CN114958803B - Phytase fermentation production method - Google Patents
Phytase fermentation production method Download PDFInfo
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- CN114958803B CN114958803B CN202210537106.XA CN202210537106A CN114958803B CN 114958803 B CN114958803 B CN 114958803B CN 202210537106 A CN202210537106 A CN 202210537106A CN 114958803 B CN114958803 B CN 114958803B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 74
- 230000004151 fermentation Effects 0.000 title claims abstract description 74
- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 61
- 229940085127 phytase Drugs 0.000 title claims abstract description 60
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 34
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 claims abstract description 44
- 229910000278 bentonite Inorganic materials 0.000 claims abstract description 41
- 239000000440 bentonite Substances 0.000 claims abstract description 41
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 229940092782 bentonite Drugs 0.000 claims description 37
- 239000002609 medium Substances 0.000 claims description 17
- 238000009630 liquid culture Methods 0.000 claims description 16
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 12
- ONCZQWJXONKSMM-UHFFFAOYSA-N dialuminum;disodium;oxygen(2-);silicon(4+);hydrate Chemical compound O.[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[Na+].[Na+].[Al+3].[Al+3].[Si+4].[Si+4].[Si+4].[Si+4] ONCZQWJXONKSMM-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 229910000280 sodium bentonite Inorganic materials 0.000 claims description 7
- 229940080314 sodium bentonite Drugs 0.000 claims description 7
- 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 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000001508 potassium citrate Substances 0.000 claims description 6
- 229960002635 potassium citrate Drugs 0.000 claims description 6
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims description 6
- 235000011082 potassium citrates Nutrition 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 6
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 6
- 235000011151 potassium sulphates Nutrition 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 4
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 229910000281 calcium bentonite Inorganic materials 0.000 claims description 3
- 229910052744 lithium Inorganic materials 0.000 claims description 3
- 241000228245 Aspergillus niger Species 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 15
- 244000005700 microbiome Species 0.000 abstract description 13
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 15
- 235000002949 phytic acid Nutrition 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 6
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 229940068041 phytic acid Drugs 0.000 description 6
- 239000000467 phytic acid Substances 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000005995 Aluminium silicate Substances 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- FENRSEGZMITUEF-ATTCVCFYSA-E [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] FENRSEGZMITUEF-ATTCVCFYSA-E 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 235000012211 aluminium silicate Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical group O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229940083982 sodium phytate Drugs 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
- INAPMGSXUVUWAF-GCVPSNMTSA-N [(2r,3s,5r,6r)-2,3,4,5,6-pentahydroxycyclohexyl] dihydrogen phosphate Chemical class OC1[C@H](O)[C@@H](O)C(OP(O)(O)=O)[C@H](O)[C@@H]1O INAPMGSXUVUWAF-GCVPSNMTSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention relates to the field of microbial fermentation, in particular to a phytase fermentation production method, wherein bentonite is added in the phytase fermentation production process. The invention aims to overcome the defect of low efficiency of preparing phytase by microbial fermentation in the prior art, and bentonite is added in the fermentation production process of phytase, so that microorganisms can be adsorbed on the surface of lamellar bentonite for growth, the activity of the microorganisms is improved, and meanwhile, the added bentonite can improve the culture environment of the microorganisms, thereby being beneficial to improving the concentration of products in a culture medium, improving the production capacity of a fermentation tank per unit volume and further improving the production efficiency of phytase.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a phytase fermentation production method.
Background
Phytase (Phytase, phy), also known as phytate hydrolase (myo-inositol hexakishydrolase phosphate), is an acidic orthophosphoric monoester phosphate hydrolase that catalyzes the hydrolysis of phytic acid to produce lower inositol phosphate derivatives and inorganic phosphate. Suzuki was equal to 1907, where phytase was first found in plants, after which scientific researchers in various countries conducted extensive and intensive research into phytase of various origins.
Because of their special catalytic properties, phytase has become a popular food and feed additive in recent years, especially as a monogastric animal feed additive, and has gained widespread acceptance and use worldwide.
