CN114058666A - Special peptone for producing inosine by fermentation and preparation method and application thereof - Google Patents
Special peptone for producing inosine by fermentation and preparation method and application thereof Download PDFInfo
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- CN114058666A CN114058666A CN202111565634.8A CN202111565634A CN114058666A CN 114058666 A CN114058666 A CN 114058666A CN 202111565634 A CN202111565634 A CN 202111565634A CN 114058666 A CN114058666 A CN 114058666A
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- 239000001888 Peptone Substances 0.000 title claims abstract description 78
- 108010080698 Peptones Proteins 0.000 title claims abstract description 78
- 235000019319 peptone Nutrition 0.000 title claims abstract description 78
- 229930010555 Inosine Natural products 0.000 title claims abstract description 49
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 title claims abstract description 49
- 229960003786 inosine Drugs 0.000 title claims abstract description 49
- 238000000855 fermentation Methods 0.000 title claims abstract description 34
- 230000004151 fermentation Effects 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 64
- 235000019764 Soybean Meal Nutrition 0.000 claims abstract description 32
- 239000004455 soybean meal Substances 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 30
- 235000014347 soups Nutrition 0.000 claims abstract description 26
- 239000000706 filtrate Substances 0.000 claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
- 241000251468 Actinopterygii Species 0.000 claims abstract description 13
- 239000000843 powder Substances 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims description 82
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- 102000004400 Aminopeptidases Human genes 0.000 claims description 27
- 108090000915 Aminopeptidases Proteins 0.000 claims description 27
- 108090000145 Bacillolysin Proteins 0.000 claims description 18
- 102000035092 Neutral proteases Human genes 0.000 claims description 18
- 108091005507 Neutral proteases Proteins 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 18
- 241000283690 Bos taurus Species 0.000 claims description 17
- 230000008569 process Effects 0.000 claims description 17
- 239000004365 Protease Substances 0.000 claims description 13
- 238000010411 cooking Methods 0.000 claims description 10
- 108090000317 Chymotrypsin Proteins 0.000 claims description 9
- 108090000526 Papain Proteins 0.000 claims description 9
- 108090000631 Trypsin Proteins 0.000 claims description 9
- 102000004142 Trypsin Human genes 0.000 claims description 9
- 229960002376 chymotrypsin Drugs 0.000 claims description 9
- 235000019834 papain Nutrition 0.000 claims description 9
- 229940055729 papain Drugs 0.000 claims description 9
- 239000012588 trypsin Substances 0.000 claims description 9
- 230000002255 enzymatic effect Effects 0.000 claims description 7
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 5
- 108010007119 flavourzyme Proteins 0.000 claims description 5
- 238000012262 fermentative production Methods 0.000 claims 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 238000009826 distribution Methods 0.000 abstract description 5
- 235000015278 beef Nutrition 0.000 abstract description 3
- 230000004060 metabolic process Effects 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 24
- 238000001816 cooling Methods 0.000 description 24
- 238000010438 heat treatment Methods 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 108090000765 processed proteins & peptides Proteins 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 13
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- 239000004744 fabric Substances 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 239000000463 material Substances 0.000 description 8
- 238000003825 pressing Methods 0.000 description 8
- 238000010025 steaming Methods 0.000 description 8
- 238000003860 storage Methods 0.000 description 8
- 239000003814 drug Substances 0.000 description 6
- 239000000796 flavoring agent Substances 0.000 description 6
- 235000019634 flavors Nutrition 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 241000186063 Arthrobacter Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 238000005086 pumping Methods 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000019264 food flavour enhancer Nutrition 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
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- 239000002994 raw material Substances 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical class [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 239000004097 EU approved flavor enhancer Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 description 1
- 206010043417 Therapeutic response unexpected Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 235000013902 inosinic acid Nutrition 0.000 description 1
- 239000004245 inosinic acid Substances 0.000 description 1
- 229940028843 inosinic acid Drugs 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
Abstract
The invention discloses a peptone special for producing inosine by fermentation, a preparation method and application thereof, belonging to the field of peptone preparation. The invention discloses a preparation method of peptone special for inosine by fermentation production, which comprises the following steps: (1) adding various enzymes into the fish bone soup to obtain fish bone peptone concentrated solution; (2) adding various enzymes into the beef bone soup to obtain fish bone peptone concentrated solution; (3) adding various enzymes into the soybean meal filtrate to obtain soybean meal peptone concentrated solution; (4) and mixing the three concentrated solutions in proportion, and drying into powder to obtain the inosine peptone specially used for producing inosine by fermentation. The peptone prepared by the method has reasonable molecular weight distribution, effectively controls the fermentation metabolism speed of the strain while promoting mass propagation and growth of the strain, prolongs the inosine synthesis time, improves the inosine yield and lays an important foundation for large-scale fermentation production of inosine.
