CN110791436B - Aspergillus niger strain capable of producing pectinase at high yield and application thereof - Google Patents
Aspergillus niger strain capable of producing pectinase at high yield and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to an aspergillus niger strain capable of producing pectinase at a high yield and application thereof. The Aspergillus niger LD-092 of the invention is obtained by performing Co treatment on Aspergillus niger AJ213 original strain60The preservation number of the strain obtained by the ray mutagenesis treatment is CGMCC No. 18559. The enzyme activity of the pectinase produced by fermenting Aspergillus niger LD-092 reaches 80000-; the pectinase obtained by fermentation has the optimum pH range of 2.5-4.0 and the optimum action temperature range of 30-50 ℃, has obvious acid resistance, can be stored for 36 hours under the pH of 3.0-4.0, keeps the enzyme activity at more than 85 percent, can be widely applied to industrial production, obviously expands the industrial application range of the pectinase and improves the application value of the pectinase.
Description
The technical field is as follows:
the invention belongs to the technical field of bioengineering, and particularly relates to an aspergillus niger strain capable of producing pectinase at a high yield and application thereof.
Background art:
pectinase is a generic name of enzymes capable of degrading pectic substances through hydrolysis and cracking, and is a complex enzyme containing multiple components, mainly comprising protopectinase, pectinesterase, polygalacturonase and pectin lyase 4. Pectinases were first discovered in 1930 for the preparation of wines and juices. In the 60's of the 20 th century, scientists had a clear understanding of the chemical nature of plant tissue components, and thus have been able to utilize pectinase more widely and effectively.
More than 40 microorganisms have been reported to produce pectinases in nature, including Aspergillus, Penicillium, Fusarium, Kluyveromyces, and certain facultative anaerobic bacteria. At present, the pectinase is prepared by adopting Aspergillus niger, Rhizopus and coniothyrium minitans fermentation methods abroad, and the Aspergillus niger fermentation is reported at home. At present, the research, development and production technology of pectinase are relatively mature abroad, and researchers in China do a lot of work in the aspects of the seed selection, production process, application and the like of pectinase. The pectinase has wide application field, not only can be used in the food industry, but also can be widely applied to the industries of hemp degumming, wood preservation, biological pulping, environmental protection, sewage softening treatment, feed and the like. However, since the enzyme has low activity and high production cost, the wide application of the enzyme is severely limited, and the enzyme is mainly used for the aspects of fruit juice manufacture, fruit wine brewing, can processing, wood preservation, paper pulp bleaching, hemp degumming, plant disease control and the like at present.
Therefore, the method for screening the high-yield acid-resistant aspergillus niger strain and obtaining the liquid fermentation of the strain has great significance for promoting industrial production on a large scale and improving the application range of domestic pectinase.
The invention content is as follows:
the invention aims to provide an Aspergillus niger strain with high pectinase yield and a liquid fermentation enzyme production method thereof, which can ensure the stability of the high pectinase yield obtained by Aspergillus niger fermentation and obviously improve the acid resistance of pectinase.
The purpose of the invention is realized by the following technical scheme:
the Aspergillus niger strain for high yield of pectinase is Aspergillus niger LD-092, and the preservation number of the strain is CGMCC No. 18559.
The Aspergillus niger LD-092 of the invention is obtained by performing Co treatment on Aspergillus niger AJ213 original strain60The aspergillus niger LD-092 obtained by the ray mutagenesis treatment is preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 10 months and 9 days, and the preservation addresses are as follows: western road No.1, north chen, west road, 3, china academy of sciences, zip code: 100101, the preservation number of the strain is CGMCC No. 18559.
