CN115677823A - Method for extracting natural active protein of selenium-rich ganoderma lucidum - Google Patents
Method for extracting natural active protein of selenium-rich ganoderma lucidum Download PDFInfo
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Abstract
The invention discloses a method for extracting natural active protein of selenium-enriched ganoderma lucidum, which adds a pretreated enzyme compound into a crude protein product water solution of a selenium-enriched fermentation liquor of ganoderma lucidum mycelia, extracts the protein in the treatment liquor again through a specific treatment procedure, greatly increases the protein content in the product obtained by secondary extraction, wherein the activity of the active protein represented by amylase and xylanase is kept above 80 percent.
Description
Technical Field
The invention relates to a method for extracting natural active protein of selenium-rich ganoderma lucidum, and belongs to the technical field of extraction and purification.
Background
Ganoderma (Ganoderma) is a traditional health edible fungus in our country. Among the edible fungi, the ganoderma lucidum has strong tolerance to selenium, and is an ideal fungus for obtaining selenium-rich food. Selenium can exist in different forms after entering into the organism, wherein the selenium element existing in the form of the composition of the bio-organic molecules is called organic selenium. Among organic selenium, selenoprotein is an important and relatively clearly studied class of selenium-containing molecules. Selenium is present in amino acid residues as a substitute for sulfur in a large part of proteins, and selenomethionine and selenocysteine are more important. Selenomethionine is a relatively stable molecule, and selenocysteine is susceptible to further reactions, e.g., selenium may leave amino acids in ionic form, thereby losing biological activity. In protein molecules, selenium in selenocysteine needs two molecules to form selenocysteine, namely a form of diselenide bond in the protein molecule is formed to exist stably. Selenium in selenocysteine is a substitute of sulfur, so the diselenide bond also has the function of a disulfide bond. Disulfide bonds are the most important structures for protein formation and maintenance of higher order structures, and disulfide bond-containing proteins generally lose activity due to structural changes after disulfide bond cleavage. In contrast, a protein can also be substantially judged to have stable disulfide bonds if it can maintain its biological activity.
The invention discloses a method for preparing ganoderma lucidum polysaccharide, which is characterized in that mycelium fermentation is an important method for preparing ganoderma lucidum polysaccharide, and the earlier-stage research of the invention discovers that culture solution obtained by ganoderma lucidum mycelium fermentation contains a large amount of extracellular polysaccharide and a large amount of protein, and if the protein can be extracted, the method can be an effective way for preparing selenium-containing protein. In the ganoderma lucidum mycelium fermentation liquor, protein is mixed with a large amount of extracellular polysaccharide, and a large amount of ganoderma lucidum polysaccharide is often brought out during protein extraction, so that extracellular protein with high purity is difficult to obtain.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for extracting high-purity protein from selenium-enriched ganoderma lucidum fermentation liquor and greatly improving the content of the protein with natural activity, wherein the pretreated enzyme compound is added into a crude protein product water solution of the selenium-enriched ganoderma lucidum fermentation liquor, the protein in the treatment solution is extracted again through a specific treatment procedure, the protein content in the product obtained by secondary extraction is greatly increased, and the activity of the active protein represented by amylase and xylanase is kept above 80%.
The first purpose of the invention is to provide a method for extracting natural active protein of selenium-rich ganoderma lucidum, which comprises the following steps:
s1, centrifuging fermentation liquor obtained after fermentation of ganoderma lucidum mycelia to obtain supernatant, and performing salting-out and desalting treatment on the obtained supernatant to obtain a crude protein solution;
s2, adding the solution 105 into the crude protein solution, mixing, adding a composite dextranase D solution for enzymolysis, and salting out and desalting to obtain the selenium-rich ganoderma lucidum natural active protein;
wherein the solution 105 comprises 50 mM-100 mM disodium hydrogen phosphate-potassium dihydrogen phosphate, 5% -10% alcohol and 1% -2% PEG-200;
the composite dextranase D solution is prepared by the following method: mixing glucanase BglA, glucanase AnEglA6 and cellulase according to the enzyme activity ratio of 0.5-1.5;
wherein, the treatment liquid T312 is prepared by the following method: comprises 1-2% of PEG-200, 0.001-0.002% of polyethylene glycol octyl phenyl ether, 0.05-0.07 g/L of CoCl 3 And 0.01 to 0.02g/L of ZnCl 2 The pH value of the solution is adjusted to 7.5-8.5, after the solution is kept for 1.5-2.5 h, the pH value is adjusted to 9.0-9.5, and the solution is kept for 20-30 h at the temperature of-5 ℃.
