CN117683081A - Antihypertensive peptide and application thereof in antihypertensive products - Google Patents
Antihypertensive peptide and application thereof in antihypertensive products Download PDFInfo
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- CN117683081A CN117683081A CN202310212595.6A CN202310212595A CN117683081A CN 117683081 A CN117683081 A CN 117683081A CN 202310212595 A CN202310212595 A CN 202310212595A CN 117683081 A CN117683081 A CN 117683081A
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- natto
- fermentation
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- bacillus subtilis
- antihypertensive
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The application discloses a antihypertensive peptide and application thereof in antihypertensive products. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.21709, and is classified and named as bacillus subtilis Bacillus subtilis subsp.natto R3, a preservation date of 2021 and 25 months 01, and a preservation address of Beijing Chaoyang area Beichen Xiyu No.1 and 3. The strain R3 has the functions of anticoagulation and ACE enzyme inhibition, can ferment leech low-temperature dried products to generate two active polypeptides, and has the functions of protecting ischemia reperfusion brain injury rats and reducing SIHR rat blood pressure, and has wide application prospects in preparing anticoagulation products or blood pressure reduction products.
Description
Technical Field
The application relates to the technical field of bacillus subtilis, in particular to bacillus subtilis natto subspecies R3 and application thereof in fermenting antihypertensive peptides.
Background
Bacillus natto (Bacillus natto) belongs to the genus Bacillus of the family Bacillus, and is a production strain of natto, and has a eating history of over 2000. The bacillus natto can decompose macromolecular substances such as protein, carbohydrate, fat and the like in the fermentation process, so that the bacillus natto fermented product contains a large amount of nutritional ingredients such as amino acid, oligosaccharide, organic acid and the like which are easy to digest and absorb by human bodies. Bacillus natto is a "recognized safety" (GRAs) microorganism, and has very practical significance in developing and researching the health care effects of resisting tumor, reducing blood pressure, resisting oxidization and thrombolysis of fermented products of the Bacillus natto.
Disclosure of Invention
In view of this, the present application aims to provide bacillus subtilis natto subspecies to open up the practical application scenes and fields thereof.
In a first aspect, the embodiment of the application discloses a bacillus subtilis natto subspecies which is preserved in China general microbiological culture Collection center (CGMCC) No.21709, named bacillus subtilis Bacillus subtilis subsp.natto R3, a preservation date of 2021, month 25 and a preservation address of North Chen Xilway No.1, no.3 in the Korean region of Beijing city.
In a second aspect, the embodiment of the application discloses application of bacillus subtilis natto subspecies in low-temperature dried products of fermented leeches.
In a third aspect, the embodiment of the application discloses application of bacillus subtilis natto subspecies in preparation of anticoagulant products.
In a fourth aspect, the embodiment of the application discloses application of bacillus subtilis natto subspecies in preparation of antihypertensive products.
In a fifth aspect, the embodiment of the application discloses a method for fermenting leech low-temperature dried product by bacillus subtilis natto subspecies, which comprises the following steps:
preparing a seed culture solution and a fermentation culture solution, wherein the seed culture solution comprises 1-8wt% of leech low-temperature dried product, and the fermentation culture solution comprises 1-5wt% of leech low-temperature dried product;
preparing a seed suspension, wherein the seed suspension is obtained by inoculating the preserved bacillus subtilis natto subspecies into the seed culture solution for culture after strain activation;
obtaining a fermentation liquor, wherein the fermentation liquor is obtained by transferring the seed suspension into the fermentation culture liquor, and carrying out ventilation fermentation under the stirring condition of 150-180 rpm at the temperature of 34-45 ℃, wherein the ratio of air quantity to tank volume is 1: 0.5-1 (v/v.m), fermenting for 36-72 h;
and (3) obtaining a dry product, wherein the dry product is obtained by purifying the fermentation broth.
In the embodiment of the application, the seed culture solution also comprises 0.5-2 g/L natto, 1.5-3 g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCL, and the pH of the seed culture solution is 7.0-7.2;
the fermentation culture solution also comprises 0.5-1.5 g/L natto, 10g/L glucose, 1.5-3 g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCL, and the pH of the fermentation culture solution is 7.0-7.2.