The invention also aims to provide the method for producing the enzyme by fermenting the Aspergillus niger LD-092, which mainly comprises the following steps:
slant culture: selecting a ring of Aspergillus niger LD-092, inoculating to a solid slant culture medium, and culturing at constant temperature of 30 deg.C for 36h to obtain first-stage seeds;
and (3) shake flask culture: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 30 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;
seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 15% (v/v) of the inoculum size, and culturing for 45h at the constant temperature of 30 ℃ and the rotating speed of 200 r/min;
culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a fermentation culture medium according to the proportion of 6-8% (v/v) of the inoculation amount, keeping the temperature at 30-31 ℃, setting the rotation speed at 200-: 1-2vvm, controlling the pH value of the fermentation broth to be 4.5 by using a supplemented medium in the whole fermentation process, ending the fermentation when the enzyme activity is slowly increased and the thallus autolysis is serious, wherein the fermentation period is 150-155 h, and obtaining the final fermentation broth; the enzyme activity of the pectinase in the fermentation liquid is 80000-85000U/mL.
And (4) extracting and refining the final fermentation liquor to obtain the finished product liquid enzyme preparation of the pectinase.
The slant culture medium (g/L): 200 parts of potato, 20 parts of glucose and 17 parts of agar, wherein the pH value is 4.5, and the potato is sterilized for 20min at 121 ℃;
the seed culture medium (g/L): glucose 20, fine bran 20, corn steep liquor 4.5, potassium dihydrogen phosphate 1.5, magnesium sulfate 1, pH4.5, sterilizing at 121 ℃ for 20 min;
the seeding tank medium (g/L): 80 parts of glucose, 30 parts of fine bran, 4.55 parts of yeast extract, 4.4 parts of corn steep liquor, 1.5 parts of ammonium sulfate, 0.8 part of magnesium sulfate, 1.5 parts of monopotassium phosphate and 1 part of calcium chloride, wherein the pH value is 4.5, and the sterilization is carried out for 30min at the temperature of 123 ℃;
the fermentation medium (g/L): 300 parts of corn starch, 30 parts of bean cake powder, 50 parts of fine bran, 27 parts of corn steep liquor, 1.2 parts of magnesium sulfate, 4 parts of ammonium sulfate, 2.5 parts of sodium dihydrogen phosphate, 0.8 part of calcium chloride, 4.5 parts of pH, and 30min of sterilization at 121 ℃;
the feed medium (g/L): maltose 600, corn steep liquor 22, magnesium sulfate 1.5, 121 ℃ sterilization for 30 min.
The extraction and refining method of the pectinase comprises the following steps:
adding 1-5% of perlite filter aid into the final fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;
carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 20000 molecular weight ultrafiltration membrane to obtain a concentrated solution;
adding 15 percent (m/v) of stabilizer and 0.45 percent (m/v) of preservative into the concentrated solution by mass percentage, adjusting the pH value to 4.5, and then filtering and sterilizing to obtain the finished product liquid enzyme preparation of pectinase.
Preferably, the stabilizing agent is glycerin, and the preservative is potassium sorbate and sodium benzoate in a mass ratio of 1: 2.
The optimum action pH range of the pectinase produced by the fermentation of the Aspergillus niger LD-092 is 2.5-4.0; the optimal action temperature range is 30-50 ℃; the enzyme activity is kept above 85% after the mixture is stored for 36 hours under the pH value of 3.0-4.0.
Has the advantages that:
the invention carries out Co treatment on the Aspergillus niger original strain AJ21360A mutant strain LD-092 with high pectinase yield is bred by ray mutagenesis treatment, and the Aspergillus niger LD-092 fermentation process is supplemented with optimized material feeding conditions to ensure that the enzyme activity reaches 80000
U/mL to 85000U/mL, while the highest enzyme activity of the Aspergillus niger AJ213 after liquid fermentation is 69200U/mL. Therefore, the enzyme activity of the Aspergillus niger LD-092 is obviously higher than that of the Aspergillus niger AJ213 original strain.
The components of the culture medium in the fermentation method of the strain are all derived from raw materials with lower cost, and the fermentation cost is greatly reduced while the fermentation productivity is improved and the production efficiency is improved, thereby being greatly helpful for production.
