CN114350549B - Polymyxin A1 production strain and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological fermentation, and discloses a polymyxin A1 production strain and application thereof. The strain is Paenibacillus sp SY20 and is deposited in the Guangdong province microorganism strain collection at 9/1 of 2021 and is numbered GDMCC No. 61909. Then, fermenting the strain by microorganism to obtain fermentation liquor, centrifuging the fermentation liquor, precipitating by ammonium sulfate, performing ion exchange, separating by a C18 reversed-phase high-performance liquid chromatographic column to obtain an active component with higher purity, and finally identifying the active component as polymyxin A1. The polymyxin A1 has good antibacterial effect on various gram-negative pathogenic bacteria, and can be applied to preparation of medicines for inhibiting the gram-negative pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a polymyxin A1 production strain, a preparation method and application of polymyxin A1.
Background
Polymyxin is a group of cyclic lipopeptid antibiotics produced by paenibacillus polymyxa (Paenibacillus polymyxa), has antibacterial effect on gram-negative bacteria, has good effect on treating infections caused by multidrug-resistant gram-negative bacteria (especially acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae), and thus attracts wide clinical attention. Presently, polymyxins are found to comprise mainly A, B, C, D, E components, and each component has multiple variants. More clinically, the sulfate and mesylate salts of polymyxin B and polymyxin E are used, and are each a mixture of variants.
Polymyxin A (polymyxin A) is an antibiotic substance isolated and identified by brown and colleagues in the wilkon physiological research laboratory in the united kingdom in 1947 from an organism identified as a bacillus pneumophilus. It was initially named Aerosporin and was subsequently found to have an antibacterial spectrum and biological activity very similar to that of polymyxin and to belong to the same family of antibiotic compounds. According to the international agreement, all antibiotics from bacillus polymyxa have been given the generic name "polymyxin" and a term was developed to describe the different antibiotics of this family. Thus, aerosporin is known as polymyxin a. Polymyxin A is divided into two variants A1 and A2 according to the fatty acid chain, A1 is 6-methylctanoyl (C 9 H 17 O) A2 is 6-methylheptanoyl (C) 8 H 15 O). Following Aerosporin, there are few reports about other polymyxin a producing strains and their isolation and identification.
Disclosure of Invention
Aiming at that polymyxin is mostly produced by Paenibacillus polymyxa, and polymyxin A1 is discovered and reported recently, the invention provides a production strain of polymyxin A1, which is identified as a strain of Paenibacillus thiomeracil (Paenibacillus thiaminolyticus) through whole genome, and simultaneously provides a preparation method of polymyxin A1.
The invention aims at realizing the following technical scheme:
a polymyxin A1 producing strain which is Paenibacillus sp SY20 deposited on month 9 of 2021 at the Cantonese microorganism strain deposit under the number GDMCC No. 61909. Then, fermenting the strain by microorganisms to obtain fermentation liquor, and carrying out centrifugation, ammonium sulfate precipitation, ion exchange and C18 reversed-phase high-performance liquid chromatography column separation on the fermentation liquor to prepare an active component with higher purity, thereby identifying the active component as polymyxin A1.
The application of the strain in fermentation production of polymyxin A1.
Preferably, after the strain is fermented and cultured, the fermentation broth is centrifuged, and then subjected to 'three-step' ammonium sulfate precipitation-cation exchange chromatography-reverse phase chromatography to obtain polymyxin A1.
The preparation of the polymyxin A1 specifically comprises the following steps:
(1) Inoculating the strain of claim 1 into LB culture medium, fermenting and culturing, centrifuging and collecting fermentation supernatant;
(2) Adding ammonium sulfate into the obtained fermentation supernatant to ensure that the saturation of the ammonium sulfate of the supernatant is 50-100%, and standing to obtain a suspension;
(3) Centrifuging the obtained suspension, collecting precipitate, and dissolving with water to obtain crude protein solution containing ammonium sulfate;
(4) And (3) dialyzing and desalting the obtained ammonium sulfate-containing crude protein solution, performing vacuum freeze drying to obtain crude protein, and separating and purifying to obtain polymyxin A1.
Preferably, the crude protein in step (4) is subjected to the following separation and purification treatments: dissolving the crude protein with a buffer solution, and performing elution separation on a cation exchange chromatographic column after sterilization, wherein the elution conditions are as follows: the balance liquid A accounts for 0-40%, the eluent B accounts for 60-100%, and the eluent is collected to obtain the partially purified active component.