Research and exploration of phytase by researchers is mainly focused on the field of microbial phytase. Commercial phytase products have been developed in various countries around the world, for example, a plurality of phytase multienzyme system products (multienzyme SP, SF, TP, etc.) are developed by the company AIKO Bio technology in finland, and have various enzymolysis effects, so that the absorption and utilization of phosphorus, cellulose and protein of livestock and poultry are promoted; alltech, U.S. developed "Allzyme Phy tase" phytase with excellent thermostability and pH stability; a novel commercial phytase product is developed by Shanghai Yongxing agricultural bioengineering company in China, and has good heat stability and low pH resistance.
Phytase has wide application, but has a few problems which need to be solved in the aspect of industrial application. On the one hand, the yield of the natural phytase is too low, the supply requirement of industrial large-scale application is difficult to meet, and the extraction and production costs of the natural phytase are high, so that the natural phytase does not have a widely applied price basis. Secondly, although the phytase has wide sources, the yield of the phytase of plant and animal sources is low and insufficient to meet the requirements of industrial production application, and the application range of the phytase of plant and animal sources is not wide enough to have the phytase of microorganism sources, and the main source of the phytase is the microorganism at present. Therefore, how to improve the efficiency of preparing phytase by microbial fermentation is really needed to be focused by researchers.
Disclosure of Invention
The invention provides a phytase fermentation production method for overcoming the defect of low efficiency of preparing phytase by microbial fermentation in the prior art.
In order to achieve the aim of the invention, the invention is realized by the following technical scheme:
The first object of the present invention is to provide the use of bentonite in the production of phytase.
The first object of the present invention is to provide a phytase fermentation production method, comprising the steps of:
Bentonite is added during the fermentation process.
The inventor of the present application has unexpectedly found in daily experiments that the addition of bentonite in the process of producing phytase by fermentation has a promoting effect on the yield of phytase.
The inventors have conducted a certain study on this. Firstly, bentonite is a mineral clay with lamellar microstructure, the thickness of single bentonite is about 1 nanometer, and meanwhile, the specific surface area is large, so that the bentonite has excellent adsorption performance. When the bentonite is added to the process of producing the phytase, microorganisms can be adsorbed on the surface of the lamellar bentonite for growth, so that the stability of the growth environment of the microorganisms can be improved, the activity of the microorganisms is effectively improved, the production efficiency of the phytase is greatly improved, the concentration of a product in a culture medium is effectively improved, and the production capacity of a unit volume fermentation tank is improved.
Preferably, the bentonite comprises: at least one or a combination of a plurality of sodium bentonite, calcium bentonite, lithium bentonite and acid modified bentonite.
Preferably, the bentonite is added in an amount of 0.5g/L to 5g/L.
The present inventors found that the addition amount of bentonite has a significant effect on the production of phytase. The inventor finds that when the addition amount of bentonite is less than 0.5g/L, the stability improving effect of the bentonite on the growth environment of microorganisms is limited, so that the production efficiency of phytase is not obviously improved. However, as the adsorption performance of the bentonite is good, if the dosage is too large, the active ingredients are adsorbed on the bentonite, the activity is reduced, the fermentation is affected, and the inventor reagent tests prove that the production efficiency of the phytase is improved most obviously when the addition amount of the bentonite is between 0.5g/L and 5 g/L.
Preferably, the method further comprises the following steps:
(S.1) primary liquid culture;
(S.2) fermenting in a fermentation tank;
(S.3) separation and purification of phytic acid.
Preferably, the primary liquid culture process in the step (s.1) is as follows:
inoculating single colony into seed culture medium, and shake culturing at 32-40 deg.C for 30-45 hr.
Preferably, the strain used is a aspergillus niger.
Preferably, the fermenter fermentation process in the step (S.2) is as follows: inoculating 6-18% of the inoculating amount into fermentation medium, culturing at 32-40deg.C for 60-84 hr, and performing submerged fermentation culture.
Preferably, bentonite is added to the fermenter along with the fermentation medium.
Preferably, the fermentation medium comprises the following components: potassium citrate, 1-4 g/L; 1-2 g/L of calcium sulfate; 35-50 g/L of monopotassium phosphate; 3-7 g/L of ammonium sulfate; 12-18 g/L of potassium sulfate; 9-15 g/L of magnesium sulfate; glucose 4-8 g/L.