Description
Technical Field
The invention relates to the field of peptone preparation, and in particular relates to a peptone special for inosine fermentation production, and a preparation method and application thereof.
Background
Inosine (also called inosine) is also called inosine and has a molecular formula of C10H12N4O5The relative molecular mass was 268.23. Inosine has very wide application value in the fields of food, medicine and the like, and in the field of food, inosine is a delicious flavoring substance in food, and the content of the inosine is often used as one of characteristic indexes for measuring the freshness of the food; it is in various tonesThe flavor food such as nutrient flavor enhancer of monosodium glutamate, soy sauce, etc., and is one of the main raw materials of inosinic acid and flavor nucleotide disodium which are effective flavor enhancers in the food industry; in the field of medicine, inosine is a cell reactivator and has functions of stimulating cells, enhancing cell metabolism, and the like. Generally, the traditional Chinese medicine composition is used for preventing and treating cell damage of organs such as heart, liver and the like in medicine, is beneficial to restoring liver function to normal, can prevent and relieve side effects on heart or liver caused by blood defense drugs, and is a good-effect drug for treating coronary heart disease, hepatitis, leukopenia and the like.
At present, the production methods of inosine can be basically classified into three major groups: chemical decomposition, enzymatic hydrolysis and microbial fermentation. The inosine produced by the chemical synthesis method has high requirements on equipment conditions, complex process and limited production input; the large-scale production of inosine is influenced by high production cost of the enzymolysis method; the microbial fermentation method is the main method for producing inosine at present.
Peptone is the main raw material of microbial culture medium, is a complex mixture and is prepared from various amino acids, polypeptides, peptone,
Different peptone exists because different organisms need specific amino acid and polypeptide, and different peptone has different effects on the inosine fermentation level, so far, no special peptone for producing inosine by fermentation is available.
Disclosure of Invention
The invention aims to provide a peptone special for inosine fermentation production and a preparation method and application thereof, which aim to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a preparation method of peptone special for inosine by fermentation, which comprises the following steps:
(1) taking fishbone, adding water for cooking, adding chymotrypsin, aminopeptidase and neutral protease into the bone soup with the oil layer removed for enzymolysis to obtain fishbone enzymatic hydrolysate, and concentrating to obtain fishbone peptone concentrated solution;
(2) taking bovine bone, adding water, cooking, adding flavourzyme, aminopeptidase and papain into the bone soup without the oil layer for enzymolysis to obtain bovine bone enzymatic hydrolysate, and concentrating to obtain bovine bone peptone concentrated solution;
(3) taking soybean meal, adding water for cooking, filtering, adding trypsin, aminopeptidase and neutral protease into filtrate for enzymolysis, obtaining soybean meal enzymatic hydrolysate after the enzymolysis is finished, and concentrating to obtain soybean meal peptone concentrated solution;
(4) and mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution according to the mass ratio of 4-5:7:2-3, and drying into powder to obtain the inosine-specific peptone for fermentation production.
Further, in the step (1), the adding amount of the chymotrypsin, the aminopeptidase and the neutral protease is 1 per mill, 2 per mill and 1 per mill of the weight of the fishbone respectively.
Further, in the step (1), the enzymolysis condition is pH 7-7.5, the temperature is 45-50 ℃, and the time is 140 min.
Further, in the step (2), the adding amount of the flavourzyme, the aminopeptidase and the papain is 1 per mill, 2 per mill and 1 per mill of the weight of the beef bones respectively.
Further, in the step (2), the enzymolysis condition is pH 6.5-7, the temperature is 45-50 ℃, and the time is 140 min.
Further, in the step (3), the adding amount of the trypsin, the aminopeptidase and the neutral protease is 1 per mill, 2 per mill and 1 per mill of the weight of the soybean meal respectively.
Further, in the step (3), the enzymolysis condition is pH 7-7.5, the temperature is 50-55 ℃, and the time is 140 min.