In an embodiment of the present application, the purification treatment comprises:
leaching: adding water with the volume of 5 times into the fermentation liquor, fully leaching for 6-8 hours at the temperature of more than 85 ℃, and centrifuging to obtain supernatant;
ultrafiltration: firstly, treating with a 5KD hollow fiber column, collecting concentrated solution, drying (or freeze-drying) at low temperature to obtain a dried product, and preserving at-20 ℃.
In embodiments of the present application, the dried product comprises an anticoagulant peptide and a antihypertensive peptide.
Compared with the prior art, the application has the following beneficial effects:
according to the embodiment of the application, natto is taken as a screening source, through primary screening, secondary screening and final screening, pulsed light radiation treatment is used in the secondary screening, and through identification, the bacillus subtilis natto subspecies R3 are finally obtained through screening, and through a lysozyme experiment and ACE activity detection, the bacillus subtilis natto subspecies R3 have anticoagulation and ACE enzyme inhibition activities at the same time, while conventional bacillus subtilis natto subspecies do not have the two activities at the same time.
Furthermore, the application also ferments leech low-temperature dried product through R3 strain to obtain a dried product, and the dried product obtained by identifying that the dried product contains two active polypeptides, which respectively and correspondingly generate the effects of anticoagulation and inhibition of ACE enzyme activity, is proved by animal experiments to have the protection effect on ischemia reperfusion brain injury rats and reduce the blood pressure level of SIHR rats. Therefore, the embodiment of the application also provides application prospect of applying the R3 strain to the preparation of anticoagulant products or the preparation of blood pressure reducing products.
Drawings
Fig. 1 is a final screen panel result graph provided in an embodiment of the present application.
FIG. 2 is a 16S rDNA electrophoresis chart of R3 strain provided in the example of the present application, wherein lane 1 is Marker, and lane 2 is 16S rDNA.
Fig. 3 is a Nano-UPLC-MS spectrum of an anticoagulant polypeptide provided in an embodiment of the present application.
Fig. 4 is a Nano-UPLC-MS spectrum of antihypertensive peptides provided in the examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
Screening and identification of bacillus subtilis natto subspecies R3
1. Screening for sample sources
Taking 500g of fresh soybeans, wrapping the fresh soybeans with fresh sun-dried lotus leaves, and fermenting the fresh soybeans in a ventilation place for 15 days in summer to obtain the natto. 0.34-1 g of prepared natto is ground and dissolved in 20ml of L0.75% physiological saline solution, and then the mixture is oscillated for 1h and kept stand, and the supernatant is used as the original bacterial suspension to be screened.
2. Primary screen
Taking original bacteria suspension original times of solution, 10 -1 ～10 -8 Respectively coating the diluted solutions on beef extract protein vein culture medium plates, wherein each treatment is provided with 3 parallel diluted solutions; culturing in a constant temperature incubator at 37 ℃ for 24 hours in an inversion way, observing the growth condition of colonies, selecting a flat plate with uniform colony distribution and colony numbers between 30 and 80, picking single colonies with different colony morphologies for gram staining, and observing under a microscope: the bacillus is repeatedly purified until pure seeds are obtained. 41 single colonies, designated as R1 to R41 in sequence, were obtained by separating and purifying from 7 natto seeds, respectively, according to the above-described method.
3. Double screen
The R1 to R41 obtained by separation are inoculated on a skim milk solid culture medium, and are inversely cultured at 37 ℃ until the colony number is not less than 200, and then the following treatment is carried out:
the plate was put into a pulsed strong light sterilization apparatus FD2000 (Shanghai rayleigh intelligent science and technology limited) to perform pulsed light irradiation treatment, and the conditions of each treatment were: pulse voltage 2220V, pulse number 65 times, and irradiation distance 5cm. The total treatment is 10 times, 1 time is carried out at intervals of 1-2 hours, colonies surviving on the plates after 10 times of treatment are transferred to fresh plates, 3 generations are transferred together, and the strains which can still survive through 3 generations transfer are R2, R3, R9, R15 and R22 finally.
4. Final screen
And respectively inoculating R2, R3, R9, R15 and R22 obtained by re-screening to respective dibbling on an anticoagulant plate, culturing for 24 hours at 37 ℃, observing whether a lysosome ring is generated, and detecting thrombin activity.