Therefore, the invention has the following beneficial effects:
(1) According to the invention, bentonite is added in the phytase fermentation production process, so that microorganisms can be adsorbed on the surface of the lamellar bentonite for growth, the activity of the microorganisms is improved, and the production efficiency of phytase is improved.
(2) According to the phytase fermentation production process provided by the invention, the added bentonite improves the culture environment of microorganisms, and is beneficial to increasing the concentration of products in a culture medium so as to increase the production capacity of a fermentation tank per unit volume.
Detailed Description
The invention is further described below in connection with specific embodiments. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
Example 1
The phytase fermentation production process comprises the following steps: primary liquid culture; fermenting in a fermentation tank; separating and purifying phytic acid;
and a primary liquid culture step, namely inoculating single colony into a seed culture medium, and performing shake culture at 35 ℃ for 36 hours to perform primary liquid culture. The strain is black koji enzyme;
a fermentation section of the fermentation tank, which is inoculated to a fermentation culture medium with an inoculum size of 8 percent, and is cultivated for 72 hours at 35 ℃ for liquid submerged fermentation cultivation;
the fermentation medium comprises the following components: potassium citrate, 2.0 g/L; calcium sulfate, 1.2 g/L; 38.0 g/L of monopotassium phosphate; ammonium sulfate 4.0 g/L; 15.0 g/L of potassium sulfate; 11.0 g/L of magnesium sulfate; glucose 5.0 g/L;
2.0 g/L sodium bentonite is added into the fermentation medium.
Example 2
The phytase fermentation production process comprises the following steps: primary liquid culture; fermenting in a fermentation tank; separating and purifying phytic acid;
and a primary liquid culture step, namely inoculating single colony into a seed culture medium, and performing shake culture at 35 ℃ for 36 hours to perform primary liquid culture. The strain is black koji enzyme;
a fermentation section of the fermentation tank, which is inoculated to a fermentation culture medium with 10 percent of inoculum size, and is cultivated for 84 hours at 35 ℃ for liquid submerged fermentation cultivation;
The fermentation medium comprises the following components: potassium citrate, 2.5 g/L; calcium sulfate, 1.0 g/L; 40.0 g/L of monopotassium phosphate; 3.5 g/L of ammonium sulfate; 13.0 g/L of potassium sulfate; 12.0 g/L of magnesium sulfate; glucose 6.0 g/L;
3.0 g/L of calcium bentonite is added into the fermentation medium.
Example 3
The phytase fermentation production process comprises the following steps: primary liquid culture; fermenting in a fermentation tank; separating and purifying phytic acid;
and a primary liquid culture step, namely inoculating single colony into a seed culture medium, and performing shake culture at 33 ℃ for 36 hours to perform primary liquid culture. The strain is black koji enzyme;
A fermentation section of the fermentation tank, which is inoculated in a fermentation culture medium with an inoculum size of 14 percent, and is cultivated for 65 hours at the temperature of 33 ℃ for liquid submerged fermentation cultivation;
the fermentation medium comprises the following components: potassium citrate, 3.5 g/L; calcium sulfate, 1.5 g/L; 45.0 g/L of monopotassium phosphate; ammonium sulfate 5.5 g/L; 16.0 g/L of potassium sulfate; 14.5 g/L of magnesium sulfate; glucose 6.0 g/L;
0.5 g/L lithium bentonite is added into the fermentation medium.
Example 4
The phytase fermentation production process comprises the following steps: primary liquid culture; fermenting in a fermentation tank; separating and purifying phytic acid;
and a primary liquid culture step, namely inoculating single colony into a seed culture medium, and performing shake culture at 38 ℃ for 45 hours to perform primary liquid culture. The strain is black koji enzyme;
A fermentation section of the fermentation tank, which is inoculated to a fermentation culture medium with an inoculum size of 18 percent, and is cultivated for 84 hours at 38 ℃ for liquid submerged fermentation cultivation;
The fermentation medium comprises the following components: potassium citrate, 1.5 g/L; calcium sulfate, 1.0 g/L; 35.0 g/L of monopotassium phosphate; 6.5 g/L of ammonium sulfate; 17.0 g/L of potassium sulfate; 10.5 g/L of magnesium sulfate; glucose 8.0 g/L;
5.0 g/L acid modified bentonite is added into the fermentation medium.