In the treatment liquid T312 of the invention, the contents of PEG-200 and polyethylene glycol octyl phenyl ether are calculated according to the volume ratio.
Further, in the composite dextranase A solution, the enzyme activity of the dextranase BglA is 400U/mL-600U/mL.
Further, the cooling is carried out in an ice water bath.
Further, the salting out is performed by adding ammonium sulfate.
Furthermore, the desalting is carried out for 2 to 4 times by adopting an ultrafiltration membrane with the molecular weight cutoff of 2.5 to 3.5 KD.
Furthermore, in the step S2, the enzymolysis treatment is carried out for 0.5 to 2 hours at the temperature of between 38 and 42 ℃.
Further, in the step S2, after the enzymolysis treatment, the pH value is adjusted to 3.5-4.5, and the heat preservation treatment is carried out for 4-6 h at the temperature of 25-35 ℃.
Further, the fermentation of the ganoderma lucidum mycelia is carried out for 5-10 days at the temperature of 28-32 ℃ and at the speed of 160-200 r/min.
Further, in the step S1, the centrifugation is 10,000-14,000r/min for 5-10 min.
The second purpose of the invention is to provide the selenium-rich ganoderma lucidum natural active protein prepared by the method.
The invention has the beneficial effects that:
the invention adds the pretreated enzyme compound into the aqueous solution of the crude protein extract of the ganoderma lucidum mycelium selenium-rich fermentation liquor, extracts the protein in the treatment solution again through a specific treatment procedure, and greatly increases the protein content in the product obtained by the second extraction, wherein the activity of the active protein represented by amylase and xylanase is kept above 80 percent.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
The preparation method of the crude protein comprises the following steps:
preparing selenium-rich fermentation liquid of Ganoderma mycelia by the method of 1, centrifuging the fermentation liquid for 5min at a speed of 12,000r/min after fermentation is finished, and collecting supernatant. The supernatant of the fermentation broth was extracted by salting out according to method 2, and further desalted by method 3, whereby a crude protein solution was obtained. The protein content of the crude protein is generally about 40%.
The preparation method of the reproduced protein comprises the following steps:
the method for treating the crude protein solution, which is established by the invention, comprises the following steps: adding the solution 105 into the crude protein solution, mixing, adding the composite dextranase D solution, and keeping the temperature at 40 ℃ for 1h. After the reaction was completed, the pH was adjusted to 4.0 with hydrochloric acid. Keeping the temperature at 30 ℃ for 5h. The reaction solution is salted out by ammonium sulfate to obtain protein precipitate, and the precipitate is dissolved by water and desalted by an ultrafiltration tube. And desalting to obtain a reproduced protein solution, and calculating the content of the protein in the reproduced protein. By the method, the content of protein in the reproduced protein is generally about 75%, and the total enzyme activity of amylase and xylanase is maintained to be more than 80% of that of the crude enzyme solution.
Composition of the above solution 105: 50mM disodium hydrogenphosphate-monopotassium phosphate, 10% ethanol, 1% PEG-200.
The preparation method of the composite dextranase solution comprises the following steps: the dextranase BglA obtained by appropriate dilution and the AnEglA6 obtained by 1000U/mL were mixed in equal volumes, and the cellulase solid powder was added to a final concentration of 500U/mL. The solution is called composite dextranase A solution, called complex enzyme A for short. When the composite dextranase D solution is prepared, the composite dextranase A solution is quickly put into an ice-water mixture after the compounding of the composite dextranase A solution is finished, and the temperature is quickly reduced by lightly shaking. One tenth volume of solution T312 was added. The temperature is increased to 45 ℃ and maintained for 20min. Quickly put into ice water and adjusted to pH4.0 with hydrochloric acid. The enzyme solution after the treatment is called as a compound dextranase D solution, which is called as compound enzyme D for short. Wherein the T312 has the following components: 1% by volume of PEG200,0.001% by volume of polyoxyethylene octylphenyl ether, 0.05g/L of CoCl,0.01g/L of ZnCl 2 Adjusting pH to 8.0 with sodium hydroxide or hydrochloric acid, maintaining for 2 hr, adjusting pH to 9.0 with sodium hydroxide, and maintaining in ice water for 24 hr.