Wherein the anticoagulant plate comprises: 3.0g/L beef extract, 10.0g/L peptone, 5.0g/L NaCL, 20g/L agar and 10v/v%20NIH/mL thrombin standard solution; wherein, 20NIH/mL thrombin standard solution was prepared with pH5.0 physiological saline, thrombin formulation was purchased from Sigma-Aldrich under accession number P00734, 150UNITS. The thrombin standard solution should be poured into the plate evenly when the plate is cooled to below 45 ℃ until the plate is completely solidified.
Inoculating R2, R3, R9, R15 and R22 obtained by re-screening into a final-screen liquid culture medium according to an inoculum size of 5wt%, transferring to a 37% shaking table at 120rpm, culturing for 30-35 h, and taking the fermentation broth to perform ACE inhibition as an index for screening. Among them, ACE inhibition activity is referred to below.
Wherein the final screening culture solution comprises 8wt% of leech low-temperature dried product, 2g/L natto, 3g/L skimmed milk powder, 0.5g/L beef extract, 5g/L peptone and 5g/L NaCL, and the pH value is 7.0-7.2.
As a result, as shown in FIG. 1, the plate on which the lysosome ring was produced had only R3, and the strains having ACE inhibition ratios had R2, R3, R9, R15 and R22.
5. ACE inhibition activity assay
The improved Cushman ultraviolet colorimetry is adopted to measure the ACE inhibition rate of the fermentation broth.
Preparation of HIL substrate solution: the ACE substrate Hip-His-Leu (HHT, sigma) was dissolved in borate buffer solution at pH8.3 containing 0.lmol/L NaCL to give a concentration of 5 mmol/L.
Mixing 100 μL of 5.0mmol/L HHT solution and 40 μL of centrifuged scallop skirt fermentation broth, keeping the mixture in a water bath at 37 ℃ for 10min, adding 20 μL of 0.l U/mL ACE enzyme solution, uniformly mixing, and reacting in a constant-temperature water bath at 37 ℃ for 35min.
Taking out from the water bath, adding 200 mu L lmol/L HCL into the reaction system to terminate the reaction, adding 1.2mL of hippuric acid generated by frozen acetic acid and acetic acid extraction, centrifuging at 3500rpm for 5min after vortex shaking and mixing uniformly, sucking 1.0mL of acetic acid and acetic acid layer, drying and cooling in a 90 ℃ oven for 1 hour, adding 4mL of distilled water for full dissolution, and measuring the absorbance at 228nm after vortex mixing. Parallel control except that 200. Mu.L mol/L HCl was added before the reaction to terminate the reaction, the remaining components, the operation steps and the reaction tube were repeated 3 times to average the absorbance values, and then the ACE inhibition ratios of the above-mentioned fermentation broths of R2, R3, R9, R15 and R22 were calculated according to the formula, resulting in fermentation broths of 48h, and ACE inhibition ratios of the fermentation broths of R2, R3, R9, R15 and R22 were 67.5%, 73.2%, 73.1%, 71.9% and 68.3%, respectively.
6. Identification of R3 Strain
Through the steps of the primary screening, the secondary screening and the final screening, only the R3 strain can generate a thrombin ring for thrombin and can also generate an ACE inhibition effect through fermentation, so that the R3 strain obtained through screening is identified.
Extracting genome of strain R3 as template, performing PCR amplification with universal primer (27F: agagttgatctggcgcctcag, shown as SEQ ID NO. 1; 1499 2R: ggttacttgtacgactt, shown as SEQ ID NO. 2), detecting by agarose electrophoresis of 1%, and displaying at 900%
A distinct band is about bp, as shown in FIG. 2, which shows that the 16SrDNA fragment of the genome of the strain has been amplified, and the fragment length obtained by sequencing is 867bp, as shown in SEQ ID NO. 3.
The sequences of strain BN-3 were aligned in NCBI using Blast, and the strains with higher homology all belonged to Bacillus, indicating that the strain R3 selected was Bacillus subtilis, bacillus natto subsp. Bacillus subtilis subsp. The strain R3 is named as Bacillus subtilis subsp.natto R3 and is preserved in China general microbiological culture Collection center (CGMCC) No.21709, and is named as bacillus subtilis Bacillus subtilis subsp.natto R3, the preservation date is 2021 month 01 and 25, and the preservation address is North Chen West Lu No.1 and No.3 in the Korean region of Beijing city.