Further, the enzymolysis process in the step (1), the step (2) and the step (3) adopts intermittent stirring, specifically, stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min.
The invention also provides the peptone special for producing inosine by fermentation, which is prepared by the preparation method.
The invention also provides application of the peptone special for producing inosine by fermentation in producing inosine by fermentation.
The invention discloses the following technical effects:
the invention provides a preparation method of inosine special peptone by fermentation, which adopts a complex enzyme technology to respectively carry out enzymolysis on different materials, simultaneously increases the enzymolysis reaction rate and avoids influence of excessive stirring on enzyme stability in an intermittent stirring mode, and finally obtains the inosine special peptone by fermentation. The peptone has reasonable molecular weight distribution, wherein the proportion of the small molecular weight polypeptide is up to 60 percent, and the small molecular weight polypeptide has moderate proportion, can effectively promote the early growth and the propagation of arthrobacter, ensure the biomass accumulation of the arthrobacter, and lay a foundation for the mass synthesis of inosine; meanwhile, the peptone also contains large molecular weight polypeptide in a certain proportion, so that the later metabolic fermentation speed of the strain can be effectively controlled, the inosine synthesis time is prolonged, and the inosine yield is improved.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
A preparation method for producing inosine special peptone by fermentation comprises the following steps:
(1) taking fresh fishbone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh fishbone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a material storage tank (self pressure in the tank), passing through an oil-water separator, putting the bone soup with an oil layer removed into an enzymolysis tank, adjusting the pH value to 7, adjusting the temperature to 50 ℃, adding 1 thousandth of chymotrypsin, 2 thousandth of aminopeptidase and 1 thousandth of neutral protease into the bone soup, slowly stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.1 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And (4) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the fish bone peptone concentrated solution.
(2) Taking fresh ox bone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh ox bone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a storage tank (self pressure in the tank), separating with an oil-water separator, placing the bone soup with oil layer removed to an enzymolysis tank, adjusting pH to 6.8, and heating to 50 deg.C. Adding 1 ‰ flavor protease, 2 ‰ aminopeptidase and 1 ‰ papain, slowly stirring, stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.1 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And after the filtration (400-mesh filter cloth) is finished, concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the bovine bone peptone concentrated solution.
3. Taking low-temperature soybean meal, adding water, cooking (the mass ratio of the low-temperature soybean meal to the water is 1:8), heating to 95 ℃, and preserving heat for 1 hour. Filtering with plate-frame filter to obtain clear liquid, and pumping to enzymolysis tank. The pH of the filtrate was adjusted to 7 and the temperature 50 ℃. Then adding 1 per mill of trypsin, 2 per mill of aminopeptidase and 1 per mill of neutral protease into the soybean meal, slowly stirring, stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min. After enzymolysis is finished, adjusting the pH value to 8 by using sodium hydroxide, heating to 90 ℃, then slowly cooling to about 50 ℃ for 2-3 hours, and stirring in the cooling process can not be started. And (3) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the soybean meal peptone concentrated solution.
(4) Mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution in a mass ratio of 4:7:2, and drying the mixture into powder by a centrifugal spray dryer to obtain the peptone.
Example 2
(1) Taking fresh fishbone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh fishbone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a material storage tank (self pressure in the tank), passing through an oil-water separator, placing the bone soup with oil layer removed into an enzymolysis tank, adjusting pH value to 7.2, adjusting temperature to 50 ℃, adding 1 thousandth of chymotrypsin, 2 thousandth of aminopeptidase and 1 thousandth of neutral protease into the bone soup, slowly stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.2 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And (4) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the fish bone peptone concentrated solution.
(2) Taking fresh ox bone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh ox bone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a storage tank (self pressure in the tank), separating with an oil-water separator, placing the bone soup with oil layer removed to an enzymolysis tank, adjusting pH to 6.7, and heating to 50 deg.C. Adding 1 ‰ flavor protease, 2 ‰ aminopeptidase and 1 ‰ papain, slowly stirring, stirring for 10min and 5min, stirring for 10min and 10min, stirring for 15min and 15min, and stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.2 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And after the filtration (400-mesh filter cloth) is finished, concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the bovine bone peptone concentrated solution.
