CN111363006B - Ganoderma lucidum mycelium antihypertensive peptide and preparation method thereof - Google Patents
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- CN111363006B CN111363006B CN202010047485.5A CN202010047485A CN111363006B CN 111363006 B CN111363006 B CN 111363006B CN 202010047485 A CN202010047485 A CN 202010047485A CN 111363006 B CN111363006 B CN 111363006B
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Abstract
The invention discloses a ganoderma lucidum mycelium antihypertensive peptide and a preparation method thereof, belonging to the technical fields of polypeptide biotechnology and antihypertensive drugs. The antihypertensive peptide has good in vivo and in vitro antihypertensive activity, low molecular weight and easy absorption by human body, and can be used as a health food additive or an antihypertensive drug; the ganoderma lucidum strain SCIM 1006 of the invention is Chinese ganoderma lucidum mycelium capable of producing antihypertensive peptide Ser-Tyr-Pro, and the natural polypeptide yield is improved to 1.52g/L by adding exogenous amino acid in a liquid fermentation culture medium and combining a low-temperature low-rotation-speed fermentation technology; meanwhile, the method for extracting and purifying the antihypertensive peptide is simple to operate, is rapid and convenient, and is easy for industrial production.
Description
Technical Field
The invention relates to a ganoderma lucidum mycelium antihypertensive peptide and a preparation method thereof, belonging to the technical field of polypeptide biotechnology and antihypertensive drugs.
Background
Hypertension is one of the main diseases threatening human health, and is a common cardiovascular and cerebrovascular disease in life. With the development of modern society and the improvement of the living standard of people, the incidence rate of hypertensive cardiovascular and cerebrovascular diseases is greatly increased and the trend is more towards the youthfulness. According to the statistics of '2018 world health statistics report', the death rate of cerebrovascular diseases is the first in non-infectious disease centers, the number of deaths reaches 1790 thousands, and the number of hypertension patients in China is more than 3 hundred million. However, some of the antihypertensive synthetic drugs such as captopril, ramipril and lisinopril have side effects such as fatigue, nausea, severe cough and pruritus in clinical application. Therefore, a blood pressure lowering medicine which is relatively safe, efficient and free of toxic and side effects is urgently sought. In recent years, active peptides derived from degradation products of food proteins have attracted attention for their advantages such as high efficiency, safety, and easy absorption by the human body.
Angiotensin I converting enzyme (ACE) belongs to vascular endothelial cell membrane binding enzyme, catalyzes Angiotensin I to Angiotensin II to cause blood pressure rise, and is an ideal target for treating hypertension. ACE inhibitory peptides are small molecular substances with potential blood pressure lowering effect, and can inhibit ACE enzyme activity, reduce damage of bradykinin and reduce degradation of angiotensin I. ACE inhibitory peptides obtained from animal products, marine organisms and plants (from animal products, marine organisms and plants, and plants-a review. food chemistry,2017,228: 506-. Furthermore, it is now common to employ directed enzymatic techniques to degrade food-derived proteins to produce ACE inhibitory peptides. However, the enzymatic hydrolysis product obtained by the directional enzymatic hydrolysis technology has relatively complex components and difficult subsequent purification; the enzymolysis conditions (enzyme adding amount, temperature, pH and the like) of the protease need to be strictly controlled in the enzymolysis process, more 'material resources + energy sources + manpower' are consumed, and the production cost is high, so that the development of large-scale production of the ACE inhibitory peptide is restricted. At present, few foreign researchers find natural ACE inhibitory peptides from traditional fermentation products, and open up a new idea for preparing the natural ACE inhibitory peptides by using microorganisms.
The ACE inhibitory peptides reported at present comprise peptide substances from different sources, such as cuttlefish muscle protein Val-Glu-Leu-Tyr-Pro, egg His-Leu-Phe-Gly-Pro-Gly-Lys-Lys-Asp-Pro-Val, silkworm pupa Gly-Ala-Met-Val-His, peanut Tyr-Leu-Val-Arg and the like, have different molecular weights, generally consist of 4-12 amino acid residues, have strong ACE inhibitory activity in vitro, but most researchers do not verify the in vivo blood pressure reducing effect of the researchers, and the in vivo blood pressure reducing mechanism is still unclear.