Low-temperature dried product of bacillus subtilis natto subspecies R3 fermented leech
The embodiment of the application also discloses a method for fermenting leech low-temperature dried product by utilizing the bacillus subtilis subsp natto R3 disclosed in the embodiment. The method specifically comprises the following steps:
1) Preparing a seed culture solution and a fermentation culture solution, wherein the seed culture solution comprises 1-8wt% of leech low-temperature dried product, and the fermentation culture solution comprises 1-5wt% of leech low-temperature dried product;
2) Preparing a seed suspension, wherein the seed suspension is obtained by inoculating the preserved bacillus subtilis natto subspecies into the seed culture solution for culture after strain activation;
3) Obtaining a fermentation liquor, wherein the fermentation liquor is obtained by transferring the seed suspension into the fermentation culture liquor, and carrying out ventilation fermentation under the stirring condition of 150-180 rpm at the temperature of 34-45 ℃, wherein the ratio of air quantity to tank volume is 1:0.5 to 1 (v/v.m) for 36 to 72 hours;
4) And (3) obtaining a dry product, wherein the dry product is obtained by purifying the fermentation broth.
According to the embodiment of the application, the low-temperature dried leech product is fermented by utilizing the bacillus subtilis natto subspecies R3, so that hirudin contained in the low-temperature dried leech product is fully utilized, and the polypeptide with anticoagulation effect is synthesized through biodegradation. In addition, since R3 strain was modified by pulsed light treatment, ACE-inhibiting polypeptides were synthesized. Thus, a dry product is obtained by the fermentation process.
In specific example 1, the preserved strain R3 was transferred to a slant medium, cultured at 37℃for 24 hours, transferred to a seed culture medium, and shake-flask cultured at 150rpm at 37℃for 24 hours. Wherein the seed culture solution comprises 6.5wt% of leech low-temperature dried product, 1.5g/L natto, 2.6g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCL, and the pH of the seed culture solution is 7.0-7.2. Measuring absorbance OD660 of seed cell suspension not lower than 0.8, and detecting viable bacteria by viable bacteria counting method, wherein viable bacteria number is not lower than 10 8 cfu/mL。
Specifically, the seed cell suspension meeting the requirement is transferred into fermentation culture solution, and is aerated and fermented under the stirring condition of 150-180 rpm at 34-45 ℃, the ratio of air quantity to tank volume is 1:0.5-1 (v/v.m), and the fermentation is carried out for 36-72 h. Wherein the fermentation culture solution comprises 3.4wt% of leech low-temperature dried product, 1.5g/L natto, 10g/L glucose, 3g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCL, and the pH of the fermentation culture solution is 7.0-7.2.
Detecting the absorbance OD660 of the fermentation liquid to be not lower than 0.8, and purifying. The purification treatment steps of specific example 1 include:
leaching: adding water with the volume of 5 times into the fermentation liquor, fully leaching for 6-8 hours at the temperature of more than 85 ℃, and centrifuging to obtain supernatant;
ultrafiltration: firstly, treating with a 5KD hollow fiber column, collecting concentrated solution, drying (or freeze-drying) at low temperature to obtain a dried product, and preserving at-20 ℃.
Comparative example 1: for this purpose, the present application also provides a method for fermenting leech low-temperature dried product using existing bacillus subtilis subspecies natto as comparative example 1. The procedure was the same as in example 1 above, except that the strain used for fermentation was Bacillus subtilis subsp.subtilis CICC23916, purchased from China industry microbiological culture Collection center. And leaching and ultrafiltering the fermentation broth to obtain a dry product.
Detection of dried articles
The product was dissolved in water and treated with a 1KD hollow fiber column, and the filtrate and concentrate were collected, respectively. The concentrated solution is dried (or freeze-dried) at low temperature to obtain the component 1. Concentrating the filtrate with hollow fiber column of 3KD, and drying (or lyophilizing) at low temperature to obtain component 2.
The component 1 and the component 2 are respectively purified by Sephadex-G25, and the main component with high absorbance at 215nm is collected, concentrated and frozen so as to carry out Nano-upgrading liquid chromatography-mass spectrometry (Nano-UPLC-MS) identification.
The Nano-UPLC-MS identification method comprises the following steps:
chromatographic conditions: c18 chromatographic column, sample injection volume of 10 μL, column temperature of 30deg.C, flow rate of 300nL/min, and detection wavelength of 215nm. Mobile phase a: containing 100% acetonitrile, mobile phase B: ultrapure water containing 0.1% (V/V) formic acid.
