CN116515795B - Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid - Google Patents
Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid Download PDFInfo
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- CN116515795B CN116515795B CN202310745627.9A CN202310745627A CN116515795B CN 116515795 B CN116515795 B CN 116515795B CN 202310745627 A CN202310745627 A CN 202310745627A CN 116515795 B CN116515795 B CN 116515795B
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- phytic acid
- aspergillus tubingensis
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- 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 title claims abstract description 74
- 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 title claims abstract description 74
- 235000002949 phytic acid Nutrition 0.000 title claims abstract description 74
- 229940068041 phytic acid Drugs 0.000 title claims abstract description 74
- 239000000467 phytic acid Substances 0.000 title claims abstract description 74
- 241000228232 Aspergillus tubingensis Species 0.000 title claims abstract description 40
- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 28
- 229940085127 phytase Drugs 0.000 title claims abstract description 28
- 230000000593 degrading effect Effects 0.000 title claims abstract description 23
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- 238000012216 screening Methods 0.000 description 30
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000012258 culturing Methods 0.000 description 12
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
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- 102000004190 Enzymes Human genes 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
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- 238000002360 preparation method Methods 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 4
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- 239000005720 sucrose Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
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- 230000000694 effects Effects 0.000 description 3
- WPEXVRDUEAJUGY-UHFFFAOYSA-B hexacalcium;(2,3,4,5,6-pentaphosphonatooxycyclohexyl) phosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([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 WPEXVRDUEAJUGY-UHFFFAOYSA-B 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
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- 238000007789 sealing Methods 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- 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 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000220215 Moringa Species 0.000 description 2
- 235000011347 Moringa oleifera Nutrition 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
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- 239000007787 solid Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 229930182490 saponin Natural products 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
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- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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Abstract
The invention discloses application of aspergillus tubingensis in preparing phytase and/or degrading phytic acid, and relates to the technical field of phytic acid degradation. At least one of living thalli, dead thalli and fermentation clear liquid of aspergillus tubingensis TPDA-1 is used for preparing a microbial inoculum, and the aspergillus tubingensis TPDA-1 is applied to preparing phytase and/or degrading phytic acid; the invention provides a new path for the production and development of phytase and solves the problem of lack of bacteria for degrading phytic acid microorganisms in the prior art.
Description
Technical Field
The invention relates to the technical field of phytic acid degradation, in particular to application of aspergillus tubingensis in preparing phytase and/or degrading phytic acid.
Background
Anti-nutritional factors are a generic term for a class of substances that affect nutrient digestion and absorption and animal body health and production. Although the common plant source feed is rich in nutrients, the absorption and the utilization of the plant source feed are restricted by some anti-nutritional factors, and the common anti-nutritional factors in the plant source feed mainly comprise tannin, phytic acid, trypsin inhibitor, saponin and the like. The phytic acid widely exists in plants and is a common anti-nutritional factor, most of phosphorus in the plants exists in the form of phytic acid phosphorus, the anti-nutritional characteristic of the phytic acid mainly passes through two ways, one way is that the phytic acid can form an insoluble complex with proteins in intestinal tracts of organisms to influence the absorption and utilization of the proteins, and the other way is that the organism of a monogastric animal can not synthesize phytase by itself, so that the absorption and utilization rate of the phytic acid phosphorus is low. Some forage plants have high content of phytic acid, the application of the forage plants in the forage is severely limited, and how to efficiently degrade the phytic acid in the forage plants has become a research difficulty and a hot spot.
At present, the method for degrading the phytic acid in the feed mainly comprises physical, chemical and microbial fermentation, wherein the physical and chemical methods have high cost, low degradation rate and easy environmental pollution; the microbial fermentation method is relatively low in cost and not easy to pollute the environment, and the phytase is mainly produced by the secretion of microorganisms to degrade the phytic acid, so that the microbial fermentation technology not only can improve the nutrition components of the feed, but also can destroy harmful factors in the feed, reduce the content of anti-nutritional factors and promote the digestion and absorption of feed raw materials. Therefore, the development of microbial strains capable of producing phytase and thus degrading phytic acid is a current problem to be solved.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide application of aspergillus tubingensis in preparing phytase and/or degrading phytic acid, so as to solve the problem of lack of a phytic acid degrading microorganism strain in the prior art.