Preferably, the balance solution A is 20-50 mM Tris-HCl, the pH value is 7.0-9.0, and the eluent B is 20-50 mM Tris-HCl+1M NaCl, the pH value is 7.0-9.0.
Further, the partially purified active component is subjected to the following separation and purification treatments to obtain polymyxin A1:
(a) Dialyzing for desalting, lyophilizing under vacuum, and dissolving with water;
(b) Purifying the dissolved active component with semi-preparative liquid phase system, and collecting peak component with retention time of 9.248 + -1.0 min;
(c) And (c) dialyzing the component obtained in the step (b) to remove salt, and performing vacuum freeze drying to obtain high-purity active component powder.
Preferably, the chromatographic conditions are: the chromatographic column is C18-H, and the mobile phase is acetonitrile: sodium sulfate solution= (20-22): (78-80), wherein the concentration of the sodium sulfate solution is 25-35 mM, and the pH value is adjusted to 2.3-2.5 by phosphoric acid; chromatographic column temperature: 30.+ -. 5 ℃.
Preferably, the saturation of the ammonium sulfate in the step (2) is 65% -100%; the fermentation culture conditions of the step (1): fermenting and culturing at 25-40 deg.c and 1-10% inoculation amount and 100-300 rpm for 24-72 hr.
Preferably, the fermentation culture conditions described in step (1): fermenting and culturing at 28-37 deg.c and 1-5% inoculation amount and 120-200 rpm for 32-48 hr; the centrifugation conditions are as follows: centrifuging at 8000-10000 rpm for 15+ -5 min; the standing condition in the step (2) is that the mixture is kept standing at 4 ℃ overnight; the centrifugation condition in the step (3) is that the suspension is centrifuged for 10-20 min at 8-10000 rpm and 4 ℃.
Preferably, the dialysis conditions in steps (4) and (a), (c) are each: the cutoff pore size of the dialysis bag was 500Da and dialysis was performed with water at 4 ℃.
The fermentation medium (LB) formula for microbial fermentation of the polymyxin A1 production strain is as follows: 10g of sodium chloride, 10g of peptone, 5g of yeast extract and the balance of deionized water are contained in each 1000mL of liquid culture medium.
Further, the purified polymyxin A1 was identified:
dissolving the active ingredient powder obtained by the purification in the step (4) by using sterile water, and carrying out LC-MS/MS and MALDI-TOF identification. And (3) combining the identification result with the whole genome sequencing result of the production strain, predicting a secondary metabolite gene cluster in the production strain by utilizing anti-SMASH online prediction software, and predicting the NRPS/PKS monomer structure. The identification result is polymyxin A1. The molecular weight is M= 1156.7279Da, and the molecular formula is shown as follows:
compared with the prior art, the invention has the following beneficial effects:
the invention provides a brand new production strain of polymyxin A1, which can ferment and produce the polymyxin A1. The prepared polymyxin A1 has good antibacterial effect on various gram-negative pathogenic bacteria.
The strain of the invention is named Paenibacillus sp.SY20 and is deposited in the microbiological strain collection center of the Guangdong province microbiological institute in 2021, 9 months, and is numbered as GDMCC No. 61909, address: the institute of microbiology, university of guangdong, china, postal code: 510070. whole genome sequencing was identified as a strain of Paenibacillus thioaminolyticus (P.thiaminolyticus SY 20).
Drawings
FIG. 1 (a) is a diagram showing the microscopic bacterial pattern of the polymyxin A1 producing strain P.thiaminolyticus SY 20; FIG. 1 (b) is a diagram showing the bacterial status under electron microscope.
FIG. 2 is a graph showing the effect of P.thiaminolyticus SY20 strain fermentation supernatant on inhibiting Escherichia coli.
FIG. 3 is a graph showing the fermentation time-growth curve, fermentation time-pH, fermentation time-bacteriostatic activity of P.thiaminolyticus SY20 strain.
FIG. 4 (a) shows the bacteriostatic effect of crude protein extracted from P.thiamine yticus SY20 fermentation supernatant by gradient ammonium sulfate precipitation on Escherichia coli; FIG. 4 (b) shows the bacteriostatic effect of the crude protein extracted from 65% -100% ammonium sulfate precipitation on Escherichia coli after dialysis and lyophilization.