Gradient separation conditions: the mobile phase solution B is increased from 5% to 45% in 0-40 min, increased from 45% to 80% linearly in 40-50 min, maintained at 80% for 5min, and finally reduced to 5% equilibrium for 15min.
Mass spectrometry conditions: nano-scale electrospray ionization ((nano-ESI), positive ion mode scanning, scanning range m/z 350-1800. Primary spectrogram scanning resolution is 120K, secondary spectrogram scanning resolution is 7.5K, cascade fragmentation energy is 30%, and fragmentation mode is high-energy collision fragmentation cleavage HCD.
The mass spectrum is shown in figures 3-4, the molecular weight of Nano-UPLC-MS is obtained from component 1 and component 2, and the amino acid sequence obtained from component 1 is IPRPQSHNDFDFEEIPEEYLQ and the molecular weight is 2637.8, the main component in component 2 is KKPE and the molecular weight is 700.83 Da.
It is understood that the fermented product provided in the embodiments of the present application comprises the two dry products.Component 1 and component 2 Activity detection of polypeptides
Test solution: a20 mg/mL 10mL solution of component 1 was prepared as described above, and a 20mg/mL 10mL solution of component 2 was prepared as described above.
In addition, a dried leech product (available from Siam Ralin Biotechnology Co., ltd.) was prepared as a 20mg/mL 10mL solution as a control.
Fibrinogen 10mg (purchased from Sigma) was weighed and dissolved to 2mL with Tris-HCl; the packed thrombin (Sigma-Aldrich, accession number P0074150 UNITS) was diluted with pure water to 1mL and shaken well to give a thrombin solution with a concentration of 40U/mL; precisely sucking 50 mu L of sample solution, placing into a test tube, adding 100 mu L of fibrinogen solution, shaking uniformly, placing into a 37 ℃ water bath, slowly adding thrombin solution, 5 mu L per minute, shaking while adding, observing coagulation condition until coagulation, recording the volume of consumed thrombin solution, and calculating the activity of a sample according to the following formula, wherein the activity U=C1×vl/C2×V2; wherein U represents U/mg per 1g of thrombin-containing activity unit; c1 represents the active concentration U/mL of thrombin; c2 represents the concentration of the sample solution of 20mg/mL; v1 represents the volume of thrombin consumed, μL; v2 represents the amount of the sample solution added in. Mu.L.
Meanwhile, the test sample is tested for ACE inhibition activity by adopting the ACE inhibition activity test method provided by the embodiment, and the test result is shown in table 1.
TABLE 1
IC of component 2 to be purified 50 Measurement of values: preparing freeze-dried samples (0.1-2.0 mg/mL) with different concentrations, measuring ACE inhibition rate of the samples according to the method provided by the embodiment, making a correlation curve of the logarithmic value of the concentration of the samples to the ACE inhibition rate, and calculating IC of the samples 50 Values.
As shown in table 1, component 1 contains a polypeptide component with a high anticoagulant activity and a molecular weight of 2637.8, and component 2 contains a polypeptide component with a high ACE inhibition rate and a molecular weight of 700.83Da, so that it is proved that the dried product provided in example 1 of the present application contains both polypeptide components, and has both high anticoagulant activity (far higher than that of leech low-temperature dried products) and high ACE inhibition rate. The dried product obtained by fermenting leech low-temperature dried product of Bacillus subtilis subsp.subtitle CICC23916 of comparative example 1 only has anticoagulation activity, but does not have ACE inhibition, and the influence on anticoagulation activity and ACE inhibition is eliminated by detecting other natto powder, glucose and skimmed milk powder in fermentation culture solution.
Animal experiment
To further verify the effect of the dry product obtained by fermentation with R3 bacteria in the examples of the present application, the following will be described in connection with animal experiments.
1. Materials and methods
1.1, laboratory animals and test drugs
120 Wistar rats, each half of which has a weight of 250-280g, were provided by the laboratory animal center of Shandong university, and were assigned the pass number SCXK (Lu) -2003004a.
Test drugs 100mg of example 1, comparative example 1 and dried leech at low temperature were dissolved in 2mL of 0.75% physiological saline, respectively, to give test samples, and Xuesaitong injection (100 mg,2mL, kunqiao group Co., ltd.) was used as positive control 1.