The technical scheme for solving the technical problems is as follows: the use of Aspergillus tubingensis for the preparation of phytase and/or for the degradation of phytic acid is provided.
Further, the aspergillus tubingensis isAspergillus tubingensis) Designated A.tubingensis TPDA-1, deposited at the collection of microorganisms and cell cultures of Guangdong province at 2023, month 03, under the accession number GDMCC No:63289; deposit unit address: guangzhou city first middle road No. 100 college No. 59 building 5.
Further, the ITS nucleotide sequence of Aspergillus tubingensis is shown as SEQ ID No. 1.
The invention also provides a microbial inoculum comprising the aspergillus tubingensis.
Further, aspergillus tubingensis is at least one of live bacteria, dead bacteria and fermentation clear liquid.
Further, the fermentation clear liquid is prepared by the following method: fermenting and culturing aspergillus tubingensis, and carrying out solid-liquid separation to obtain fermentation clear liquid.
Further, in the fermentation culture, the inoculum size was 1X 10 6 -1×10 8 CFU/mL, rotational speed of 100-120rpm, temperature of 20-30deg.C, and time of 4-10d.
Further, solid-liquid separation was performed at 12000rpm and 4℃for 20min.
The aspergillus tubingensis provided by the invention can generate a large amount of viable aspergillus tubingensis after being cultivated, the invention is not particularly limited to a cultivation method, as long as the aspergillus tubingensis can be greatly proliferated by the cultivation method, for example, the aspergillus tubingensis can be cultivated in a flat plate culture medium until spores are produced, the spores are eluted by normal saline and fully oscillated and diluted to obtain spore suspension, and the spore suspension can be stored at a refrigerator of 4 ℃ for standby or directly inoculated into a fermentation culture medium for fermentation cultivation.
The invention also provides a method for degrading phytic acid, which comprises the following steps:
the aspergillus tubingensis and the sample containing phytic acid are mixed according to the volume ratio of 1-3:1, fermenting.
Further, fermenting at pH 4-6, 20-30deg.C and 100-120rpm for 4-10d.
Further, in the sample containing phytic acid, the phytic acid content is 3-8g/L.
After fermentation, methanol can be added to terminate degradation, and after fermentation, the product obtained by degradation of phytic acid can be further purified, for example, extraction, crystallization, filtration and the like are adopted; the sample containing phytic acid may be a solution containing phytic acid; the phytic acid-containing sample can also be obtained by subjecting a phytic acid-containing plant material to an extraction treatment.
The fermentation may be performed by mixing fermentation cells (living cells or dead cells) of A.tubingensis with a sample containing phytic acid so that the phytase in A.tubingensis degrades phytic acid, or by mixing fermentation products of A.tubingensis (fermentation clear liquid containing phytase, or phytase extracted and purified from fermentation liquid of A.tubingensis) with the sample containing phytic acid to degrade phytic acid.
The invention has the following beneficial effects:
1. the aspergillus tubingensis TPDA-1 capable of degrading the phytic acid is obtained through screening and then is used for preparing the phytase and/or degrading the phytic acid, a new path is provided for the production and development of the phytase, and the production problem of the biodegradable anti-nutritional factor phytic acid is effectively solved.
2. The aspergillus tubingensis TPDA-1 has good effects in preparing phytase and degrading phytic acid, and the phytase in the fermentation clear liquid obtained by fermentation culture has higher enzyme activity and strong specific conversion capability on the phytic acid; the highest phytic acid degradation rate is 94%, and the method has good application value. Meanwhile, the phytase in the aspergillus tubingensis TPDA-1 is convenient to use when being used for degrading phytic acid, has wide pH and temperature tolerance, and has high enzymolysis efficiency.