The cellulase is a product of Nanning Pompe bioengineering Co., ltd, and is a solid cellulase of 10 ten thousand U/g. Other reagents were purchased from the national pharmaceutical group chemical reagents, ltd. The ganoderma lucidum strain for selenium-rich fermentation is purchased from an industrial microbial resource platform of China colleges and universities of south Jiangnan university, and the strain number is as follows: CICIM F7085.
The related method comprises the following steps:
method 1 preparation method of Ganoderma mycelia selenium-rich fermentation broth:
inoculating selenium-rich Ganoderma fermentation broth to Ganoderma mycelia grown on solid PDA culture medium plate, and shake-culturing at 30 deg.C at a rotation speed of 180r/m. The culture was carried out for 7 days. The formula of the selenium-rich ganoderma lucidum fermentation liquid is the same as that of the liquid fermentation primary culture medium in the literature [ a high-selenium-resistant ganoderma lucidum mutant strain and application thereof, ZL202111524579, polemonglian, shenwei, simulan, chengxiaozui, yanhaiquan, quality in summer and Chenyilian ], but the glucose in the liquid fermentation primary culture medium is changed to 50g/L, and 0.5g/L of ammonium nitrate and 0.3g/L of sodium selenite are added.
Method 2 salting-out method for protein-containing solution:
and taking the protein-containing solution without the precipitate, continuously stirring the solution, and simultaneously gradually adding solid ammonium sulfate to ensure that the ammonium sulfate is continuously added and continuously dissolved, and the solid ammonium sulfate is not accumulated as the standard for adding the ammonium sulfate. Transferring the fermentation liquid added with the ammonium sulfate into a triangular flask, and slowly shaking the shaking table at the temperature of 4 ℃ and at the speed of 60 r/min. After 12h, centrifuging and collecting precipitate. The precipitate was dissolved in water.
Method 3 ultrafiltration desalination of salted-out protein:
the protein was concentrated using Millipore ultrafiltration concentration tubes with a molecular weight cut-off of 3 KD. Taking 15mL of supernatant, placing the supernatant in a 3kD ultrafiltration centrifuge tube, and centrifuging the tube at 12,000rpm for 10min. In this process, small molecule species with molecular weights less than 3kD are filtered into the collection tube. Enzyme molecules, etc. larger than 3kD are trapped in the upper ultrafiltration tube. After concentration, pure water is added to restore the volume, and ultrafiltration concentration is carried out again, so that crude protein solution which does not contain ammonium sulfate basically can be obtained after about 3 times.
Method 4 detection of total protein content:
the content of protein in the solution was determined using a Bradford solution protein concentration detection kit from pecu-yunna corporation, according to the kit instructions.
Method 5 detection of protein to dry matter ratio:
the solution to be tested is divided into two parts in equal amount. One of the aliquots was used for the determination of the total protein content, and the result was assumed to be m. And removing water by adopting a freeze-drying method to obtain dry matter, weighing, and assuming that the result is n. The method for protein to dry matter total amount can be written as: m1/n1 (100%).
Method 6 detection of amylase Activity:
the amylase detection method is the same as the literature [ Shen xi, lin Li Zhen, huang Wen, guo Yu Wan, chen donates faithfulness, fan you, wang Zheng Xiang, heterologous expression and recombinant enzyme property of acid fungus alpha-amylase, food and fermentation industry, 2013, 39 (8): 5-10 ], because the extracellular amylase produced by the ganoderma lucidum used in the invention is an acid enzyme, the detection is carried out in a citric acid buffer solution with the pH value of 4.0 according to the enzyme activity detection method under the condition of the pH value of 4.0 in the detection.
Method 7 xylanase detection method:
the xylanase detection method is the same as the literature [ Pyrolusitum, poplar silk, chengxing Ling, dongxiao, jinpeng, liuxiao, wangzhengxiang, aspergillus niger xynC gene encodes a low-temperature acidic xylanase [ J ]. The food and fermentation industry, 2017,43 (11): 44-50 ], the pH value is 4.5, and the detection temperature is 40 ℃.