1.2, establishing MCAO model rats
Rat surgical modeling: rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (350 mg/kg body weight), fixed supine, incision in the middle of the neck, incised the skin, blunt-separated layers of tissue, separated right Common Carotid Artery (CCA), internal Carotid Artery (ICA) and External Carotid Artery (ECA), ligated ECA and CCA, and after occlusion of the ICA distal end with a venous clip, an incision was made rapidly at the common carotid artery approximately 0.5cm from the ECA and ICA bifurcation, and a nylon wire (0.32 mm) coated with paraffin at one end was inserted to an insertion depth of 1.85±0.5mm, achieving middle cerebral artery occlusion leading to cerebral ischemia. Ligating the entrance, suturing the incision, and retaining the nylon thread head and tail outside the body. After 1h of ischemia, the rats were again anesthetized, pulling the nylon wire extra-retaining ends to slightly resistance to achieve middle cerebral artery reperfusion. The artificial operation is similar to other model mice in separating common carotid artery, internal carotid artery and external jugular vein, but ligating right common carotid artery CCA, preserving heat of the operated rat, and completing the establishment of MCAO model rat. Neurological examination scores for established MCAO model rats were referenced to the Zea Longa 5 score, which reached 3 or 4 score, indicating successful modeling.
1.3 effects on MCAO rats
And (3) observing neurological symptoms and tissue biochemical index measurement tests: wistar rats were divided into a blank group, a model group and a dosing group. The established MCAO model mice are subjected to intraperitoneal injection, and the test products are prepared by the low-temperature dried products of the example 1, the comparative example 1 and the leech and the positive control product 1. The injection accumulation was 20mg/kg body weight. The blank and model groups were given equal amounts of saline, and the blank group machine was used for normal healthy Wistar rats not subjected to the above-described surgery.
1.4, determination of cerebral infarction scope:
the animals are subjected to head breaking after 1 hour of ischemia reperfusion for 23 hours, are rapidly placed on an ice tray for taking brains, the olfactory bulb, the cerebellum and the low-level brains are removed, are frozen at the temperature of minus 20 ℃ for 20 minutes, are taken out, are uniformly cut into 6 pieces, are placed in a bottle containing TTC, are closed, are incubated in a 37C incubator for 30 minutes, and are transferred into a 4% paraformaldehyde solution for fixation. The non-ischemic portion was stained rose-red, and the ischemic portion was white. After fixing the brain tissue, the ischemic portion was carefully separated and weighed, and the cerebral infarction range (%) =infarct area weight/whole brain weight×100% was determined as follows.
1.5, determination of Biochemical index of brain tissue
The index comprises superoxide dismutase (SOD), malondialdehyde (MDA), lactate Dehydrogenase (LDH), glutathione (GSH), glutathione peroxidase (GSH-PX), nitric Oxide (NO), na + -K + ATPase and Ca + -Mg + -ATPase。
The detection method comprises the following steps: 10% brain homogenate obtained according to the method is centrifuged at 4000rpm for 10min, 20 mu L of supernatant is taken, and the LDH content is measured according to the instructions of an LDH measuring kit (Abcam China); respectively taking 30 mu L, and measuring SOD and GSH content according to the specification of SOD and GSH measuring kit (Abcam China); taking 100 mu L of supernatant, and determining MDA content according to the specification of an MDA determination kit (Beijing Box manufacturing technology Co., ltd.); 200 mu L of supernatant is taken and GSH-PX content is measured according to the specification of GSH-PX measuring kit (Shanghai Shangzun biotechnology Co., ltd.); 500. Mu.L of the supernatant was used to determine the NO content according to the instructions of the NO determination kit (Shanghai Redsi Biotech Co., ltd.).
1.6 establishment of stress-induced hypertension rat (SIHI) animal model
And molding by adopting a high-salt diet composite cold stress method. The Wistar rats were fed with a high-salt rat feed (synergistic) containing 10% sodium chloride and a 0.85% sodium chloride solution every day, and the rats were placed in an exposure box containing water at 5±2 ℃ with a glass plate as the face wall and a certain amount of water every day for 4 hours, and after continuous treatment for 2 weeks, blood pressure of the rats was measured with a BP-6 type animal noninvasive blood pressure test system and continuously measured 3 times, and the average value was taken until the blood pressure reached the highest (compared with normal Wistar rats), and experiments were started after the blood pressure was maintained relatively stable at a high level, and the criterion was that the blood pressure after stress should have a significant difference from the blood pressure before stress (P < 0.01).