Ganoderma belongs to fungi, has high pharmacological value, and has effects of resisting tumor, regulating immunity, protecting liver, and lowering blood pressure. Triterpenoids and polysaccharides are two main active ingredients of ganoderma lucidum, and reports on ganoderma lucidum active oligopeptides are few. The cultivation period of the ganoderma lucidum fruiting body is long (4-9 months), and the ganoderma lucidum fruiting body is easily influenced by seasons and environments, so that the biological stability of active substances of the fruiting body is poor, and the method for preparing ganoderma lucidum mycelia to obtain the active substances by separating ganoderma lucidum cells and utilizing a liquid fermentation technology is a more effective means. Therefore, the ganoderma lucidum mycelia serving as a new resource for preparing the active peptide has important practical significance for development and utilization. In the previous research process, 3 ACE inhibitory peptides are obtained from ganoderma lucidum mycelia by an inventor team, the amino acid sequences of the ACE inhibitory peptides are Gln-Leu-Asp-Leu, Gln-Leu-Val-Pro and Gln-Asp-Val-Leu, and the ACE inhibitory IC50 of the ACE inhibitory peptides are 21.0 mu mol/L, 24.6 mu mol/L and 23.8 mu mol/L respectively. However, there is still a great need to develop more ACE inhibiting peptides, as well as more active ACE inhibiting peptides.
Disclosure of Invention
In order to solve at least one technical problem, the invention separates a new Ganoderma lucidum strain SCIM 1006 which can produce novel antihypertensive peptide and is identified as Chinese Ganoderma lucidum (Ganoderma lingzhi), and carries out structural and functional verification on the produced antihypertensive peptide; compared with the ACE inhibitory peptides reported at present, the ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro obtained by the invention has stronger ACE inhibitory activity, IC50 is 52.5 mu g/mL, the molecular weight is only 366Da, and the peptide can be easily absorbed by human bodies when entering intestinal tracts; meanwhile, the antihypertensive drug has a good in-vivo antihypertensive effect, and has an effect of reducing both arterial systolic pressure and systolic pressure by blocking an ACE-Ang I-AT1R pathway to exert the antihypertensive effect.
On the basis, a method for separating and preparing the high-efficiency antihypertensive polypeptide from the Chinese ganoderma lucidum mycelia is obtained through a large number of grope experiments. The method comprises the steps of adding 17 mixed amino acids to stimulate ganoderma lucidum mycelia to generate polypeptide, performing ultrafiltration by adopting a 3kDa membrane to prepare a pure natural, low-molecular-weight and high-activity ganoderma lucidum mycelia antihypertensive peptide crude product, and finally performing high-purification on the antihypertensive peptide by utilizing a high performance liquid chromatography technology to prepare a pure natural, low-molecular-weight, high-activity and high-purity ganoderma lucidum mycelia antihypertensive peptide product.
The first purpose of the invention is to provide a peptide, wherein the amino acid sequence of the peptide is Ser-Tyr-Pro (shown as SEQ ID NO: 1).
The peptide is antihypertensive peptide, and belongs to competitive inhibitory peptide for inhibiting ACE activity. The compound has the advantages that a salt bond is formed by Ser and Lys472 of an ACE active area, three hydrogen bonds are formed by Ser and Lys472, Tyr and Gln242 and Pro and Lys415 of the ACE active area, and hydrophobic effects are generated by Tyr, Pro and Tyr484 and Phe488 of the ACE active area, so that the active area of ACE is occupied, and the compound cannot act on a substrate to be inactivated.
The antihypertensive peptide can be used for remarkably reducing the arterial diastolic pressure through intraperitoneal injection and intravenous injection; in vivo, angiotensin I induces eNOS phosphorylation of vascular endothelial cells, inhibits endothelin-1 expression, and exerts hypotensive activity.