The pectinase obtained by fermenting the Aspergillus niger LD-092 has the optimum pH range of 2.5-4.0 and the optimum action temperature range of 30-50 ℃, has obvious acid resistance, is stored for 36 hours under the pH of 3.0-4.0, keeps the enzyme activity above 85 percent, can be widely applied to industrial production, obviously expands the industrial application range of the pectinase and improves the application value of the pectinase.
Description of the drawings:
FIG. 1: relative enzyme activity at different pH;
FIG. 2: relative enzyme activity at different temperatures;
FIG. 3: acid resistance curve.
The specific implementation mode is as follows:
the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
Example 1 Aspergillus niger LD-092 fermentation enzyme production and extraction method of pectinase produced thereby
The fermentation enzyme production method of the Aspergillus niger LD-092 mainly comprises the following steps:
slant culture: selecting a ring of Aspergillus niger LD-092, inoculating to a solid slant culture medium, and culturing at constant temperature of 30 deg.C for 36h to obtain first-stage seeds;
and (3) shake flask culture: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 30 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;
seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 15% (v/v) of the inoculum size, and culturing for 45h at the constant temperature of 30 ℃ and the rotating speed of 200 r/min;
culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a fermentation tank culture medium according to the proportion of 6% (v/v) of the inoculation amount, keeping the temperature at 30 ℃, rotating at the speed of 200r/min, and setting the ventilation: 1-2vvm, controlling the pH value of the fermentation broth to be 4.5 by using a supplemented medium in the whole fermentation process, ending the fermentation when the enzyme activity is slowly increased and the thallus autolysis is serious, wherein the fermentation period is 155h, and obtaining the final fermentation broth; the enzyme activity of the pectinase in the fermentation liquor is 83210U/mL;
and (4) extracting and refining the final fermentation liquor to obtain the finished product liquid enzyme preparation of the pectinase.
The slant culture medium (g/L): 200 parts of potato, 20 parts of glucose and 17 parts of agar, wherein the pH value is 4.5, and the potato is sterilized for 20min at 121 ℃;
the seed culture medium (g/L): glucose 20, fine bran 20, corn steep liquor 4.5, potassium dihydrogen phosphate 1.5, magnesium sulfate 1, pH4.5, sterilizing at 121 ℃ for 20 min;
the seeding tank medium (g/L): 80 parts of glucose, 30 parts of fine bran, 4.55 parts of yeast extract, 4.4 parts of corn steep liquor, 1.5 parts of ammonium sulfate, 0.8 part of magnesium sulfate, 1.5 parts of monopotassium phosphate and 1 part of calcium chloride, wherein the pH value is 4.5, and the sterilization is carried out for 30min at the temperature of 123 ℃;
the fermenter medium (g/L): 300 parts of corn starch, 30 parts of bean cake powder, 50 parts of fine bran, 27 parts of corn steep liquor, 1.2 parts of magnesium sulfate, 4 parts of ammonium sulfate, 2.5 parts of sodium dihydrogen phosphate, 0.8 part of calcium chloride, 4.5 parts of pH, and 30min of sterilization at 121 ℃;
the feed medium (g/L): maltose 600, corn steep liquor 22, magnesium sulfate 1.5, 121 ℃ sterilization for 30 min.
The extraction and refining method of pectinase comprises the following steps:
adding 1-5% of perlite filter aid into the final fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;
carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 20000 molecular weight ultrafiltration membrane to obtain a concentrated solution;
adding 15 percent (m/v) of glycerol, 0.15 percent (m/v) of potassium sorbate and 0.3 percent (m/v) of sodium benzoate by mass percent into the concentrated solution, adjusting the pH to 4.5, and then filtering and sterilizing to obtain the finished product liquid enzyme preparation of pectinase.