1.7 effects in rats greater than SIHI
SIHR rats were randomly divided into dosing and positive control groups and model groups. The administration group administers the test products provided in example 1, comparative example 1 and leech cryogenically dried product of the stomach lavage of SIHR rats, respectively, at a dose of 20mg/kg body weight. SIHR rats were dosed with gavage irbesartan (Sainofil France) at a dose of 16.5mg/kg. The model group is SIHR rats, and no test sample is given. Normal Wistar rats were also established as a blank. The administration was stopped for 4 consecutive weeks, and after the rat blood pressure was stabilized, the rat tail artery blood pressure was measured by indirect pressure measurement and the measurement result was recorded.
1.8 data processing
The experimental data are subjected to data analysis by Excel 2013 and SPSS 22.0 statistical software for statistical arrangement, each data is measured for a plurality of times and represented by an average value and a standard deviation thereof, and single-factor analysis of variance (One-way ANOVA) and DunCan's multiple comparison are respectively carried out by SPSS 22.0, and a significant difference mark is carried out.
2. Results
TABLE 2
As shown in table 2, the brain tissue of the rats in the blank group is normal, and the rats in the model group and the administration group have different degrees of infarct, and the cerebral infarction range of the rats in the administration group is obviously reduced relative to that of the model group after the administration of the test sample, wherein the test sample provided in example 1, namely the dried product obtained by fermenting the leech low-temperature dried product by adopting the R3 strain disclosed in the embodiment of the application, has the most obvious effect of reducing the infarct degree of the rats.
TABLE 3 Table 3
As shown in tables 2 and 3, after the rats are refilled by cerebral middle artery ischemia, MDA, GSH, SOD, GSH-PX, LDH and NO in brain tissues are obviously changed, LDH, MDA and NO contents of rats in a model group are obviously higher than those of rats in a pseudo-blank group, and SOD, GSH and GSH-PX are obviously lower than those of rats in a blank group. In the dosing group, the test article provided in example 1 significantly reduced LDH, MDA and NO levels in rat brain tissue, while SOD, GSH and GSH-PX levels were significantly increased after dosing the model rats, and wherein SOD and GSH-PX levels were nearly restored to be comparable to the blank group. In the administration group, after the test sample provided by the low-temperature dried leech products of comparative example 1 is administered to the model rat, the MDA, GSH, SOD, GSH-PX, LDH and NO contents in the brain tissue of the rat are partially changed, but the normal level is difficult to recover. Moreover, the test samples provided by the low-temperature dried leech products in comparative example 1 hardly significantly change the SOD, GSH-PX and LDH contents in brain tissues of the model rats. From this, it is shown that the test product provided in example 1 of the present application, namely, the dried product obtained by fermenting leech low-temperature dried product with the R3 strain disclosed in the examples of the present application, has a protective effect against acidosis and free radical damage to brain tissue of model rats.
TABLE 4 Table 4
As is clear from Table 4, na in brain tissue after reperfusion of rat by middle cerebral artery ischemia + -K + ATPase and Ca 2+ -Mg 2 + ATPase activity was evident in the blank. In the administration group, after the test sample administration model rats provided in example 1, na in brain tissue of the rats + -K + ATPase and Ca 2+ -Mg 2+ ATPase activity was significantly elevated in rats from the model group and significantly higher than in the positive control group. In the administration group, after the test sample provided by the low-temperature dried leech products of comparative example 1 is administered to the model rat, na in the brain tissue of the rat + -K + ATPase and Ca 2+ -Mg 2+ ATPase activity was not significantly altered from model rats. Thus, comparative example 1 and dried leech at low temperature provided a test pair greater than Na + -K + ATPase and Ca 2+ -Mg 2+ The ATPase ischemic injury has no obvious effect, and the dry product obtained by fermenting leech low-temperature dry product by using the R3 strain disclosed in the example of the application can provide obvious improvement.