The second purpose of the invention is to provide a Ganoderma lucidum strain SCIM 1006(Ganoderma lingzhi), which is preserved in China general microbiological culture Collection center (CGMCC) in 11 and 25 days in 2019, with the preservation number of CGMCC No.18819 and the preservation address of No. 3 Hospital No.1 of North Chen West Lu of the sunward area in Beijing.
The ganoderma lucidum strain SCIM 1006 can produce antihypertensive peptide with the amino acid sequence of Ser-Tyr-Pro.
It is a third object of the present invention to provide a method for producing the peptide.
In one embodiment, the method is produced by artificial synthesis or by biological fermentation or by enzymatic hydrolysis.
In one embodiment, the method comprises fermentative production using the Ganoderma strain SCIM 1006 of the present invention.
In one embodiment, the fermentation uses a medium,contains components required for normally culturing Ganoderma. For example, the catalyst contains 0.5 to 0.6% NH4Cl, 4.1-4.2% soluble starch, 0.005% vitamin B1, 0.15% K2HPO4。
In one embodiment, the fermentative production further comprises utilizing a fermentation medium comprising mixed amino acids.
In one embodiment, the mixed amino acids in the fermentation medium include two or more of the following amino acids: ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg, Ala.
In one embodiment, the mixed amino acids in the fermentation medium are a mixture of 17 amino acids: ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg, Ala.
In one embodiment, the fermentation medium has a single amino acid added in an amount of 0.02g/L or more.
In one embodiment, the method of preparing the fermentation medium comprises: dissolving the components of the culture medium except amino acid in water, adjusting the pH to 6.8-7.0, and sterilizing; preparing amino acid into high concentration mother liquor, filtering with 0.22 μm filter membrane in sterile environment according to the addition amount of final concentration of above 0.02g/L, and adding into sterilized solution.
In one embodiment, the fermentation production is to inoculate the mycelium seed liquid of the ganoderma lucidum strain SCIM 1006 into a fermentation medium in an inoculation amount of 10-15% for fermentation.
In one embodiment, the fermentation is performed at 20-28 ℃ and 140r/min for 6-8 days.
In one embodiment, the fermentation is carried out at a low temperature and a low rotation speed of 120r/min at a temperature of 24 ℃.
In one embodiment, the fermentative production is, in particular:
(1) inoculating Ganoderma lucidum strain SCIM 1006(Ganoderma lingzhi) to KH strain containing glucose as carbon source, peptone and yeast extract as nitrogen source2PO4And MgSO4In the seed culture medium which is a mineral element, the seed culture medium comprises the following components: 4-4.6% of glucose, 0.3-0.5% of peptone, 0.2-0.3% of yeast extract and KH2PO40.15％，MgSO40.05 percent; then fermenting for 7-9 days in a shake flask with the temperature of 28-30 ℃ and the rotating speed of 150-180 r/min to obtain ganoderma lucidum seed liquid;
(2) preparing ganoderma lucidum seed liquid into inoculation liquid with the thallus concentration of 1.7-2.0 g/L, inoculating the inoculation liquid into a liquid fermentation culture medium according to the volume ratio of 12-15%, and performing submerged fermentation for 6-8 days at the temperature of 27-30 ℃ and the rotating speed of 160-190 r/min; wherein the liquid fermentation medium contains 3.9-4.3% soluble starch and 0.5-0.7% NH4Cl, 0.005% vitamin B1, 0.15% K2HPO4And mixed amino acids.
The fourth purpose of the invention is to provide a method for extracting and purifying the peptide from the ganoderma lucidum fermentation liquor. The Ganoderma fermentation broth is fermentation broth of Ganoderma strain SCIM 1006.
The method comprises the following steps: extracting and separating the antihypertensive peptide in the ganoderma lucidum mycelia by using a homogenate buffer solution and an ultrafiltration membrane; purifying by high performance liquid chromatography to obtain Ganoderma mycelium antihypertensive peptide pure product.
