Method for producing pectinase from trachycarpus racemosus based on single-factor test and application

Abstract

The invention discloses a method for producing pectinase by using trachycarpus racemosus based on a single factor test and application, and the method comprises the following steps: selecting a fermentation strain, setting a solid state fermentation culture medium, setting fermentation culture conditions, inoculating the fermentation strain into the solid state fermentation culture medium, testing the performance of pectinase, and determining the content of pectinase according to the test result. And optimizing the solid-state fermentation culture medium and the fermentation culture condition to obtain the optimal solid-state fermentation culture medium setting condition and the optimal fermentation culture condition. Compared with the prior art, the method has the advantages that the quantity and vitality of spores are remarkably improved by selecting the trachycarpus racemosus strains as the fermentation strains, so that the vitality and production efficiency of the strains to be fermented are improved, and the optimal solid-state fermentation culture medium setting conditions and the optimal fermentation culture conditions are obtained by optimizing the solid-state fermentation culture medium setting conditions and the fermentation culture conditions; the optimized pectinase product is obtained under the optimized condition, and the pectinase product has high activity and excellent high temperature resistance and acid resistance, so that the application range of pectinase is widened.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation engineering, and in particular to a method and application of producing pectinase by Transverse Rhizoctonia racemosus based on a single factor test. Background Art

[0002] Pectinases are a class of enzymes that specifically decompose pectin. They are part of the hydrolase family. These enzymes include polygalacturonase (PG), protopectinase, pectin methylesterase (PE), pectin lyase (PL), and cellulase. They mainly work by breaking glycosidic bonds, removing branches, or modifying pectin. Specifically, protopectinase can convert protopectin into soluble pectin by breaking the chemical bond between arabinose and polymethoxygalacturonic acid. Pectin methylesterase and pectin acetylesterase are responsible for removing methoxy and acetyl residues from pectin to generate polygalacturonic acid. Polygalacturonase breaks the glycosidic α-(1,4) bonds between galacturonic acid residues through hydrolysis and trans-elimination reactions, breaking them into smaller molecules, thereby promoting coagulation and sedimentation. Cellulase promotes the decomposition of macromolecules such as pectin by reducing viscosity and increasing the diffusion of endogenous enzymes, increasing the contact area between enzymes and other components.

[0003] Pectinases are widely used in the food industry, such as in juice clarification, winemaking and juicing processes. In addition, they also play an important role in environmental management, feed processing, pharmaceutical manufacturing and biofuel production. However, the current production methods of pectinases produce pectinases with poor activity and poor acid and high temperature resistance.

[0004] The defects of the existing pectinase production method are: Patent document CN110885760A discloses a method for producing pectinase. The strain Aspergillus niger LD-092 used in the method for producing pectinase by liquid fermentation is obtained by subjecting the original strain AJ213 of Aspergillus niger to Co60 ray mutagenesis treatment, and the strain deposit number is CGMCC No.18559. The activity of pectinase produced by fermentation of Aspergillus niger LD-092 reaches 80000-85000U / mL; the optimum pH range of the fermented pectinase is 2.5-4.0, the optimum action temperature range is 30-50°C, it has significant acid resistance, and the enzyme activity is maintained at more than 85% when stored at pH 3.0-4.0 for 36 hours. It can be widely used in industrial production, significantly expanding the industrial application range of pectinase and improving the application value of pectinase; but the problem that the application range of the prepared pectinase is small is not taken into account, which reduces the application value of pectinase. Summary of the invention

[0005] The object of the present invention is to provide a method and application of producing pectinase by Transverse Rhizoctonia racemosa based on a single factor test, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for producing pectinase by Transverse Rhizoctonia racemosa based on a single factor test, comprising the following steps: S1, select fermentation bacteria; S2, setting solid-state fermentation medium; S3, setting fermentation culture conditions, inoculating fermentation bacteria into the solid fermentation medium; S4, performing a performance test on the pectinase obtained in S3; S5. Optimize the solid-state fermentation medium using a single factor experimental method according to the test results, and repeat steps S3 and S4 to obtain the optimal solid-state fermentation medium setting conditions; S6. Under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, and steps S3 and S4 are repeated to obtain the optimal fermentation culture conditions.

[0007] Preferably, the fermentation strain in S1 is Lichtheimia sp. X-8.

[0008] Preferably, the solid-state fermentation medium comprises a carbon source, a nitrogen source, metal ions and water.