3. Taking low-temperature soybean meal, adding water, cooking (the mass ratio of the low-temperature soybean meal to the water is 1:8), heating to 95 ℃, and preserving heat for 1 hour. Filtering with plate-frame filter to obtain clear liquid, and pumping to enzymolysis tank. The pH of the filtrate was adjusted to 7.1 and the temperature 50 ℃. Then adding 1 per mill of trypsin, 2 per mill of aminopeptidase and 1 per mill of neutral protease into the soybean meal, slowly stirring, stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min. After enzymolysis is finished, adjusting the pH value to 8 by using sodium hydroxide, heating to 90 ℃, then slowly cooling to about 50 ℃ for 2-3 hours, and stirring in the cooling process can not be started. And (3) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the soybean meal peptone concentrated solution.
(4) Mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution in a mass ratio of 4:7:3, and drying the mixture into powder by a centrifugal spray dryer to obtain the peptone.
Example 3
(1) Taking fresh fishbone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh fishbone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing the bone soup into a material storage tank (self pressure in the tank), passing through an oil-water separator, putting the bone soup with an oil layer removed into an enzymolysis tank, adjusting the pH value to 7.4, adjusting the temperature to 45 ℃, adding 1 thousandth of chymotrypsin, 2 thousandth of aminopeptidase and 1 thousandth of neutral protease into the bone soup, slowly stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.3 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And (4) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the fish bone peptone concentrated solution.
(2) Taking fresh ox bone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh ox bone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a storage tank (self pressure in the tank), separating with an oil-water separator, placing the bone soup with oil layer removed to an enzymolysis tank, adjusting pH to 7, and keeping the temperature at 47 deg.C. Adding 1 ‰ flavor protease, 2 ‰ aminopeptidase and 1 ‰ papain, slowly stirring, stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.3 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And after the filtration (400-mesh filter cloth) is finished, concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the bovine bone peptone concentrated solution.
3. Taking low-temperature soybean meal, adding water, cooking (the mass ratio of the low-temperature soybean meal to the water is 1:8), heating to 95 ℃, and preserving heat for 1 hour. Filtering with plate-frame filter to obtain clear liquid, and pumping to enzymolysis tank. The pH of the filtrate was adjusted to 7.3 and the temperature was 55 ℃. Then adding 1 per mill of trypsin, 2 per mill of aminopeptidase and 1 per mill of neutral protease into the soybean meal, slowly stirring, stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min. After enzymolysis is finished, adjusting the pH value to 8 by using sodium hydroxide, heating to 90 ℃, then slowly cooling to about 50 ℃ for 2-3 hours, and stirring in the cooling process can not be started. And (3) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the soybean meal peptone concentrated solution.
(4) Mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution in a mass ratio of 5:7:2, and drying the mixture into powder by a centrifugal spray dryer to obtain the peptone.
Example 4
(1) Taking fresh fishbone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh fishbone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing the bone soup into a material storage tank (self pressure in the tank), passing through an oil-water separator, putting the bone soup with an oil layer removed into an enzymolysis tank, adjusting the pH value to 7.5, adjusting the temperature to 43 ℃, adding 1 thousandth of chymotrypsin, 2 thousandth of aminopeptidase and 1 thousandth of neutral protease into the bone soup, slowly stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.3 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And (4) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the fish bone peptone concentrated solution.
(2) Taking fresh ox bone, adding water, steaming at high temperature and high pressure (the mass ratio of the fresh ox bone to the water is 1:6), and preserving heat for 1 hour under 3 standard atmospheric pressures. Pressing bone soup to a storage tank (self pressure in the tank), separating with an oil-water separator, placing the bone soup with oil layer removed to an enzymolysis tank, adjusting pH to 6.5, and keeping the temperature at 45 deg.C. Adding 1 ‰ flavor protease, 2 ‰ aminopeptidase and 1 ‰ papain, slowly stirring, stirring for 10min and 5min, stirring for 15min and 10min, stirring for 15min and 15 min. And after the enzymolysis is finished, adding 500mL of acetic acid, adjusting the pH value to 5.3 by using hydrochloric acid, heating to 90 ℃, slowly cooling to about 50 ℃ for 2-3 hours, and not stirring in the cooling process. And after the filtration (400-mesh filter cloth) is finished, concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the bovine bone peptone concentrated solution.