Comparative example 1:
The composition is the same as in example 1, except that: bentonite was not used.
Comparative example 2
The composition is the same as in example 1, except that: 0.2g/L sodium bentonite is added into the fermentation medium.
Comparative example 3
The composition is the same as in example 1, except that: 6g/L sodium bentonite is added into the fermentation medium.
Comparative example 4
The composition is the same as in example 1, except that: 10g/L sodium bentonite is added into the fermentation medium.
Comparative example 5
The composition is the same as in example 1, except that: sodium bentonite is replaced with kaolin.
Implementation results: see table below
Results of phytase fermentation
| Yield is improved | Enzyme activity U/g | Viability of | |
| Example 1 | 17% | 10010 | 97% |
| Example 2 | 21% | 10030 | 98% |
| Example 3 | 19% | 10020 | 97% |
| Example 4 | 12% | 9990 | 98% |
| Comparative example 1 | / | 9940 | 96% |
| Comparative example 2 | 5% | 10010 | 97% |
| Comparative example 3 | 9% | 9980 | 97% |
| Comparative example 4 | 6% | 9990 | 96% |
| Comparative example 5 | 1% | 9970 | 95% |
The enzyme activities in the above table are defined as follows: the sample releases 1 mu mol of inorganic phosphorus from sodium phytate per minute under the conditions that the concentration of the sodium phytate is 5.0 mmol/L, the temperature is 37 ℃ and the pH value is 5.50, namely, the sample is a phytase activity unit which is expressed as U.
The results show that the phytase fermentation process with bentonite can improve the activity, the biological activity and the yield of phytase compared with the process without bentonite in the comparison document 1.
Meanwhile, when examples 1 to 4 are compared with comparative examples 2 to 4, the inventors found that the addition amount of bentonite has a significant effect on the production of phytase. When the addition amount of bentonite is less than 0.5g/L (comparative example 2), the stability improvement effect of the bentonite on the growth environment of microorganisms is limited, so that the improvement of the production efficiency of phytase is not obvious. In addition, since bentonite has good adsorption performance, if the dosage is too large (comparative example 3 and comparative example 4), the active ingredients are adsorbed on the bentonite, the activity is reduced, the fermentation is affected, and the inventor reagent tests prove that the production efficiency of phytase is improved most obviously when the addition amount of the bentonite is between 0.5g/L and 5 g/L.
Comparing example 1 with comparative example 5, we found that the replacement of bentonite with kaolin having closer properties did not result in closer performance tests, indicating that bentonite had an effect of improving phytase production efficiency that was previously unexpected by those skilled in the art.
Finally, what has been disclosed is merely a specific embodiment of the invention. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (5)
1. A phytase fermentation production method is characterized in that,
The method comprises the following steps:
(S.1) primary liquid culture;
(S.2) fermenting in a fermentation tank; the bentonite in the step (S.2) is added into a fermentation tank along with a fermentation medium, the addition amount of the bentonite is 0.5g/L-5g/L, and the adopted strain is aspergillus niger;
(S.3) isolation and purification of phytase.
2. The method for producing phytase according to claim 1, wherein,
The bentonite comprises: at least one or a combination of a plurality of sodium bentonite, calcium bentonite, lithium bentonite and acid modified bentonite.
3. The method for producing phytase according to claim 1, wherein,
The primary liquid culture process in the step (S.1) is as follows:
inoculating single colony into seed culture medium, and shake culturing at 32-40 deg.C for 30-45 hr.
4. The method for producing phytase according to claim 1, wherein,
The fermentation process of the fermentation tank in the step (S.2) is as follows: inoculating 6-18% of the inoculating amount into fermentation medium, culturing at 32-40deg.C for 60-84 hr, and performing submerged fermentation culture.
5. The method for producing phytase according to claim 1, wherein,
The fermentation medium comprises the following components: potassium citrate, 1-4 g/L; 1-2 g/L of calcium sulfate; 35-50 g/L of monopotassium phosphate; 3-7 g/L of ammonium sulfate; 12-18 g/L of potassium sulfate; 9-15 g/L of magnesium sulfate; glucose 4-8 g/L.
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