[0009] Preferably, the carbon source comprises at least one of bran, pomace, and rice husk, the nitrogen source comprises at least one of yeast extract, soybean meal, and ammonium sulfate, and the metal ions comprise K + , Mn 2+ Mg 2+ , Fe 3+ 、Na + At least one of .

[0010] Preferably, the ratio of carbon source:nitrogen source in the solid-state fermentation medium is (1-7):1.

[0011] Preferably, the fermentation conditions include the solid fermentation medium charge amount, the fermentation strain inoculation amount, the fermentation temperature, the fermentation pH and the culture time.

[0012] Preferably, the inoculation amount of the fermentation bacteria is 0.5-2.5 ml.

[0013] The invention relates to the application of Pectinase produced by Transverse Rhizoctonia racemosus based on single factor experiment, and the application comprises the application of pectinase in catalyzing pectin hydrolysis reaction.

[0014] Preferably, when the pectinase catalyzes the pectin hydrolysis reaction, the pH of the reaction system is 2.5-7.5 and the reaction temperature is 30-80°C.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention significantly improves the number and activity of spores by selecting the high-yielding pectinase strain Lichtheimia sp. X-8, which is beneficial to improving the activity of the fermented bacteria, thereby greatly improving the production efficiency. The optimal solid-state fermentation medium setting conditions and the optimal fermentation culture conditions are obtained by optimizing the solid-state fermentation medium setting conditions and the optimal fermentation culture conditions. Under the optimized conditions, an optimized pectinase product is obtained, and the optimized pectinase product has high activity and excellent high temperature resistance and acid resistance, and can maintain a high relative enzyme activity within the temperature range of 30°C to 80°C and within the pH range of 2.5 to 7.5, thereby improving the applicable range of pectinase.

[0016] 2. The present invention adopts solid fermentation technology to replace liquid fermentation technology, which reduces the complexity of operation, simplifies the production process of pectinase, shortens the fermentation and culture cycle of pectinase, and improves the product quality of pectinase, so that pectinase can be industrially produced.

[0017] 3. The present invention realizes the recycling of resources and reduces the production cost to a certain extent by utilizing low-cost solid waste as the carbon source for bacterial growth.