The extraction: collecting Ganoderma fermentation broth, collecting mycelium, pouring liquid nitrogen, grinding to fine particle shape, transferring into precooled homogenate buffer solution, homogenizing on ice bath, and centrifuging to obtain supernatant; collecting ultrafiltrate with molecular weight less than 3kDa from the supernatant, and removing homogenate buffer solution in the filtrate to obtain the crude product of the ganoderma lucidum mycelium antihypertensive peptide.
In one embodiment, the homogenization buffer is 10mM Tris-HCl buffer, pH 7.0, containing 2mM EDTA, 1% enzyme inhibitor Roche, 10mM beta-mercaptoethanol, 5-10% glycerol; the mixture was kept in a refrigerator at 4 ℃ for 2 hours before use.
In one embodiment, the tissue is homogenized for 5s per 15s to prevent degradation of the target peptide as much as possible.
And (3) purifying: dissolving the crude product of the ganoderma lucidum mycelium antihypertensive peptide in acetonitrile water solution, loading the solution to a C18 solid phase extraction column, collecting the eluted adsorption part after flowing and eluting, removing acetonitrile, and then loading the solution to RP-HPLC for further purification.
In one embodiment, the purifying comprises: dissolving the crude product of the ganoderma lucidum mycelium antihypertensive peptide in 10% acetonitrile water solution, loading the crude product to a C18 solid phase extraction column, washing the crude product with 10% acetonitrile water solution, eluting the crude product with 70% acetonitrile water solution, collecting the eluted adsorption part, concentrating the adsorption part at low temperature in vacuum to remove acetonitrile, and loading the sample to RP-HPLC for further purification.
In one embodiment, the chromatographic conditions for RP-HPLC are: chromatographic column Znertsil ODS-3 (4.6X 250mm), flow rate 1.0mL/min, detection wavelength 220nm, mobile phase of acetonitrile and ultrapure water, 0-3min, 15% acetonitrile; 3-51min, 15-55% acetonitrile. Collecting the components with retention time of 8-9 min and purity of more than 95%, namely the pure product of the ganoderma lucidum mycelium antihypertensive peptide.
The fifth purpose of the invention is to provide a medicine with the function of reducing blood pressure, wherein the medicine contains the peptide with the amino acid sequence of Ser-Tyr-Pro or the ganoderma lucidum strain SCIM 1006 of the invention.
In one embodiment, the drug contains a peptide with an amino acid sequence of Ser-Tyr-Pro or the Ganoderma lucidum strain SCIM 1006 of the present invention as a main active ingredient.
In one embodiment, the drug is a drug having an effect of reducing arterial diastolic pressure.
In one embodiment, the medicament further comprises adjuvants required for preparing the medicament.
In one embodiment, the medicament can be a pharmaceutical composition, and can be prepared into capsules, tablets, granules, pills, powder or oral liquid which are commonly used in clinic according to a conventional preparation process.
It is a sixth object of the present invention to provide a method for regulating the expression of eNOS and endothelin-1, which comprises using a peptide having an amino acid sequence of Ser-Tyr-Pro or a strain having an amino acid sequence of Ser-Tyr-Pro for diagnosis and treatment of non-diseases.
In one embodiment, the method utilizes Ser-Tyr-Pro to upregulate eNOS phosphorylation levels or to inhibit endothelin-1 protein expression levels.
The invention also claims a nucleotide sequence for coding the Ser-Tyr-Pro peptide, a carrier or a recombinant bacterium for expressing the Ser-Tyr-Pro peptide, and an arbitrary kit containing the Ser-Tyr-Pro peptide or the ganoderma lucidum strain SCIM 1006 capable of producing the Ser-Tyr-Pro peptide.