FIG. 5 (a) shows the separation of crude P.thiamine yticus SY20 active protein on CM weak cation exchange column. The abscissa in the graph represents the elution time (min), and the ordinate represents the ultraviolet absorbance (mAU); 2-19 represents the number of tubes collected to elute; c1 (tubes 2-6) are peaks eluted at 0% B; c2 (7-9 tubes) 30% B eluted peak; c3 (tube 12), C4 (tubes 14, 15) was 60% of the peak eluted with B; FIG. 5 (b) is a partially enlarged view of the C4 component (14 th and 15 th tubes); FIG. 5 (C, d) shows the inhibition of E.coli by the 2-17 th tube fraction, wherein only the C4 peak (14 th and 15 th tubes) is the active component peak, and the UV absorption of this peak at 214nm is extremely small, which was not shown in FIG. 5 (a). This part is enlarged and as shown in fig. 5 (b), there is a peak at this position of C4.
FIG. 6 (a) is a graph showing the separation of C4 active components on a reversed phase C18-H column, wherein the abscissa indicates the elution time (min) and the ordinate indicates the ultraviolet absorbance (mAU); FIG. 6 (b, c) shows the inhibitory activity of the eluted fraction against E.coli at each retention time of 9.248min as the active fraction.
FIGS. 7 (a) and (b) are LC-MS/MS diagrams of active components after separation by a C18-H chromatographic column, wherein the left side and the right side of the diagrams (a) and (b) are respectively primary mass spectra, and the right side of the diagrams (a) and (b) are respectively secondary mass spectra of corresponding peaks; FIG. 7 (C) is a MALDI-TOF diagram of the active component after separation by a C18-H column.
FIG. 8 (a) is a graph showing predicted polymyxin synthesis gene clusters based on the whole genome of the production strain; FIG. 8 (b) shows the prediction of NRPS/PKS monomer structures for three key genes of the gene cluster; FIG. 8 (c) is a secondary mass spectrum of the active ingredient fragment, deduced to be polymyxin A1.
FIG. 9 shows the bacteriostatic effect of the prepared polymyxin A1 on various pathogenic bacteria and food-borne bacteria; (a) enterobacter sakazakii, (b) salmonella enteritidis, (c) vibrio parahaemolyticus, (d) bacillus cereus, (e) klebsiella pneumoniae, and (f) escherichia coli.
Detailed Description
The invention discloses a polymyxin A1 producing strain identified as Paenibacillus thioaminolyticus P.thiaminolyticus SY20 through genome-wide, and a preparation and identification method of the polymyxin A1, which are specifically shown in the following examples.
Example 1
1. Production Strain acquisition
Paenibacillus producing polymyxin A1 is obtained by screening according to the following method: the sour bamboo shoot sample is diluted to 10 in a gradient way in a sterile test tube by using sterile water -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Seven gradients. As the screened bacteria are limited to bacillus, the samples with various gradients are respectively placed in a water bath kettle with the temperature of 70 ℃ and heated for 20min so as to remove partial mixed bacteria of non-bacillus. After cooling, 10 is selected -2 、10 -4 、10 -6 Three gradients, respectively sucking 200. Mu.L of the three gradients on the bacillus enrichment medium, NA medium and GPY medium, coating, sealing with sealing film, and culturing in an incubator at 28 ℃. After bacterial colonies grow out on the culture medium, a single bacterial colony is picked up by a sterile gun head, streaked and purified on an LB plate for multiple times, and the antibacterial activity is detected. The active strain is identified as Paenibacillus thioaminolyticus (P.thiamine SY 20), and the colony is milky white and has a smoother surface; the thalli are in a long rod shape under a microscope, and are gram-positive; the electron microscope is in a slender rod shape, the two ends are pointed, and the length is varied from 4.5 mu m to 9.3 mu m. The bacterial pattern under the microscope and the electron microscope is shown in FIG. 1 (a, b).
Wherein: the bacillus enrichment medium contains the following components per 1000 mL: 10g of peptone, 3g of yeast extract, 3g of starch, 2g of disodium hydrogen phosphate, 1.5g of dipotassium hydrogen phosphate, 0.1g of magnesium sulfate heptahydrate, and the balance of water, wherein the pH value is 7.8.