[0018] 4. The present invention optimizes the setting conditions of the solid-state fermentation medium and the fermentation culture conditions by adopting a single-factor experimental method, which facilitates the intuitive acquisition of the effects of different factors on the pectinase activity, and further facilitates the acquisition of the optimal setting conditions of the solid-state fermentation medium and the optimal fermentation culture conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram showing the effect of the solid matrix ratio in the solid-state fermentation medium of the present invention on the pectinase activity; Figure 2 It is a schematic diagram showing the effect of the types of metal ions on the activity of pectinase in the solid-state fermentation medium of the present invention; Figure 3 It is a schematic diagram showing the effect of the amount of metal ions added to the solid-state fermentation medium on the activity of pectinase; Figure 4 It is a schematic diagram showing the effect of the water content in the solid-state fermentation medium on the activity of pectinase of the present invention; Figure 5 It is a schematic diagram showing the effect of the inoculation amount on the pectinase activity in the fermentation culture conditions of the present invention; Figure 6It is a schematic diagram showing the effect of the loading amount on the pectinase activity in the fermentation culture conditions of the present invention; Figure 7 It is a schematic diagram showing the effect of culture temperature on pectinase activity in the fermentation culture conditions of the present invention; Figure 8 It is a schematic diagram of the effect of pH on pectinase activity in the fermentation culture conditions of the present invention; Fig. 9 It is a schematic diagram showing the effect of temperature on the relative enzyme activity of pectinase before and after optimization in the detection process of the present invention; Fig.10 This is a schematic diagram of the effect of pH on the relative enzyme activity of pectinase before and after optimization in the detection process of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Embodiment 1: The selection of fermentation strains in S1 comprises the following steps: Selected strains: The strain used is Lichtheimia racemosus strain, which is classified as Lichtheimia sp. X-8, and the strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 7, 2023, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, and the deposit number is CGMCC NO.40909; The strain is activated to obtain a fermentation strain to be cultured: the strain Lichtheimia sp.X-8 with a preservation number of CGMCC NO.40909 is activated and cultured on a PDA solid medium (the PDA solid medium comprises: 200 g / L potato, 20 g / L glucose, 20 g / L agar, water is added to make the volume 1000 mL, and sterilized at 115°C for 30 min), cultured for 3 days, eluted the spores of the racemose mold with sterile physiological saline, placed in a sterilized centrifuge tube containing glass beads, fully shaken to break up the spores, filtered the spore suspension with sterile gauze, counted the number of spores using a hemocytometer, and diluted with physiological saline to make the spore concentration 1×106 spores / mL, thereby obtaining a fermentation strain to be cultured; The solid-state fermentation medium is set in S2 and comprises the following steps: Setting the initial fixed fermentation medium: in the initial solid fermentation medium, the carbon source is bran, and the carbon source mass is 21 g, the nitrogen source is yeast extract, and the yeast extract mass is 9 g, and the water is 18.6 mL; In S3, the fermentation culture conditions are set, and the fermentation strains are inoculated into the solid fermentation medium, including the following steps: The material in S2 is loaded into the culture medium, and the loading amount of the solid fermentation culture medium is 30 g. The fermentation strain to be cultured obtained in S1 is inoculated into the solid fermentation culture medium, and the inoculation amount is 1 mL. The culture is carried out at a natural pH and a fermentation temperature of 25° C. for 3 days to obtain pectinase 1, and the fermentation culture conditions for obtaining pectinase 1 are the initial fermentation culture conditions; The enzyme activity performance test of pectinase 1 is carried out, and the test method specifically includes the following steps: The pectinase activity of different strains screened was determined using pectin as a substrate. Take 5 mL of pectin solution and add it to two colorimetric tubes A and B, and place them in a constant temperature water bath at 50℃±0.2℃ to preheat for 8 minutes; add 5 mL of pH3.5 citric acid-sodium citrate buffer to tube A (blank group); add 1 mL of diluted crude enzyme solution and 4 mL of citric acid-sodium citrate buffer to tube B (experimental group), shake well and time immediately, and after an accurate reaction of 30 minutes, take it out immediately, take 1 mL of the reaction solution and add 3 mL of DNS to shake well, boil it in a water bath for 10 minutes, cool it to 25 mL, and measure the absorbance at a wavelength of 540 nm. The absorbance of group A (blank control group) is recorded as A1, and the absorbance of group B (experimental group) is A2. Definition of enzyme activity unit: Under the reaction conditions of 50℃ and pH3.5, 1 g of solid enzyme powder (1 mL of liquid enzyme) decomposes pectin in 1 hour to produce 1 mg of galacturonic acid as one activity unit, expressed as U / g or U / mL; The activity performance of pectinase 1 was tested in the above steps, and the pectinase 1 was measured to be 15791 U / g dry basis.

[0022] Embodiment 2: In S5, the single factor method is used to optimize the solid-state fermentation medium, which specifically includes the following steps: S511: selecting bran and soybean meal as solid matrix, and the masses of bran and soybean meal are 21 and 19 respectively, and water is 18.6mL to obtain pectinase II under initial fermentation culture conditions, and pectinase II is tested for enzyme activity performance, and recording pectinase II is 15519U / g dry basis; S512: Selecting pomace and yeast extract as solid matrices, wherein the masses of pomace and yeast extract are 21 and 19, respectively, and water is 18.6 mL, obtaining pectinase III under initial fermentation culture conditions, and performing enzyme activity performance test on pectinase III, and measuring pectinase II to be 15421 U / g dry basis; S513: pomace and soybean meal were selected as solid matrices, and the masses of pomace and soybean meal were 21 and 19 respectively, and water was 18.6 mL. Pectinase IV was obtained under initial fermentation culture conditions, and the enzyme activity performance of pectinase IV was tested, and the pectinase IV was measured to be 15489 U / g dry basis.

[0023] Based on the above performance test results, it can be seen that when bran and yeast extract are selected as the solid matrix of the fermentation medium as the carbon source and nitrogen source, the activity of the obtained pectinase is higher. Therefore, bran is selected as the carbon source and yeast extract as the nitrogen source, and they are recorded as the best solid matrix in the optimal solid-state fermentation culture setting conditions.

[0024] Embodiment three: S5 uses a single factor method to optimize the solid-state fermentation medium, and also includes the following steps: S52: Yeast extract and bran are selected as the solid matrix of the culture medium, and the ratio of bran to yeast extract is a single factor variable, and water is 18.6 mL. Then, steps S3 and S4 are repeated. Under the premise of a loading amount of 30 g and an inoculation amount of 1 mL, the culture is carried out at a natural pH and a fermentation temperature of 25°C for 3 days to obtain a second group of pectinase, and the activity performance of each pectinase in the second group of pectinase is determined respectively. The ratio of bran to yeast extract in the solid fermentation medium of the second group of pectinase is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1, respectively. The test results of the activity performance of the second group of pectinase are as follows: Figure 1 shown.