The invention has the beneficial effects that:
compared with the existing active peptide, the antihypertensive peptide has the following advantages: 1) the antihypertensive peptide has good in vivo and in vitro antihypertensive activity, exerts the antihypertensive effect by blocking an ACE-Ang I-AT1R channel, and has effects on reducing both arterial systolic pressure and systolic pressure, wherein the effect on reducing diastolic pressure is better; 2) the antihypertensive peptide has the characteristics of low molecular weight and easy absorption by human body, and the pure product Ser-Tyr-Pro has the molecular weight of only 366Da and can be directly absorbed after entering intestinal tracts; 3) the crude product and pure product of the antihypertensive peptide can be used as health food additive or antihypertensive drug;
the Ganoderma lucidum strain SCIM 1006 of the invention is Chinese Ganoderma lucidum (Ganoderma lingzhi) mycelium which can produce antihypertensive peptide Ser-Tyr-Pro.
Compared with the prior art, the method for preparing the antihypertensive peptide by fermenting the mycelium of the ganoderma lucidum SCIM 1006 has the following advantages: 1) the fermentation technology is strong in pertinence, and particularly aims at the modern fermentation technology of the ganoderma lucidum mycelium antihypertensive peptide, but not the traditional ganoderma lucidum mycelium fermentation technology; 2) by adding exogenous amino acid into the liquid fermentation medium and combining with a low-temperature low-rotation-speed fermentation technology, the yield of the natural polypeptide of the ganoderma lucidum mycelia is improved to 1.52g/L, which is far higher than the yield of the traditional fermentation technology without adding exogenous amino acid.
According to the extraction and purification method of the antihypertensive peptide, the reagents such as EDTA, enzyme inhibitor Roche, beta-mercaptoethanol, glycerol and the like are added into the homogenate buffer solution in the extraction and separation process of the antihypertensive peptide from the ganoderma lucidum mycelia, so that the endogenous protease is prevented from degrading the target peptide, the product quality is ensured, and the proportion of the polypeptide with the molecular weight of less than 2kDa in the obtained crude product accounts for 86.8%; the ganoderma lucidum mycelium antihypertensive peptide is purified by adopting a solid phase extraction column and an RP-HPLC two-step chromatography, the purity is over 95 percent, the operation is simple, the method is rapid and convenient, and the industrial production is easy to realize.
Biological material preservation information
A Ganoderma strain SCIM 1006 is classified and named as Ganoderma Ganoderma lingzhi, and has been preserved in China general microbiological culture Collection center (CGMCC) in 11 month and 25 days 2019, with the preservation number of CGMCC No.18819, and the preservation address of No. 3 Hospital No.1 of Beijing Kogyo-Hongyao.
Drawings
FIG. 1 is the Ganoderma strain SCIM 1006ITS sequence.
FIG. 2 is a molecular phylogenetic tree of Ganoderma strain SCIM 1006.
FIG. 3 is an RP-HPLC chromatogram of antihypertensive peptide from ganoderma lucidum mycelia; collecting component (a); ACE inhibition assay (b).
FIG. 4 shows the ACE inhibitory mechanism of the peptide antihypertensive from mycelia of Ganoderma lucidum; wherein,
(a) competitive inhibition model of the antihypertensive peptide (100. mu.g/mL: (●)，50μg/mL(○) And 0 μ g/mL (. tangle-solidup)); (b) steady state simulation analysis of the antihypertensive peptide (top left diagram: energy minimization of potential energy analysis; top right diagram: temperature analysis NVT; bottom left diagram: density; bottom right diagram: complexity of the antihypertensive peptide-ACE complex and protein scaffold RMSD analysis of NPT); (c) simulation of molecular dynamics of the binding of antihypertensive peptides to ACE.
FIG. 5 shows the gastrointestinal simulated digestion analysis of the peptide of ganoderma lucidum mycelium. The antihypertensive peptide (1mg/mL) is digested at 37 deg.C for 30min in a digestion solution containing pepsin (0.5%, w/w) and having a pH of 2.0, the pH of the digestion solution is further adjusted to 7.5 with NaOH, and pancreatin (0.5%, w/w) is added to digest at 37 deg.C for 90 min.
FIG. 6 is the evaluation of the in vivo blood pressure lowering effect of ganoderma lucidum mycelium antihypertensive peptide; wherein,
(a) arterial systolic pressure changes before and after administration; (b) arterial diastolic pressure changes before and after administration. Intraperitoneal injection at 10mg/kg body weight dose: (○) Intravenous injection of (A)●) And control (. DELTA.) and arterial blood pressure was measured 0-8h after administration.