Method 8 preparation of dextranase BglA:
the preparation method of the glucanase BglA is the same as the literature [ Chenyuanjuan, shenwei, chengxiaozi, wangzhengxiang, high-efficiency expression of bacillus amyloliquefaciens beta-1, 3-1, 4-glucanase, biotechnology, 2011, 21 (2): 22-26]. The recombinant bacterium Bacillus amyloliquefaciens (pUB-PQ-GM-bglA) reported in the literature is adopted for fermentation preparation, the fermentation method is consistent with the literature, and the enzyme activity of the fermentation liquid is about 1200U/mL. The recombinant bacterium Bacillus amyloliquefaciens (pUB-PQ-GM-bglA) is preserved in the biological engineering institute of south Jiangnan university by the author of the document, and the strain is preserved on an industrial microbial resource information platform in colleges and universities in China again when the recombinant bacterium Bacillus amyloliquefaciens is used, and the preservation number is as follows: CICIM B6930. When the strain is preserved, because the name of the strain is considered to be not in accordance with the naming rule of the current genetic engineering strain by workers of the industrial microorganism resource information platform of colleges and universities in China, the name is changed into: bacillus amyloliquefaciens 7658/pUB-PQ-GM-bglA.
Method 9 preparation method of dextranase AnEglA 6:
the preparation method of the glucanase AnEglA6 is the same as the literature [ Hanbing, wang Shilan, de Qing Meiduo, shen, chen fai, yan you, cloning, expression and recombinase property analysis of a high heat-resistant Aspergillus niger beta-glucanase gene, food and fermentation industry, 2018, 44 (11): 55-62 ]. Is obtained by fermentation of Pichia pastoris GS115/pPIC9K-eglA6b reported in the literature. The strain is preserved in an industrial microorganism resource information platform of colleges and universities in China when the strain is published in the paper, and the number of the strain is as follows: CICIM Y1489. The AnEglA6 enzyme solution is a crude enzyme solution obtained by shake flask fermentation of the strain, the fermentation method is the same as the above document, the fermentation level is about 2500U/mL, and the result is basically consistent with the report in the above document.
The invention is further illustrated by the examples:
example 1: comparison of crude enzyme solution reprocessing methods
The extraction method of the natural active protein of the selenium-rich ganoderma comprises the following steps:
(1) The preparation method of the crude protein comprises the following steps:
preparing selenium-rich fermentation broth of Ganoderma mycelia according to method 1 (taking Ganoderma mycelia growing on solid PDA culture medium plate, inoculating selenium-rich Ganoderma fermentation broth, shake-flask culturing at 30 deg.C with rotation speed of shake flask machine of 180r/m for 7 days), centrifuging the fermentation broth for 5min at 12,000r/min after fermentation is finished, and collecting supernatant.
The obtained supernatant of the fermentation broth was subjected to salting-out treatment by method 2: and continuously stirring the supernatant, and simultaneously gradually adding solid ammonium sulfate to ensure that the ammonium sulfate is continuously added and continuously dissolved, and the solid ammonium sulfate is not accumulated to be the standard for adding the ammonium sulfate. Transferring the supernatant added with ammonium sulfate into a triangular flask, and slowly shaking the shaking table at 4 ℃ for 60 r/min. And centrifuging after 12 hours, collecting precipitate, and dissolving the precipitate with water for later use.
The dissolved precipitate was further desalted by method 3: taking 15mL of the precipitated aqueous solution to a 3kD ultrafiltration centrifugal tube, centrifuging for 10min at 12,000rpm, adding pure water after concentration to restore the volume, and performing ultrafiltration concentration again, wherein about 3 times of ultrafiltration concentration can obtain a crude protein solution which does not contain ammonium sulfate basically, namely a crude enzyme solution.
The total amount of the prepared crude enzyme solution is 500mL, the enzyme activity of amylase is 36U/mL, the enzyme activity of xylanase is 15U/mL, and the protein content is 46%.
(2) The preparation method of the reproduced protein comprises the following steps:
the method for treating the crude protein solution, which is established by the invention, comprises the following steps: to the above crude enzyme solution, solution 105 (50 mM disodium hydrogenphosphate-monopotassium phosphate, 10% ethanol, 1% PEG-200) was added, mixed, and then, complex dextranase D was added, and the mixture was incubated at 40 ℃ for 1 hour. After the reaction is finished, hydrochloric acid is used for adjusting the pH value to 4.0, and the temperature is kept at 30 ℃ for 5 hours. And (3) dissolving the obtained protein precipitate in water by using the method 2, desalting by using an ultrafiltration tube by using the method 3 to obtain a reproduced protein solution, and calculating the content of the protein in the reproduced protein. By the method, the content of protein in the reproduced protein is about 75 percent generally, and the total enzyme activity of amylase and xylanase is maintained to be more than 80 percent of the crude enzyme solution.