[0025] Further, according to Figure 1 It can be seen that when the ratio of bran to yeast extract is 4:1, the pectinase activity reaches its maximum value. Therefore, the bran: yeast extract ratio of 4:1 is selected as the optimal solid matrix ratio in the optimal solid-state fermentation medium setting conditions.

[0026] Embodiment 4: S5 uses a single factor method to optimize the solid-state fermentation medium, and also includes the following steps: S53: Under the conditions of optimal solid matrix and optimal solid matrix ratio, metal ions are added to the solid fermentation medium, and the type of metal ions is a single factor variable, the amount of metal ions added is 1.1%, and the water in the solid fermentation medium is 18.6 mL. Then, steps S3 and S4 are repeated. Under the initial fermentation culture conditions, that is, under the premise of a charging amount of 30 g and an inoculation amount of 1 mL, at a natural pH and a fermentation temperature of 25°C, the third group of pectinases is obtained, and the activity performance of each pectinase in the third group of pectinases is determined respectively, and the metal ions are K + , Mn 2+ Mg 2+ , Fe 3+ 、Na+ The third group of pectinase includes 5 kinds of pectinase products. The activity performance test results of the third group of pectinase are as follows: Figure 2 shown.

[0027] Further, according to Figure 2 It can be seen that when the added metal ion is Mg 2+ When Mg is 2+ As the optimal addition of metal ions in the solid-state fermentation medium setting conditions.

[0028] Embodiment five: S5 uses a single factor method to optimize the solid-state fermentation medium, and also includes the following steps: S54: Under the conditions of optimal solid matrix, optimal solid matrix ratio and optimal added metal ion, a solid fermentation medium was prepared with the amount of metal ion added as a single factor variable, and the water in the solid fermentation medium was 18.6 mL, and the amounts of metal ions added were 0.7%, 0.9%, 1.1%, 1.3% and 1.5%, respectively, to obtain 5 solid fermentation mediums, and then steps S3 and S4 were repeated. Under the initial fermentation culture conditions, that is, under the premise of a charging amount of 30 g and an inoculation amount of 1 mL, at a natural pH and a fermentation temperature of 25°C, a fourth group of pectinases was obtained, and the activity performance of each pectinase in the fourth group of pectinases was determined respectively. The activity performance test results of the fourth group of pectinases are as follows. Figure 3 shown.

[0029] Further, according to Figure 3 It can be seen that Mg 2+ When the addition amount is 1.3%, the pectinase activity reaches the maximum value. 2+ The addition amount was 1.3% as the optimal metal ion addition amount in the optimal solid-state fermentation medium setting conditions.

[0030] Embodiment six: S5 uses a single factor method to optimize the solid-state fermentation medium, and also includes the following steps: S55: Under the conditions of optimal solid matrix, optimal solid matrix ratio, optimal added metal ions and optimal ion addition amount, solid fermentation medium was prepared with water content in solid fermentation medium as single factor variable, and the water content in solid fermentation medium was 45%, 50%, 55%, 60% and 65% respectively, to obtain 5 kinds of solid fermentation medium, and then steps S3 and S4 were repeated, under the initial fermentation culture conditions, i.e., under the premise of charging amount of 30 g and inoculation amount of 1 mL, at natural pH and fermentation temperature of 25°C, static culture was carried out for 3 days to obtain the fifth group of pectinase, and the activity performance of each pectinase in the fifth group of pectinase was determined respectively. The activity performance test results of the fifth group of pectinase are as follows: Figure 4 shown.

[0031] Further, by Figure 4 It can be seen that when the water content is 60%, the pectinase activity reaches its maximum value. Therefore, a water content of 60% is selected as the optimal water content in the setting conditions of the optimal solid-state fermentation medium.

[0032] By optimizing the solid-state fermentation medium, the optimal solid-state fermentation medium setting conditions were obtained, which were to use bran and yeast extract as solid matrices, with a bran to yeast extract ratio of 4:1, and Mg 2+ As the added metal ions, the added amount of the metal ions is 1.3%, and the water content in the solid fermentation medium is 60%.