FIG. 7 is the in vitro blood pressure lowering activity assay of Ganoderma lucidum mycelia; wherein,
(a) mRNA expression level of eNOS; (b) mRNA expression level of endothelin-1; (c) phosphorylated p-eNOS protein expression levels; (d) endothelin-1 protein expression levels; (e) the expression of p-eNOS and endothelin-1 proteins was analyzed by the Wester-blot technique.
Detailed Description
1. ACE inhibitory activity assay method:
mu.L of substrate HHL solution (100mmol/L borate buffer, pH8.3, containing 0.3mol/L sodium chloride) was added to 500. mu.L centrifuge tubes, 50. mu.L of the sample was again preheated at 37 ℃ for 5min, 50. mu.L of ACE solution was added to initiate the reaction, and the reaction was carried out in a 37 ℃ water bath for 60 min. After the reaction, 125. mu.L of 1mol/L hydrochloric acid was added to terminate the reaction, and 750. mu.L of ethyl acetate was further added thereto and mixed well. After centrifugation at 1000g for 5min, 500. mu.L of the upper ethyl acetate extract was taken up in a clean centrifuge tube, ethyl acetate was evaporated by a vacuum concentration apparatus, 1.5mL of deionized water was added to dissolve the extracted hippuric acid sufficiently, and the absorbance was measured at 228nm UV wavelength. Wherein, the control group uses buffer solution to replace the sample, and the blank group uses buffer solution to replace ACE. ACE inhibition was calculated as follows:
ACE inhibition ratio (%) ═ a1–A2)/(A1-A0)×100
In the formula: a. the1Measuring absorbance values for the control group;
A2measuring absorbance values for the experimental groups;
A0measuring absorbance values for the blank set;
IC50the concentration of the sample corresponding to 50% ACE inhibition, i.e. IC, is expressed50The smaller the value, the stronger the ACE inhibitory activity of this sample. Preparing sample solutions with different concentrations, drawing a smooth curve by taking the sample concentration as a horizontal coordinate and the ACE inhibition rate as a vertical coordinate, and calculating IC according to the curve50。
2. ESI-MS identification method:
the lyophilized samples were dissolved in an aqueous solution containing 1% acetic acid and 50% methanol and mass spectrometric identification of the amino acid sequence of the antihypertensive peptide in the purified fraction of ganoderma SCIM 1006 mycelia was performed by a HESI-II ESI probe using LTQ Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Sample loading, experimental setup and data collection were performed using LTQ Tune software operations and data was analyzed using Xcalibur software. The mass spectrum conditions were set as: setting the injection pump to be in an off-line sample loading mode, wherein the flow rate is 2.5-15.0 mu L/min; the temperatures of the heat source heater and the capillary tube are respectively 60 ℃ and 300 ℃; the gas flow of the sheath with the aux/gas sweeper is 5-10 arb; the positive detection mode is adopted, and the spraying voltage is 3.5 kV. And (3) carrying out collision induced dissociation on the target peptide ions, wherein the collision energy is 28-35%, the activation Q value is 0.25, and the activation time is 10 ms. The amino acid sequence of the target peptide is mainly resolved by tracing a b-type cleavage pathway, and is cross-validated with all the obtained major product ions.
The following examples are intended to further illustrate the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims appended hereto.