NA medium, per 1000mL, contained the following components: 10g of peptone, 3g of beef extract, 5g of sodium chloride and the balance of water.
GPY medium contains the following components per 1000 mL: 10g of glucose, 3g of peptone, 7g of yeast extract, 1g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, and the balance of water, wherein the pH value is 7.0.
2. Antibacterial activity detection of fermentation supernatant of production strain
The agar plate diffusion method is adopted, and the specific steps are as follows:
(1) Preparing a coliform indicator bacterium mixed bacterium plate: the indicator strain culture medium is LB culture medium, and is inoculated with Escherichia coli at a ratio of 1:100, and shaken to OD at 37 DEG C 600 About 1.0, diluted to OD with LB medium 600 At a concentration of 0.5, 160 mu L/100mL of diluted staphylococcus aureus is inoculated into an aseptic LB culture medium with an agar concentration of 0.75%, the inoculation temperature is preferably about 45-50 ℃,pouring the mixture after fully and uniformly mixing, sealing the mixture by using a sealing film after solidification, and refrigerating the mixture in a refrigerator at the temperature of 4 ℃ for standby.
(2) Bacteriostasis experiment: punching holes on the bacteria-mixing plate of the indicator bacteria by using a sterilized puncher, picking out a culture medium block by using a sterile toothpick, adding 120 mu L of fermentation supernatant to be detected into each hole, drying in a super clean bench, placing in a 37 ℃ incubator for culturing for 12 hours, and observing a bacteria inhibition zone, as shown in figure 2.
3. Culture time optimization of bacteriostatic substances produced by production strains
The twice activated producer strains were inoculated at an inoculum size of 1:100 into 1L Erlenmeyer flasks containing 500mL LB and placed in an incubator at 37℃and 180rpm for cultivation. Samples were taken every four hours to determine their pH and OD 600 pH, OD with time on the abscissa 600 The values are plotted on the ordinate as fermentation time-pH curve and fermentation time-growth curve, respectively. 1mL of each sample was centrifuged at 13,000rpm for 10min and sterilized by a 0.22 μm filter. The antibacterial activity of the sample on the escherichia coli is tested by using an agar diffusion method, the size of the antibacterial circle radius is measured by using a ruler, the time is taken as an abscissa, and the antibacterial circle radius is taken as an ordinate to draw the fermentation time-antibacterial curve of the antibacterial circle radius on the escherichia coli, which is shown in figure 3.
4. Isolation of crude protein from fermentation supernatants of production strains
After the production strain is fermented in LB culture medium for 24-72 h, the thalli in the obtained fermentation broth is separated by a method of centrifugation at 8000rpm for 15min to obtain fermentation supernatant. Slowly adding 291g of ammonium sulfate powder into 1L of fermentation supernatant, stirring while adding to ensure that the saturation degree of ammonium sulfate is 50%, standing the fermentation supernatant at 4 ℃ overnight after the ammonium sulfate is completely dissolved, centrifuging the suspension at 8000rpm and 4 ℃ for 20min, and collecting precipitate, dissolving with 20mL of deionized water to obtain a crude protein extract under 50% ammonium sulfate gradient precipitation; adding 107g of ammonium sulfate powder into the centrifuged supernatant containing 50% ammonium sulfate to ensure that the saturation degree of the ammonium sulfate is 65%, standing at 4 ℃ for overnight, centrifuging the suspension at 8000rpm and 4 ℃ for 20min, collecting precipitate, and dissolving the precipitate with 20mL of deionized water to obtain a crude protein extract deposited by 50% -65% ammonium sulfate gradient; and finally, adding 300g of ammonium sulfate powder into the centrifuged supernatant containing 65% ammonium sulfate to ensure that the saturation degree of the ammonium sulfate is 100%, standing at 4 ℃ for overnight, centrifuging the suspension at 8-000 rpm and 4 ℃ for 20min, collecting precipitate, and dissolving the precipitate with 20mL of deionized water to obtain the crude protein extract under the gradient of 65-100% ammonium sulfate. Meanwhile, the antibacterial activity of each part of crude protein was detected by an agar plate diffusion method, as shown in FIG. 4a. Finally, the part with the best activity is taken for dialysis and desalination and freeze drying, thus obtaining crude protein powder. The active crude protein powder was dissolved in 20mL of 20mM Tris-HCl, pH 7.0-9.0 buffer and tested for bacteriostatic activity as shown in FIG. 4b.