3. Taking low-temperature soybean meal, adding water, cooking (the mass ratio of the low-temperature soybean meal to the water is 1:8), heating to 95 ℃, and preserving heat for 1 hour. Filtering with plate-frame filter to obtain clear liquid, and pumping to enzymolysis tank. The pH of the filtrate was adjusted to 7 at a temperature of 52 ℃. Then adding 1 per mill of trypsin, 2 per mill of aminopeptidase and 1 per mill of neutral protease into the soybean meal, slowly stirring, stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min. After enzymolysis is finished, adjusting the pH value to 8 by using sodium hydroxide, heating to 90 ℃, then slowly cooling to about 50 ℃ for 2-3 hours, and stirring in the cooling process can not be started. And (3) filtering (400-mesh filter cloth) after the end, and concentrating the obtained filtrate in vacuum until the solid content is 50% to obtain the soybean meal peptone concentrated solution.
(4) Mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution in a mass ratio of 5:7:3, and drying the mixture into powder by a centrifugal spray dryer to obtain the peptone.
Comparative example 1
The difference from the example 1 is that 1 per thousand chymotrypsin, 2 per thousand aminopeptidase and 1 per thousand neutral protease are used in the steps (1), (2) and (3).
Comparative example 2
The difference from the example 1 is that 1 per mill of flavourzyme, 2 per mill of aminopeptidase and 1 per mill of papain are used in the steps (1), (2) and (3).
Comparative example 3
The difference from example 1 is that 1 ‰ trypsin, 2 ‰ aminopeptidase, and 1 ‰ neutral protease are used in steps (1), (2), and (3).
Comparative example 4
The difference from the embodiment 1 is that the stirring mode in the enzymolysis process of the step (1), the step (2) and the step (3) is as follows: stirring for 10min and stopping for 1min, stirring for 15min and stopping for 3min, stirring for 20min and stopping for 5min, stirring for 20min and stopping for 10min, and stirring for 30min and stopping for 10 min.
The molecular weight distributions of the peptone powder finished products of examples 1-4 and comparative examples 1-4 were measured using the SDS-PAGE method, and the results are shown in Table 1:
TABLE 1 molecular weight distribution results (%)
Effect example 1
1. Arthrobacter ACCC12070, provided by university of Shandong science and technology engineering laboratories.
2. Culture medium
Slant and seed activation medium: a beef extract culture medium;
fermentation medium: 2% glucose, 1% peptone of examples 1-4 and comparative examples 1-4, 0.2% disodium hydrogen phosphate, 0.1% magnesium sulfate, 0.01% manganese sulfate, pH 7.0.
3. The test process comprises the following steps:
(1) activating strains: inoculating the preserved strain on a slant culture medium, and culturing at 37 deg.C for 18 h.
(2) Seed culture: taking a loopful of strain from the slant of 18h of activation, transferring the loopful of strain into a 250mL triangular flask containing 25mL of seed culture medium, and culturing at 220r/min and 34 ℃ for 18 h.
(3) Seed fermentation culture: fresh seed liquid cultured for 18h is taken and inoculated into a 250mL triangular flask filled with 25mL fermentation medium by the inoculation amount of 5 percent, and the seed liquid is cultured for 72h at 220r/min and 34 ℃.
(4) Measurement of inosine production
The measurement was carried out by High Performance Liquid Chromatography (HPLC).
Chromatographic conditions are as follows:
chromatograph: agilent 1200 model hplc
A chromatographic column: SymmetryC18Reversed phase column (4.6X 150mm, 3.5 μm)
Mobile phase: a: ultrapure water (containing 0.5% phosphoric acid); b: methanol
Detection wavelength: 248 nm; flow rate: 0.6 mL/min; sample introduction amount: 20 mu L of the solution; column temperature: at 30 ℃.