Example 1: screening of Ganoderma strains and discovery of functional peptides
1. Screening of Ganoderma strains
In places such as shrubs, fallen leaves or weeds and the like which are less affected by human activities and have more humus in the deep mountains of the Yuenu mountain of Changsha city in Hunan province, fungus fruiting bodies of suspected lucid ganoderma in shapes are searched, packaged by sterile sealed bags and immediately brought back to a laboratory. Washing collected fungus fruiting bodies with clear water to remove silt and dirt, wiping water with gauze, wiping 75% alcohol to sterilize the surfaces, longitudinally cutting and tearing the middle parts of the mushroom stems of the fruiting bodies with a scalpel, picking a small tissue at the junction of the mushroom cap and the mushroom stems, transferring the small tissue to a PDA slant culture medium, plugging a cotton plug, carrying out standing culture at 25 ℃, grinding 0.5g of each mycelium in liquid nitrogen in a mortar when the mycelium is fully paved on the whole slant, adding 4 times of volume (v/w) of pure water tissue to homogenate, centrifuging (4 ℃, 12000g) for 20min, and taking supernatant to respectively measure ACE inhibitory activity and polypeptide concentration. Wherein, the ACE inhibition ratio of the water extract of the ganoderma lucidum strain SCIM 1006 is measured to be 46.85%, and the polypeptide concentration is 1.52 mg/mL.
In addition, 0.5g of each hyphae was ground in a mortar with liquid nitrogen for genomic DNA extraction, and 600. mu.L of TE, 250. mu.L of 10% SDS, and 3. mu.L of 20 ng/. mu.L proteinase K were added in this order, followed by water bath at 37 ℃ for 1 hour. Subsequently 150. mu.L of 5mol/L NaCl and then 150. mu.L of 2% CTAB were added and water bath was carried out at 65 ℃ for 20 min. Centrifuging at high speed for 20min, collecting supernatant, adding equal volume of isopropanol, and standing at room temperature for 30 min. Centrifuging at high speed for 10min, and adding 750 μ L70% ethanol into the precipitate. Centrifuging at high speed for 2min, collecting precipitate, adding 30 μ L pure water, and dissolving at 4 deg.C overnight. The ITS universal primer is adopted to carry out PCR amplification on DNA of each strain, the amplification product is sent to Beijing Huada Gene company for sequencing, and homologous sequence alignment (BLAST) is carried out in a GenBank nucleic acid sequence database to determine the species.
As can be seen from FIG. 1, the molecular weight of the PCR amplified product of the Ganoderma strain SCIM 1006 is 621bp (the sequence is shown as SEQ ID NO:2), and the sequence is submitted to the GenBank nucleic acid sequence database for sequence alignment, so that the strain has 100% sequence similarity with G.lingzhi sp.nov. (EastAsia), and has relatively low homology with G.multipileum (tropicalAsia) and other strains. Thus, the ganoderma strain SCIM 1006 genus was identified as g.
The ITS sequence of Ganoderma strain SCIM 1006 is as follows:
GGGTGACCTGCGGAAGGATCATTATCGAGTTTTGACCGGGTTGTAGCTGGCCTTCCGAGGCATGTGCACGCCCTGCTCATCCACTCTACACCTGTGCACTTACTGTGGGCTTCAGATTGCGAGGCACGCTCTTTACCGGGCTTGCGGAGCATATCTGTGCCTGCGTTTATCACAAACTCTATAAAGTAACAGAATGTGTATTGCGATGTAACACATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATCTTCAACCTACAAGCTTTTGTGGTTTGTAGGCTTGGACTTGGAGGCTTGTCGGCCGTTATCGGTCGGCTCCTCTTAAATGCATTAGCTTGGTTCCTTGCGGATCGGCTCTCGGTGTGATAATGTCTACGCCGCGACCGTGAAGCGTTTGGCGAGCTTCTAACCGTCTTATAAGACAGCTTTATGACCTCTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATA。
the Ganoderma lucidum strain SCIM 1006 is classified and named as Ganoderma lucidum Ganoderma lingzhi, and is preserved in China general microbiological culture Collection center (CGMCC) in 11 month and 25 days 2019, the preservation number is CGMCC No.18819, and the preservation address is No. 3 of Beijing city Shangyang district Beichen West Lu No. 1.
2. Fermentation culture of ganoderma lucidum mycelia
The strains used were: the Ganoderma strain SCIM 1006 has a preservation number of CGMCC No. 18819.