Comparative example: direct processing method
The direct treatment was carried out by adding 50. Mu.l of each enzyme solution to 20mL of the crude enzyme solution prepared in step (1) in the example, and treating the mixture at 130 ℃ for 1 hour. Wherein the single enzyme is an enzyme solution which is diluted or dissolved and has an estimated enzyme activity of 500U/mL.
The results of the direct treatment and the treatment according to the invention using different enzymes are shown in tables 1 and 2, respectively.
The treatment method in Table 2 was carried out in the same manner as in example 1 of the present invention, except that the complex glucanase D was changed to a different enzyme, and the amount of the enzyme added was the same as that in the above-mentioned direct enzyme addition method.
TABLE 1 direct treatment of crude enzyme solutions with different enzymes
TABLE 2 method for treating crude enzyme solutions by the method of the invention with different enzymes
As can be seen from Table 1, the treatment of crude enzyme solution with the three enzymes according to the present invention has little effect on the total yield of protein in the reproduced protein. The total yield of the protein in various treatment methods is about 85-88%, which shows that the loss of the protein mainly comes from salting out in the process of reproducing the crude protein by a salting out method. Although the treatment of several enzymes has little influence on the yield of the reproduced protein, the treatment has larger influence on the purity of the obtained reproduced protein, wherein the compound glucanase A and the compound glucanase D can greatly improve the protein content of the reproduced protein, but the activities of two active enzymes after the treatment of the compound glucanase A are almost completely lost, and the activities of two enzymes after the treatment of the compound glucanase D are more reserved. The result is shown in table 2, and it can be seen from table 2 that the damage to the active protein is still serious even if the method of the present invention is adopted, most of amylase and xylanase lose activity, and only the specially treated compound enzyme, i.e. compound glucanase D, is adopted to treat the crude protein solution by the method of the present invention, the active components in the obtained re-extracted protein are greatly increased, more than 80% of enzyme activity is retained, and the purity of the extracted protein is also greatly increased.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A method for extracting natural active protein of selenium-enriched ganoderma lucidum is characterized by comprising the following steps:
s1, centrifuging fermentation liquor obtained after fermentation of ganoderma lucidum mycelia to obtain supernatant, and performing salting-out and desalting treatment on the obtained supernatant to obtain a crude protein solution;
s2, adding the solution 105 into the crude protein solution, mixing, adding a composite dextranase D solution for enzymolysis, and salting out and desalting to obtain the selenium-rich ganoderma lucidum natural active protein;
wherein the solution 105 comprises 50 mM-100 mM disodium hydrogen phosphate-potassium dihydrogen phosphate, 5% -10% alcohol and 1% -2% PEG-200;
the composite dextranase D solution is prepared by the following method: mixing glucanase BglA, glucanase AnEglA6 and cellulase according to the enzyme activity ratio of 0.5-1.5;
wherein, the treatment liquid T312 is prepared by the following method: the content of PEG-200 is 1-2%, 0.001-0.002% of polyethylene glycol octyl phenyl ether, 0.05-0.07 g/L C DEG C 3 And 0.01 to 0.02g/L of ZnCl 2 The pH value of the solution is adjusted to 7.5-8.5, after the solution is kept for 1.5-2.5 h, the pH value is adjusted to 9.0-9.5, and the solution is kept for 20-30 h at the temperature of-5 ℃.
2. The extraction method according to claim 1, wherein the enzyme activity of the dextranase BglA in the composite dextranase A solution is 400U/mL-600U/mL.
3. The extraction process according to claim 1, wherein the cooling is carried out in an ice-water bath.
4. The extraction method according to claim 1, wherein the salting out is performed by adding ammonium sulfate.
5. The extraction method of claim 1, wherein the desalting is performed by using an ultrafiltration membrane with a molecular weight cutoff of 2.5-3.5 KD for 2-4 times.
6. The extraction method according to claim 1, wherein in the step S2, the enzymolysis treatment is carried out at 38-42 ℃ for 0.5-2 h.
7. The extraction method according to claim 1, wherein in the step S2, after the enzymolysis treatment, the pH is adjusted to 3.5-4.5, and the temperature is kept at 25-35 ℃ for 4-6 h.
8. The extraction method according to claim 1, wherein the fermentation of the ganoderma lucidum mycelia is carried out at 28-32 ℃ and 160-200 r/min for 5-10 days.
9. The method of claim 1, wherein in the step S1, the centrifugation is 10,000 to 14,000r/min for 5 to 10min.
10. A selenium-rich ganoderma lucidum natural active protein prepared by the extraction method of any one of claims 1 to 9.
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