(5) Results of the experiment
Fermentation media were prepared by using peptones prepared in examples 1 to 4 and comparative examples 1 to 4, respectively, and fermentation culture experiments were performed in sequence, and after the end of fermentation, the yield of inosine in each group was measured, and the results are shown in table 2:
TABLE 2 inosine production by each fermentation broth group
| Group of | Inosine production (g/L) |
| Example 1 | 32 |
| Example 2 | 30 |
| Example 3 | 33 |
| Example 4 | 32 |
| Comparative example 1 | 26 |
| Comparative example 2 | 25 |
| Comparative example 3 | 23 |
| Comparative example 4 | 24 |
By combining the experimental results shown in the table 1-2, it can be known that the peptone obtained by the preparation method of the present invention has a high ratio of small molecular weight polypeptides, and the ratio of 0-5kDa polypeptides in examples 1-4 is about 62%, and the low molecular weight polypeptides are easy to absorb, and can promote the early-stage mass proliferation of arthrobacter, and ensure the sufficient biomass thereof, and meanwhile, the ratio of 5-50kDa polypeptides with large molecular weight in examples 1-4 is about 30%, and ensure a certain ratio of large molecular weight polypeptides, and in the late stage of the mass propagation of arthrobacter, the metabolic fermentation speed of the strain can be effectively controlled, the inosine synthesis time can be prolonged, and the inosine yield can be increased; compared with the comparative examples 1-3, the proportion of the small molecular weight polypeptide is about 70 percent, the proportion is too high, the proportion of the large molecular weight polypeptide is reduced, the growth and propagation speed of the strain is excessively improved, and the synthesis of inosine is not facilitated; comparative example 4 the proportion of the small molecular weight polypeptide is only 40%, the proportion of the large molecular weight polypeptide reaches 55%, the proportion is too much, the stirring time is increased in the peptone preparation process, the down time is shortened, the stirring strength is enhanced, the stirring shearing force is increased, the enzymatic activity is unstable, the enzymolysis effect is reduced, the proportion of the finally obtained small molecular weight polypeptide of the peptone is too little, the early-stage propagation and growth of strains are not facilitated, and the synthesis of inosine is influenced. Therefore, the molecular weight distribution of the peptone prepared by the method is reasonable, the fermentation and metabolism speed of the strain is effectively controlled while the mass propagation and growth of the strain are promoted, and the yield of inosine is improved.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A preparation method of peptone special for inosine fermentation production is characterized by comprising the following steps:
(1) taking fishbone, adding water for cooking, adding chymotrypsin, aminopeptidase and neutral protease into the bone soup with the oil layer removed for enzymolysis to obtain fishbone enzymatic hydrolysate, and concentrating to obtain fishbone peptone concentrated solution;
(2) taking bovine bone, adding water, cooking, adding flavourzyme, aminopeptidase and papain into the bone soup without the oil layer for enzymolysis to obtain bovine bone enzymatic hydrolysate, and concentrating to obtain bovine bone peptone concentrated solution;
(3) taking soybean meal, adding water for cooking, filtering, adding trypsin, aminopeptidase and neutral protease into filtrate, performing enzymolysis to obtain soybean meal enzymatic hydrolysate, and concentrating to obtain soybean meal peptone concentrated solution;
(4) and mixing the fish bone peptone concentrated solution, the cattle bone peptone concentrated solution and the soybean meal peptone concentrated solution according to the mass ratio of 4-5:7:2-3, and drying into powder to obtain the inosine-specific peptone for fermentation production.
2. The preparation method according to claim 1, wherein in the step (1), the chymotrypsin, aminopeptidase and neutral protease are added in an amount of 1%, 2% and 1% by weight of the fishbone, respectively.
3. The method according to claim 1, wherein in the step (1), the enzymolysis condition is pH 7-7.5, the temperature is 45-50 ℃, and the time is 140 min.
4. The method according to claim 1, wherein in the step (2), the flavourzyme, the aminopeptidase and the papain are added in an amount of 1%, 2% and 1% by weight, respectively, to the bovine bone.
5. The method according to claim 1, wherein in the step (2), the enzymolysis condition is pH 6.5-7, the temperature is 45-50 ℃, and the time is 140 min.
6. The preparation method according to claim 1, wherein in the step (3), the trypsin, the aminopeptidase and the neutral protease are added in an amount of 1%, 2% and 1% by weight of the soybean meal, respectively.
7. The method according to claim 1, wherein in the step (3), the enzymolysis condition is pH 7-7.5, temperature is 50-55 ℃, and time is 140 min.
8. The preparation method according to claim 1, wherein the enzymolysis process in step (1), step (2) and step (3) adopts intermittent stirring, specifically stirring for 10min and stopping for 5min, stirring for 15min and stopping for 10min, stirring for 15min and stopping for 15min, and stirring for 15min and stopping for 15 min.
9. The inosine-specific peptone produced by fermentation by the production method according to any one of claims 1 to 8.
10. Use of the fermentative production of inosine-specific peptone according to claim 9 for fermentative production of inosine.
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