as follows Figure 1 As shown, the optimal reaction temperature is 45°C. Compared with the wild-type β-glucanase, the mutant can still maintain more than 80% of its activity at 55°C.

[0100] Example 5 Thermal Stability

[0101] The UN-101 fermentation supernatant obtained in batch 1 of Example 3 was taken as a mutant β-glucanase sample, and the MHU-1 fermentation supernatant prepared in the same way was taken as a wild-type control sample. The enzyme solution was placed at 60, 65, 70, 75, 80, 85, 90, 95, and 100° C. for 2 h. After the insulation, the enzyme activity was determined using the method described in Example 2, and the relative enzyme activity was calculated with the untreated original enzyme activity as 100%. The experimental results are shown in FIG. Figure 2When kept at 80℃ for 2h, the enzyme activity can still be kept above 85%. Compared with wild-type β-glucanase, it has better thermal stability and can meet the requirements of feed and other industrial enzymes.

[0102] Example 6 Optimal reaction pH

[0103] The UN-101 fermentation supernatant obtained in batch 1 of Example 3 was taken as a mutant β-glucanase sample, and the MHU-1 fermentation supernatant prepared in the same way was taken as a wild-type control sample. According to the enzyme activity determination method described in Example 2, at a temperature of 40°C, the β-glucanase activity at pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 was determined, and the highest activity was taken as 100%, and the enzyme activities measured at other temperatures were compared with it to obtain the relative enzyme activity at that temperature. The determination results are as follows: Figure 3 As shown, the activity of the mutated β-mannanase is highest at pH 3.0, and the relative activity can reach more than 85% in the pH range of 2.5-6.5. It is a good acid glucanase and is more suitable for playing a role in the animal digestive tract than the wild type.

[0104] Example 7 pH Stability

[0105] The UN-101 fermentation supernatant obtained in batch 1 of Example 3 was taken as a mutant β-glucanase sample, and the MHU-1 fermentation supernatant prepared in the same way was taken as a wild-type control sample, and was placed in sodium hydrogen phosphate-citrate buffer at pH 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. After standing at room temperature for 2 hours, the enzyme activity was determined by the method described in Example 2, and the relative enzyme activity was calculated with the untreated original enzyme activity as 100%. The determination results are shown in FIG. Figure 4 As shown, after being treated for 2 hours under the condition of pH 2.0-6.5, the relative enzyme activity still remained above 80%. Compared with the wild-type β-glucanase, the acid resistance of the mutated β-glucanase was greatly improved, and it was more suitable for application in the feed industry.

[0106] Example 8 Determination of the resistance of β-glucanase to digestion with pepsin, trypsin and chymotrypsin

[0107] 0.1 mL of the β-glucanase finished enzyme solution prepared in batch 1 of Example 3 was taken, and 0.5 mL of pepsin (pH 2.0 concentration 100 U / mL), 0.5 mL of trypsin (pH 7.0 concentration 150 U / mL) and 0.5 mL of chymotrypsin (pH 7.5 concentration 10 U / mL) were added respectively, and the enzyme activity was measured after treatment at 37°C for different time periods, and the relative enzyme activity was calculated with the original enzyme activity without treatment as 100%. The results are shown in FIG. Figure 5 , Figure 6 and Figure 7 As shown, after being treated with the above three high-concentration proteases for 2 hours, the mutant β-glucanase can maintain more than 90% of its activity, indicating that the β-glucanase of the present invention has a very good ability to resist hydrolysis by pepsin, trypsin and chymotrypsin. Therefore, the β-glucanase of the present invention is easier to play a role in the stomach and intestines of animals and poultry than the wild type.

[0108] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A viride strain, characterized in that Specifically, Trichoderma viride ( Trichoderma viride )UN-101, the deposit number is CGMCC NO.40414.

2. The use of Trichoderma viride UN-101 according to claim 1, characterized in that: It is used in the production of β-glucanase.

3. The use according to claim 2, characterized in that The method for producing β-glucanase using Trichoderma viride UN-101 is as follows: Fermentation process conditions of the fermenter: inoculation amount 1.5-2.5%, tank pressure 0.05-0.08 MPa, culture temperature 29-31℃, rotation speed 200-800 r / min, start feeding when the pH drops to the lowest and then rises to 6.5-7.0, control the pH at 6.0-6.3, culture to 115-125h, release the tank when the enzyme activity increases slowly and the bacteria begin to partially autolyze.

4. The use according to claim 3, characterized in that The feeding method is as follows: the initial feeding amount is 200 g / h, the feeding amount is reduced when the pH is lower than 6.0, and the feeding amount is increased when the pH is higher than 6.3, and the feeding amount is adjusted according to the pH conditions.

5. The use according to claim 3, characterized in that The composition of the fermentation tank culture medium is as follows: 2.5-3.5% corn flour, 1.5-2.5% corn steep liquor, 3.0-4.0% barley flour, 2.5-3.0% soybean cake powder, 2-2.5% ammonium nitrate, 1-1.5% potassium dihydrogen phosphate, 0.3-0.5% magnesium sulfate, and the rest is water, pH 6.0-6.

3.

6. The use according to claim 4, characterized in that The composition of the feed medium is as follows: 10-15% barley flour, 3.5-4% corn steep liquor, 1.5-2.0% magnesium sulfate, 2-2.5% potassium dihydrogen phosphate, and the rest is water, pH 6.0-6.3.

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