5. Separation and purification of crude protein on CM cation exchange column
Dissolving active crude protein powder with 20mM Tris-HCl buffer solution with pH of 7.0-9.0, performing gradient elution and separation on a cation exchange chromatographic column (HiPrep 16/10CM FF, GE) by using an AKTA pure protein separation system after filtering, and monitoring the wavelength to be 214nm and 280nm; the gradient elution conditions were: 30% of B, 60% of B and 100% of B, wherein the balance solution A consists of 20mM Tris-HCl, the pH value is 7.0-9.0, and the eluent B consists of 20mM Tris-HCl+1M NaCl, the pH value is 7.0-9.0; the loading was 8mL of the crude protein solution described above, and the flow rate was 2mL/min. First, equilibrium elution with 0% b (i.e., 100% a) gave peak C1; after the collection of the peak is finished, the concentration of the eluent is adjusted to 30% B (namely 70% A) to obtain a peak C2; after the collection is completed, the concentration of the eluent is adjusted to 60% B (namely 40% A) to obtain peaks C3 and C4; finally, the eluent concentration was adjusted to 100% b (i.e. 0% a) without other elution peaks, see fig. 5 (a). Each tube automatically collects 8-10 mL of the eluted fraction, and the activity of each tube is detected, see FIG. 5 (c, d).
6. Separating and purifying active component C4 on reversed phase C18-H column
The active component C4 is placed in a dialysis bag with the interception aperture of 500Da, dialyzed by deionized water at the temperature of 4 ℃ and then vacuum freeze-dried to prepare active component powder, and the active component powder is dissolved by the deionized water and filtered. The fractions were further refined in a C18 column by a semi-preparative liquid phase system, and fractions of different retention times were collected by an agar plate diffusion method, see FIG. 6 (a, b, C).
Chromatographic conditions: chromatographic column: C18-H (4.6X105 mm,5 μm, amethylst); the mobile phase is acetonitrile: sodium sulfate solution=20 to 22:78 to 80, wherein the concentration of the sodium sulfate solution is 31.4mM, and the pH is adjusted to 2.3 to 2.5 by phosphoric acid; chromatographic column temperature: 30 ℃; flow rate: 1mL/min; sample loading amount: 100. Mu.L; the monitoring wavelength is 214nm and 280nm, and the gradient elution separation is carried out.
7. Component LC-MS/MS and MALDI-TOF identification after refining
And (3) placing the refined active part in a vacuum drying oven to volatilize and remove acetonitrile, placing in a dialysis bag with a interception aperture of 500Da, dialyzing with deionized water at 4 ℃, changing water for multiple times, and vacuum freeze-drying after salt ions are dialyzed cleanly to obtain high-purity refined powder. After dissolution with double distilled water, LC-MS/MS and MALDI-TOF molecular weight and molecular composition identification were performed, see FIG. 7 (a, b, c). In fig. 7 (a, b), M/z= 579.3710 is a multi-charge ion peak [ m+2h ]] 2+ M= 1156.724; m/z= 1157.7359Da as parent ion peak [ m+h ]] + M= 1156.7279Da. FIG. 7 (C) is a MALDI-TOF diagram of the active component after separation by a C18-H column, wherein M/z= 1179.805 is [ M+Na ]] + M= 1156.818Da. The molecular weight of the active ingredient was therefore deduced to be m= 1156.7279Da.
8. Identification of active Components in combination with genome-wide information of production strains
The LC-MS/MS and MALDI-TOF identification results show that the molecular weight of the active components is: 1156.7359Da. The production strain was subjected to whole genome sequencing, and the secondary metabolite synthetic gene cluster encoded in the whole genome was subjected to predictive analysis using the anti smash (https:// anti map. Second symetabolite. Org/# |/start) on-line predictive software. Thiaminolyticus SY20 contains a gene cluster encoding polymyxin which has a very high similarity to polymyxin E (Colistin), see FIG. 8 (a). The NRPS/PKS monomer structure prediction was performed on this gene cluster, as shown in FIG. 8 (b). Thus, based on whole genome information, combining primary and secondary mass spectrum information, the active component was presumed to be polymyxin A1, see fig. 8 (c).