Example 2 Aspergillus niger LD-092 fermentation Performance validation
A50L fermentation tank verification experiment is carried out according to the fermentation enzyme production method of Aspergillus niger LD-092 in example 1, the fermentation period is 155h, the average enzyme activity of pectinase in fermentation liquor is 82882U/mL in 7 batches of fermentation enzyme production conditions, and Table 1 shows that the strain not only can produce pectinase with high yield, but also has remarkable stability in fermentation performance and enzyme activity of the produced pectinase.
TABLE 17 fermentation enzyme production of high-yield pectinase batches
Example 3 method for determining enzyme Activity of pectinase
(1) Preparation of enzyme solution: and (4) centrifuging the fermentation liquor at 6000r/min for 15min, and collecting supernatant fluid, namely the crude enzyme liquid.
(2) The determination method comprises the following steps: the 3, 5-dinitrosalicylic acid method (DNS method) is used. Taking 0.5mL of appropriately diluted crude enzyme solution into a 25mL colorimetric tube, adding 2.0mL of 0.4% pectin solution (pH4.0) serving as a substrate solution, carrying out water bath reaction at 45 ℃ for 30min, adding DNS solution, boiling for 5min, cooling, fixing the volume to 25mL, and measuring the light absorption value at 520 nm.
(3) Definition of enzyme activity: under certain conditions, the enzyme amount required for decomposing pectin to generate 1 mu mol galacturonic acid per minute is one enzyme activity unit (U).
Example 4 optimum pH Range of pectinases
The pectinase with the enzyme activity of 80000U/mL, which is produced by the invention and is measured under the conditions of the temperature of 40 ℃ and the pH value of 3.5, is taken as the relative enzyme activity of 100 percent, and the enzyme activity is measured by respectively using corresponding pH buffer solutions and keeping other conditions unchanged. The enzyme solution is respectively placed in buffer solutions with pH values of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, the measured relative enzyme activity change curve is shown in figure 1, the enzyme activity of the pectinase is the highest when the pH value is 3.5, and the action pH value is wider at 2.5-4.0.
Example 5 optimum temperature Range for pectinases
The pectinase with the enzyme activity of 80000U/mL, which is produced by the invention and is measured under the conditions of the temperature of 40 ℃ and the pH value of 3.5, is 100 percent of relative enzyme activity, the enzyme activity is measured under the conditions of the pH value of 3.5 and different temperatures (20, 25, 30, 35, 40, 45, 50, 55, 60 and 65), the change curve of the measured relative enzyme activity is shown in figure 2, and the optimal action temperature range of the enzyme is 30-50 ℃.
Example 6 acid resistance assay of pectinase
The pectinase with the enzyme activity of 80000U/mL, which is produced by the invention and is measured under the conditions of the temperature of 40 ℃ and the pH value of 3.5, is 100 percent of relative enzyme activity, enzyme liquid is respectively placed in buffer solutions with the pH values of 3.0, 3.5 and 4.0, the enzyme liquid is kept stand at 40 ℃, the enzyme activity is respectively measured after the enzyme liquid is kept stand for 6 hours, 12 hours, 18 hours, 24 hours, 30 hours and 36 hours, the change curve of the measured relative enzyme activity is shown in figure 3, the relative enzyme activity under each pH condition is still more than 85 percent, and the pectinase has better acid resistance.
Claims (4)
1. The Aspergillus niger strain capable of highly producing pectinase is characterized in that the Aspergillus niger is specifically Aspergillus niger LD-092, and the strain preservation number is CGMCC No. 18559.
2. Use of the aspergillus niger according to claim 1 for the production of pectinase.
3. A pectinase produced by the Aspergillus niger according to claim 1.
4. The pectinase of claim 3 where the enzymatic properties are as follows:
the optimum action pH range is 2.5-4.0; the optimal action temperature range is 30-50 ℃; the enzyme activity is kept above 85% after the mixture is stored for 36 hours under the pH value of 3.0-4.0.
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