Drawings
FIG. 1 is a schematic diagram of morphology of a primary strain;
FIG. 2 is a schematic diagram of the morphology of the fermentation hyphae of the rescreened strain;
FIG. 3 is a schematic representation of a strain development tree.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: separation and screening of aspergillus tubingensis TPDA-1
The method for separating and screening microorganisms from soil samples under moringa trees in the university of agricultural in south China comprises the following specific steps:
s1, sampling: collecting soil samples (North latitude 23 DEG 07', east longitude 113 DEG 19') under moringa trees of the forest college of agricultural university in south China;
s2, enrichment: 10g of the soil sample was taken in 90mL of sterile water and incubated overnight at 28℃and 120 rpm. The overnight culture solution is diluted to 10 by adopting a gradient dilution method -1 -10 -5 The enrichment medium is selected from potato glucose medium (main components are potato extract and glucose), and the bacterial liquid after gradient dilution is inoculated into the potato glucose medium with an inoculum size of 2vt percent for enrichment culture to obtain an enrichment strain;
s3, primary screening: selecting a solid culture medium of Nahnikovia, adding phytic acid as a primary screening culture medium, adding 0.04g/L bromophenol blue as an indicator, inoculating 200 mu L of the enriched strain obtained in the S2 into the primary screening culture medium for coating, culturing for 4d at 37 ℃, and screening out a primary screening strain through whether the bromophenol blue indicator in the primary screening culture medium has a color reaction or not, wherein the specific reference can be seen in figure 1;
s4, re-screening: selecting a solid culture medium of a Sonchi type and additionally adding phytic acid as a rescreening culture medium, inoculating 200 mu L of the primary screening strain obtained in the step S3 into the rescreening culture medium for coating, culturing for 4D at 37 ℃, and screening out the rescreening strain by judging whether transparent rings are formed in the rescreening culture medium and the ratio (D/D) of the diameters of the transparent rings to the diameters of thalli;
s5, re-screening seed culture: selecting a liquid culture medium of Morse, adding phytic acid, glucose and NaCl as a re-screening seed culture medium, culturing the re-screening strain obtained in the step S4 until spores are produced, eluting the spores with sterile water to obtain spore liquid, and obtaining the spore liquid with the inoculation amount of 10 6 -10 8 Inoculating CFU/mL into a re-screening seed culture medium, and culturing the re-screening seeds for 4d under the conditions that the temperature is 30 ℃ and the rotating speed is 120rpm to obtain re-screening seed liquid; inoculating the re-screened seed liquid into a re-screened fermentation medium at an inoculum size of 2vt percent, and culturing the re-screened seed liquid for 4d under the conditions that the temperature is 30 ℃ and the rotating speed is 120rpm to obtain a re-screened fermentation liquid; taking supernatant obtained by centrifuging the re-screening fermentation liquor for 20min at the temperature of 4 ℃ and the rotating speed of 12000rpm as re-screening fermentation liquor, measuring the protein content in the re-screening fermentation liquor and the centrifugal sediment by adopting a BCA method, and calculating to obtain that the extracellular protein in the re-screening fermentation liquor reaches 3.41 mug/mu L, and the intracellular protein in the re-screening fermentation sediment reaches3.39 mug/mug, see in particular fig. 2;
s6, inoculating the final screening strain obtained in the S5 into a PDA culture medium, carrying out PCR amplification on the gene of the re-screening strain by using primers ITS4 (TCCTCCGCTTATTGATATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGG), sequencing a product obtained after the PCR amplification, and carrying out sequence determination by Shanghai biological engineering company, wherein a sequence sequencing result is shown as SEQ ID NO:1, submitting the sequenced sequence to NCBI (National Center for Biotechnology Information), and comparing the sequenced sequence with NCBI database, wherein the result shows that the homology of the sequenced sequence and the Aspergillus tubingensis is 100%, and the strain for degrading the phytic acid, which is separated by the invention, is Aspergillus tubingensis, and can be shown in a specific figure 3;
wherein the primary screening culture medium comprises 30g/L sucrose, 5g/L calcium phytate and 3g/L NaNO 3 K1 g/L 2 HPO 4 MgSO of 0.5g/L 4 ·7H 2 O, 0.5g/L KCl, 0.01g/L FeSO 4 0.04g/L bromophenol blue and 1.8% agar;
the re-screening culture medium comprises 30g/L sucrose, 5g/L calcium phytate and 3g/L NaNO 3 K1 g/L 2 HPO 4 MgSO of 0.5g/L 4 ·7H 2 O, 0.5g/L KCl, 0.01g/L FeSO 4 0.04g/L bromophenol blue and 1.8% agar;
the components of the re-screening seed culture medium and the culture medium in the re-screening fermentation culture are as follows: 30g/L sucrose, 5g/L calcium phytate and 3g/L NaNO 3 K1 g/L 2 HPO 4 MgSO of 0.5g/L 4 ·7H 2 O, 0.5g/L KCl and 0.01g/L FeSO 4 The initial pH of the fermentation was adjusted to 5 with 40% NaOH solution.