[0033] Embodiment seven: In S6, under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, including the following steps: S61: Under the optimal solid-state fermentation medium setting conditions, the inoculation amount in the fermentation culture conditions was used as a single factor variable. Under the premise that the solid-state fermentation medium charge amount was 30 g, the inoculation amounts were set to 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 2.5 mL, respectively. The culture was statically cultured at a natural pH and a fermentation temperature of 25°C for 3 days to obtain the sixth group of pectinase, which included 5 pectinase products. The activity performance of each pectinase in the sixth group of pectinase was determined respectively. The activity performance test results of the sixth group of pectinase are as follows: Figure 5 as shown. Further, according to Figure 5 It can be seen that under the optimal solid-state fermentation medium setting conditions, when the inoculation size in the fermentation culture conditions is 1.0 mL, the pectinase activity performance test results have a maximum value. Therefore, the inoculation size of 1.0 mL is selected as the optimal inoculation size in the optimal fermentation culture conditions.

[0034] Embodiment eight: In S6, under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, including the following steps: S62: Under the optimal solid-state fermentation medium setting conditions and the optimal inoculation amount conditions, the charging amount in the fermentation culture conditions was used as a single factor variable, and the charging amounts were set to 10g, 20g, 30g, 40g, and 50g, respectively, to obtain 5 solid-state fermentation culture media, and then inoculated into the 5 solid-state fermentation culture media, respectively, under the premise of an inoculation amount of 1mL, at a natural pH, and a fermentation temperature of 25°C, and cultured for 3 days to obtain the seventh group of pectinase, and the activity performance of each pectinase in the seventh group of pectinase was determined respectively. The activity performance test results of the seventh group of pectinase are as follows: Figure 6 shown.

[0035] Further, according to Figure 6 It can be seen that under the optimal solid-state fermentation medium setting conditions, the inoculation amount in the fermentation culture conditions is 1.0mL. When the loading amount in the fermentation culture conditions is 50g, the pectinase activity performance test result has a maximum value. Therefore, the loading amount of 50g is selected as the optimal loading amount in the optimal fermentation culture conditions.

[0036] Embodiment nine: In S6, under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, including the following steps: S63: Under the optimal solid-state fermentation medium setting conditions, the optimal inoculation amount and the optimal loading amount, the temperature in the fermentation culture conditions is used as a single factor variable, the fermented bacteria are fermented at 21°C, 24°C, 27°C, 30°C and 33°C under natural pH conditions for 3 days to obtain the eighth group of pectinase, and the activity performance of each pectinase in the eighth group of pectinase is measured respectively. The activity performance test results of the eighth group of pectinase are as follows: Figure 7 shown.

[0037] Further, according to Figure 7 It can be seen that under the optimal solid-state fermentation medium setting conditions, the inoculation volume in the fermentation culture conditions is 1.0 mL and the loading volume is 50 g. When the fermentation culture temperature in the fermentation culture conditions is 27°C, the pectinase activity performance test result reaches a maximum value. Therefore, the fermentation culture temperature of 27°C is selected as the optimal fermentation culture temperature in the optimal fermentation culture conditions.

[0038] Embodiment ten: In S6, under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, including the following steps: S64: Under the optimal solid-state fermentation medium setting conditions, the optimal inoculation amount, the optimal loading amount and the optimal fermentation culture temperature conditions, the pH in the fermentation culture conditions is taken as a single factor variable, the fermented bacteria are respectively fermented at pH 3.5, 4.5, 5.5, 6.5, and 7.5 for 3 days to obtain the ninth group of pectinase, and the activity performance of each pectinase in the ninth group of pectinase is respectively measured. The activity performance test results of the ninth group of pectinase are as follows: Figure 8 shown.

[0039] Further, according to Figure 8 It can be seen that under the optimal solid-state fermentation medium setting conditions, the inoculation volume in the fermentation culture conditions is 1.0 mL, the loading volume is 50 g, and the fermentation culture temperature is 27 ° C. When the pH in the fermentation culture conditions is 5.5, the pectinase activity performance test results have a maximum value. Therefore, pH 5.5 is selected as the optimal fermentation culture pH in the optimal fermentation culture conditions.

[0040] By optimizing the fermentation culture conditions, the best fermentation culture conditions were obtained as follows: a charge amount of 50 g, an inoculation amount of 1.0 mL, a fermentation temperature of 27°C, a pH of 5.5, and a fermentation culture time of 3 days.