TABLE 5
As can be seen from table 5, the stable systolic pressure and diastolic pressure of the established SIHR model rats are significantly higher than those of the blank group, which indicates that the modeling was successful, while the positive control group significantly reduced both the diastolic pressure and the systolic pressure after the irbesartan was given to the SIHR model rats, which did play a role in reducing blood pressure, but the blood pressure of the rats was even lower than that of normal Wistar rats, and the rats may have side effects of hypotension. In the administration group, the diastolic pressure and the systolic pressure of the SIHR model rats administered with the dried product obtained by fermenting the leech low-temperature dried product by the R3 strain disclosed in the embodiment of the application provided in the embodiment 1 are obviously reduced, and the diastolic pressure and the systolic pressure of the SIHR model rats are equivalent to the blood pressure level of normal Wistar rats in the blank group, which indicates that the dried product obtained by fermenting the leech low-temperature dried product by the R3 strain disclosed in the embodiment of the application has the effect of reducing blood pressure of the rats and has no obvious hypotensive side effect. In the administration group, the test samples provided by the low-temperature dried leech products of comparative example 1 do not have obvious blood pressure reducing effect after being administered to SIHR model rats.
In summary, the natto of example 1 of the present application was used as a screening source, and through a primary screening, a secondary screening and a final screening, and pulse light irradiation treatment was used in the secondary screening, and through identification, a bacillus subtilis natto subspecies R3 was finally obtained by screening, and through a lysozyme experiment and ACE activity detection, it was found that it had both anticoagulation and inhibition of ACE enzyme activity, whereas the conventional bacillus subtilis natto subspecies (comparative example 1) did not have both activities.
Furthermore, the application also ferments leech low-temperature dried product through R3 strain to obtain a dried product, and the dried product obtained by identifying that the dried product contains two active polypeptides, which respectively and correspondingly generate the effects of anticoagulation and inhibition of ACE enzyme activity, is proved by animal experiments to have the protection effect on ischemia reperfusion brain injury rats and reduce the blood pressure level of SIHR rats. Therefore, the embodiment of the application also provides application prospect of applying the R3 strain to the preparation of anticoagulant products or the preparation of blood pressure reducing products.
While the invention has been described with respect to the preferred embodiments, it is to be understood that the scope of the invention is not limited thereto, and that any person skilled in the art will appreciate that the invention may be practiced with other modifications and variations as fall within the spirit and scope of the appended claims,
modifications and substitutions that can be easily conceived are intended to be encompassed within the scope of the present application.
Claims (7)
1. A antihypertensive peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 5.
2. The method for producing antihypertensive peptide of claim 1.
3. The method for producing antihypertensive peptide according to claim 2, wherein the antihypertensive peptide is prepared by fermentation using bacillus subtilis subspecies natto (Bacillus subtilis subsp. Natto) R3, wherein the date of preservation of bacillus subtilis subspecies natto R3 is 2021 month 01, and the preservation address is 1 st-day western road 1, no.3 in the morning sun area of beijing city.
4. A method for producing antihypertensive peptides according to claim 3, comprising the steps of:
preparing a seed culture solution and a fermentation culture solution, wherein the seed culture solution comprises 1-8wt% of leech low-temperature dried product, and the fermentation culture solution comprises 1-5wt% of leech low-temperature dried product;
preparing a seed suspension, wherein the seed suspension is obtained by inoculating the preserved bacillus subtilis natto subspecies according to claim 1 into the seed culture solution for culture after strain activation;
the fermentation liquor is obtained by transferring the seed suspension into the fermentation culture liquor, ventilating and fermenting under the stirring condition of 150-180 rpm at 34-45 ℃, wherein the ratio of air quantity to tank volume is 1:0.5-1 (v/v.m), and fermenting for 36-72 h;
and (3) obtaining a dry product, wherein the dry product is obtained by purifying the fermentation broth.
5. The method according to claim 4, wherein the seed culture solution further comprises 0.5 to 2g/L natto, 1.5 to 3g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCl, and the pH of the seed culture solution is 7.0 to 7.2;
the fermentation culture solution also comprises 0.5-1.5 g/L natto, 10g/L glucose, 1.5-3 g/L skimmed milk powder, 0.5g/L beef extract, 15g/L peptone and 5g/L NaCL, and the pH of the fermentation culture solution is 7.0-7.2.
6. The method of claim 5, wherein the purification treatment comprises:
leaching: adding water with the volume of 5 times into the fermentation liquor, fully leaching for 6-8 hours at the temperature of more than 85 ℃, and centrifuging to obtain supernatant;
ultrafiltration: firstly, treating with a 5KD hollow fiber column, collecting concentrated solution, drying at low temperature or lyophilizing to obtain a dried product, and preserving at-20deg.C.
7. The use of a antihypertensive peptide of claim 1 in a antihypertensive preparation.
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