Illustratively, the conditions for rescreening seed culture at least satisfy: the temperature is 35-39 ℃, the speed is 100-200rpm, and the time is 4-10d; the conditions of the re-screening fermentation culture at least meet the following conditions: the inoculation amount is 1-3vt%, the temperature is 25-35 ℃, the rotating speed is 100-200rpm, and the time is 4-10d.
Example 2: preparation of phytase and/or degradation of phytic acid
The process for preparing phytase and/or degrading phytic acid comprises the following steps:
(1) Inoculating Aspergillus tubingensis obtained in example 1 into plate culture medium, coating, culturing at 37deg.C until spore is produced, adding about 10mL physiological saline, eluting, shaking thoroughly, diluting to obtain concentration of 10 7 CFU/mL spore suspension is preserved in a refrigerator at 4 ℃ for standby;
(2) Inoculating the spore suspension obtained in the step (1) into a fermentation culture medium with the inoculum size of 2vt percent, shaking uniformly, sealing with four layers of gauze, and fermenting and culturing in a shaking table with the temperature of 30 ℃ and the rotating speed of 120rpm for 10d to finish the fermentation enzyme production process, thus obtaining fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) for 20min at the temperature of 4 ℃ and the rotating speed of 12000rpm to obtain supernatant serving as fermentation liquor, and mixing the fermentation liquor with a phytic acid aqueous solution with the substrate concentration of 5g/L according to the volume ratio of 1:1, fermenting for 10d at 30 ℃ under the conditions of pH 6 and rotating speed of 120rpm to obtain degradation products. And adding methanol into degradation products to terminate degradation, mixing uniformly, measuring the content of phytic acid, and calculating to obtain the degradation rate of the phytic acid reaching 94%.
Example 3: preparation of phytase and/or degradation of phytic acid
The process for preparing phytase and/or degrading phytic acid comprises the following steps:
(1) Inoculating Aspergillus tubingensis obtained in example 1 into plate culture medium, coating, culturing at 37deg.C until spore is produced, adding about 10mL physiological saline, eluting, shaking thoroughly, diluting to obtain concentration of 10 7 CFU/mL spore suspension is preserved in a refrigerator at 4 ℃ for standby;
(2) Inoculating the spore suspension obtained in the step (1) into a fermentation culture medium with the inoculum size of 2vt percent, shaking uniformly, sealing with four layers of gauze, and fermenting and culturing in a shaking table with the temperature of 20 ℃ and the rotating speed of 100rpm for 8d to finish the fermentation enzyme production process, thus obtaining fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) for 20min at the temperature of 4 ℃ and the rotating speed of 12000rpm to obtain supernatant serving as fermentation liquor, and mixing the fermentation liquor with phytic acid aqueous solution with the substrate concentration of 8g/L according to the volume ratio of 3:1, fermenting for 8d at 20 ℃ under the conditions of pH 4 and rotating speed of 100rpm to obtain degradation products. And adding methanol into degradation products to terminate degradation, mixing uniformly, measuring the content of phytic acid, and calculating to obtain the degradation rate of the phytic acid reaching 94%.