[0041] Furthermore, under the optimal solid-state fermentation medium setting conditions and the optimal fermentation culture conditions, pectinase was fermented and cultured to obtain an optimized pectinase product. After the optimized pectinase product was tested for activity, the enzyme activity of the optimized pectinase product was 25630U / g dry basis. Compared with the pectinase 1 in Example 1, the enzyme activity of the pectinase was increased by 62.3%, which greatly explored the production potential of the strain, achieved the ideal goal, and contributed to the industrial application of pectinase.

[0042] The optimized pectinase products obtained under the optimal solid-state fermentation medium setting conditions and the optimal fermentation culture conditions, and the pectinase 1 in the embodiment were taken, and the two groups of pectinase crude enzyme solutions were precipitated with ammonium sulfate, dialyzed and concentrated, and filtered through a filter membrane before loading on an anion exchange column (Q Beads 6FF) for chromatography, and gradient eluted to obtain purified optimized pectinase and optimized pre-pectinase, and the relative enzyme activity of the optimized pre-pectinase and the optimized pectinase was detected, and the temperature and pH in the detection process were used as single factor variables, respectively, to obtain the relative enzyme activity detection results as shown in the figure. Fig. 9 and Fig.10 As shown; Further, according to Fig. 9 It can be seen that the relative enzyme activity of the optimized Pectinase produced by Aglaonema racemosus X-8 reaches a peak at 60°C, which has a certain heat-resistant advantage and is a heat-resistant pectinase. With the further increase of temperature, the relative enzyme activity shows a downward trend, and in the temperature range of 30°C to 80°C, the relative enzyme activity of pectinase remains above 80%, so that the optimized pectinase still has a high enzyme activity in a larger temperature range. according to Fig.10 It can be seen that the relative enzyme activity of the pectinase produced by the optimized Acropora racemosus X-8 reaches a peak value at a pH of 4.0, and in the pH range of 2.5 to 7.5, the relative activity of the enzyme can be maintained at more than 75%, that is, the pectinase produced by the optimized Acropora racemosus X-8 has a good adaptability to the acidic environment, and can still maintain a high relative enzyme activity under acidic conditions. The optimized pectinase product was obtained by optimizing the setting conditions of the solid-state fermentation medium and the fermentation culture conditions, and the optimized pectinase product can adapt to higher temperatures and a larger temperature range, as well as acidic conditions.

[0043] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for producing pectinase by using Aglaonema racemosus based on a single factor test, characterized in that: The steps include: S1, select fermentation bacteria; S2, setting solid-state fermentation medium; S3, setting fermentation culture conditions, inoculating fermentation bacteria into the solid fermentation medium; S4, performing a performance test on the pectinase obtained in S3; S5. Optimize the solid-state fermentation medium using a single factor experimental method according to the test results, and repeat steps S3 and S4 to obtain the optimal solid-state fermentation medium setting conditions; S6. Under the optimal solid-state fermentation medium setting conditions, the fermentation culture conditions are optimized, and steps S3 and S4 are repeated to obtain the optimal fermentation culture conditions.

2. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 1, characterized in that: The fermentation strain in S1 is Lichtheimia sp. X-8.

3. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 1, characterized in that: The solid-state fermentation medium comprises a carbon source, a nitrogen source, metal ions and water.

4. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 3, characterized in that: The carbon source comprises at least one of bran, pomace and rice husk; the nitrogen source comprises at least one of yeast extract, soybean meal and ammonium sulfate; the metal ions comprises K + , Mn 2+ Mg 2+ , Fe 3+ 、Na + At least one of .

5. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 4, characterized in that: The ratio of carbon source to nitrogen source in the solid-state fermentation medium is (1-7):

1.

6. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 5, characterized in that: The fermentation culture conditions include the solid fermentation medium charge amount, the fermentation strain inoculation amount, the fermentation culture temperature, the fermentation culture pH and the culture time.

7. The method for producing pectinase by Transverse Rhizoctonia racemosa based on single factor test according to claim 6, characterized in that: The inoculation amount of the fermentation bacteria is 0.5-2.5 ml.

8. Application of the method for producing pectinase by Transverse Rhizoctonia racemosa based on a single factor test according to claim 7, wherein the pectinase produced by the method is characterized in that: The application includes the application of pectinase in catalyzing pectin hydrolysis reaction.

9. The use of Aglaonema racemosus for producing pectinase based on a single factor test according to claim 8, characterized in that: When the pectinase catalyzes the hydrolysis of pectin, the pH of the reaction system is 2.5-7.5 and the reaction temperature is 30-80°C.

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