9. Prepared colistin A1 bacteriostasis spectrum
The prepared active components are tested to have antibacterial activity on Escherichia coli (ATCC 25922), salmonella enteritidis Salmonella Enteritidis (CCTCC AB 94018), klebsiella pneumoniae Klebsiella pneumonia (ATCC 10031), enterobacter sakazakii Enterobacter Sakazakii (ATCC 29544), bacillus cereus (CMCC (B) 63301) and vibrio parahaemolyticus Vibrio parahaemolyticus (ATCC 10031) by using an agar plate diffusion method, and the prepared polymyxin A1 has good antibacterial effect on various gram-negative pathogenic bacteria as shown in figure 9.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. A polymyxin A1 producing strain is characterized in that the strain is Paenibacillus spPaenibacillus spSY20, 9.1.2021, deposited with the Guangdong province microorganism strain collection under the accession number GDMCC No. 61909.
2. Use of the strain of claim 1 for the fermentative production of polymyxin A1.
3. Use according to claim 2, characterized in that after fermentation culture of the strain according to claim 1, the fermentation broth is centrifuged and subjected to ammonium sulphate precipitation-cation exchange chromatography-reverse phase chromatography to obtain polymyxin A1.
4. A use according to claim 3, comprising the steps of:
(1) Inoculating the strain of claim 1 into LB culture medium, fermenting and culturing, centrifuging and collecting fermentation supernatant;
(2) Adding ammonium sulfate into the obtained fermentation supernatant to ensure that the saturation of the ammonium sulfate of the supernatant is 50% -100%, and standing to obtain a suspension;
(3) Centrifuging the obtained suspension, collecting precipitate, and dissolving with water to obtain crude protein solution containing ammonium sulfate;
(4) And (3) dialyzing and desalting the obtained ammonium sulfate-containing crude protein solution, performing vacuum freeze drying to obtain crude protein, and performing separation and purification twice to obtain polymyxin A1.
5. The use according to claim 4, wherein the crude protein of step (4) is isolated and purified: dissolving the crude protein with a buffer solution, and performing elution separation on a cation exchange chromatographic column after sterilization, wherein the elution conditions are as follows: the proportion of the balance liquid A is 0-40%, the proportion of the eluent B is 60-100%, and the eluent is collected to obtain a partially purified active component; the balance solution A is 20-50 mM Tris-HCl, the pH value is 7.0-9.0, and the eluent B is 20-50 mM Tris-HCl+1M NaCl,pH 7.0~9.0.
6. The use according to claim 5, wherein the partially purified active component is subjected to the following separation and purification treatments:
(a) Dialyzing for desalting, lyophilizing under vacuum, and dissolving with water;
(b) Purifying the dissolved active component with semi-preparative liquid phase system, and collecting peak component with retention time of 9.248 + -1.0 min;
(c) Dialyzing the component obtained in the step (b) to remove salt, and performing vacuum freeze drying to obtain high-purity active component powder;
the chromatographic conditions: the chromatographic column is C18-H, and the mobile phase is acetonitrile: sodium sulfate solution= (20-22): (78-80), wherein the concentration of the sodium sulfate solution is 25-35 mM, and the pH value is adjusted to 2.3-2.5 by phosphoric acid; chromatographic column temperature: 30.+ -. 5 ℃.
7. The use according to claim 4 or 5 or 6, wherein the saturation of ammonium sulphate in step (2) is 65% -100%; the fermentation culture conditions of the step (1): fermenting and culturing at 25-40 ℃ for 24-72 hours at 100-300 rpm with 1-10% of inoculation amount.
8. The use according to claim 7, wherein the fermentation culture conditions of step (1): fermenting and culturing for 32-48 h at 28-37 ℃ with 1-5% of inoculum size at 120-200 rpm; the centrifugation conditions are as follows: centrifuging at 8000-10000 rpm for 15+ -5 min; the standing condition in the step (2) is that the mixture is kept standing at 4 ℃ overnight; and (3) centrifuging the suspension at 8-10000 rpm and at 4 ℃ for 10-20 min.
9. The use according to claim 6, wherein the dialysis conditions in steps (4) and (a), (c) are each: the dialysis bag had a retention pore size of 500Da and was dialyzed against water at 4 ℃.
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| 环脂肽的研究进展;黎循航;张言周;魏志文;管政兵;蔡宇杰;廖祥儒;;中国酿造(第12期);全文 * |
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| CN114350549A (en) | 2022-04-15 |
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