Example 4: preparation of phytase and/or degradation of phytic acid
The process for preparing phytase and/or degrading phytic acid comprises the following steps:
(1) Inoculating Aspergillus tubingensis obtained in example 1 into plate culture medium, coating, culturing at 37deg.C until spore is produced, adding about 10mL physiological saline, eluting, shaking thoroughly, diluting to obtain concentration of 10 7 CFU/mL spore suspension is preserved in a refrigerator at 4 ℃ for standby;
(2) Inoculating the spore suspension obtained in the step (1) into a fermentation culture medium with the inoculum size of 2vt percent, shaking uniformly, sealing with four layers of gauze, and fermenting and culturing in a shaker with the temperature of 25 ℃ and the rotating speed of 110rpm for 4d to finish the fermentation enzyme production process, thus obtaining fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) for 20min at the temperature of 4 ℃ and the rotating speed of 12000rpm to obtain supernatant serving as fermentation liquor, and mixing the fermentation liquor with a phytic acid aqueous solution with the substrate concentration of 3g/L according to the volume ratio of 2:1, and fermenting for 4d at 25 ℃ under the conditions of pH 5 and rotational speed of 110rpm to obtain degradation products. And adding methanol into degradation products to terminate degradation, mixing uniformly, measuring the content of phytic acid, and calculating to obtain the degradation rate of the phytic acid reaching 94%.
Comparative example 1:
the phytase is prepared and/or the phytic acid is degraded by adopting a conventional method, and the steps are as follows:
(1) The components and the contents of the fermentation medium are as follows: sucrose 30g/L, naNO 3 2g/L、K 2 HPO 1g/L、KCI 0.5g/L、MgSO 4 0.5g/L、FeSO 4 0.01g/L, the balance being water; immediately adding Aspergillus niger (Aspergillus niger) after sterilization, and performing shaking culture at 30deg.C and rotation speed of 120rpm for 96 hr to obtain fermentation broth;
(2) The enzymolysis culture medium comprises the following components in percentage by weight: phytic acid 5g/L, naNO 3 2g/L、K 2 HPO 1g/L、KCI 0.5g/L、MgSO 4 0.25g/L、FeSO 4 0.01g/L, and adjusting the pH value to 5 by using 40% NaOH solution; transferring the fermentation broth obtained in the step (1) into an enzymolysis culture medium with an inoculum size of 2vt percent, and performing shake culture for 4 days at a temperature of 30 ℃ and a rotating speed of 120rpm to obtain an enzymolysis broth;
(3) And (3) centrifuging the enzymolysis liquid obtained in the step (2), measuring the content of phytic acid, and calculating to obtain the degradation rate of the phytic acid of 91%.
From the above, examples 1 to 4 and comparative example 1 show that Aspergillus tubingensis has good application effect in degrading phytic acid, the degradation rate of phytic acid reaches over 94%, and the method has good application prospects in preparing phytase and degrading phytic acid.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. The application of aspergillus tubingensis in preparing phytase and/or phytic acid degradation products is characterized in that the aspergillus tubingensis isAspergillus tubingensis) Designated A.tubingensis TPDA-1, deposited at the collection of microorganisms and cell cultures of Guangdong province at 2023, month 03, under the accession number GDMCC No:63289.
2. the use according to claim 1, wherein the aspergillus tubingensis has the ITS nucleotide sequence set forth in SEQ ID No: 1.
3. A microbial agent comprising aspergillus tubingensis according to claim 1 or 2.
4. A microbial agent according to claim 3, wherein the aspergillus tubingensis is a viable microbial organism.
5. A method for degrading phytic acid, comprising the steps of:
mixing aspergillus tubingensis and a sample containing phytic acid according to any one of claims 1-2 in a volume ratio of 1-3:1, fermenting.
6. The method for degrading phytic acid according to claim 5, wherein the sample containing phytic acid is fermented at pH 4-6, 20-30 ℃ and 100-120rpm for 4-10d, and the phytic acid content is 3-8g/L.
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| KR20010081175A (en) * | 2000-02-10 | 2001-08-29 | 김형락 | Novel Aspergillus sp. 5990 producing thermostable phytase and method for degradation of phytic acid using the same |
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| KR20010081175A (en) * | 2000-02-10 | 2001-08-29 | 김형락 | Novel Aspergillus sp. 5990 producing thermostable phytase and method for degradation of phytic acid using the same |
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