Preparing a fermentation medium, wherein the formula of the culture medium is as follows: NH (NH)4Cl of 0.5 percent, 17 mixed amino acids (Ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg and Ala, the addition amount of each amino acid is 0.02g/L, the addition amount of soluble starch is 4.1 percent, the addition amount of vitamin B1 is 0.005 percent, and the addition amount of K is 4.1 percent2HPO4Is 0.15%. The fermentation medium ingredients except amino acids were dissolved in distilled water, the pH was adjusted to 7.0, and autoclaved at 121 ℃ for 30 min. The 17 kinds of mixed amino acids are compounded into high concentration mother liquid, which is mixed with sterilized fermentation culture medium in a sterilized super clean bench in 0.22 micron filtering film and mixed homogeneously.
Inoculating Ganoderma lucidum strain SCIM 1006(Ganoderma lingzhi) to KH strain containing glucose as carbon source, peptone and yeast extract as nitrogen source2PO4And MgSO4In the seed culture medium which is a mineral element, the seed culture medium comprises the following components: glucose 4.2%, peptone 0.4%, yeast extract 0.25%, KH2PO40.15％，MgSO40.05 percent; then fermenting in a shaking flask at 28 deg.C and rotation speed of 150r/min for 8 days to obtain Ganoderma seed solution;
preparing Ganoderma seed solution into inoculation solution with thallus concentration of 2.0g/L, inoculating to liquid fermentation culture medium according to volume ratio of 15%, and fermenting at 24 deg.C and low rotation speed of 120r/min for 7 d. By adding exogenous amino acid into liquid fermentation culture medium and combining low-temperature low-rotation-speed fermentation technology, the yield of the natural polypeptide of the ganoderma lucidum mycelia reaches 1.52 g/L.
3. Extraction of ganoderma lucidum mycelium antihypertensive peptide
Taking the Ganoderma fermentation culture solution, filtering with 8 layers of gauze to remove upper layer fermentation liquid, rinsing mycelium pellet with distilled water for 4 times, collecting mycelium pellet, sufficiently absorbing surface water with filter paper, placing into a beaker, weighing, and calculating Ganoderma mycelium biomass. It was transferred to a mortar and poured into 2 volumes (v/w) of liquid nitrogenGround to fine particles and quickly transferred to 4 volumes (v/w) of pre-chilled homogenization buffer. The homogenization buffer is 10mM Tris-HCl buffer, pH 7.0, contains 2mM EDTA, 1% enzyme inhibitor Roche, 10mM beta-mercaptoethanol and 5-10% glycerol, and is placed in a refrigerator at 4 ℃ for 2 hours before use. Homogenizing Ganoderma mycelia homogenate for 20min on ice bath, stopping for 5s every 15s, and preventing target peptide degradation as much as possible. After homogenization, centrifugation (4 ℃, 12000g) was carried out for 20min and twice. And (3) taking the supernatant, respectively carrying out ultrafiltration by using 100kDa and 3kDa ultrafiltration membranes, collecting ultrafiltrates with the molecular weight of less than 3kDa, and replacing a homogenate buffer solution matrix contained in the ultrafiltrates by using a desalting column to obtain distilled water so as to remove 2mM EDTA, 1% enzyme inhibitor Roche, 10mM beta-mercaptoethanol and 5% glycerol, wherein the obtained replacement solution is the ganoderma lucidum mycelium antihypertensive peptide crude product. The ACE inhibitory activity and the molecular weight distribution ratio are measured, and the ACE inhibitory activity IC is found50480.0 μ g/mL, polypeptide content of 1.52mg/mL, molecular weight<The proportion of polypeptides below 2kDa is 86.8%.
4. Purification of ganoderma lucidum mycelium antihypertensive peptide
Dissolving the crude product of the ganoderma lucidum mycelium antihypertensive peptide in 10% acetonitrile aqueous solution to prepare a solution with the concentration of 100mg/mL, loading the solution on a C18 solid-phase extraction column, eluting 2 column volumes by using 10% acetonitrile aqueous solution, eluting 2 column volumes by using 70% acetonitrile aqueous solution, collecting the eluted adsorption part, concentrating at low temperature in vacuum to remove acetonitrile, loading the sample on RP-HPLC for further purification, wherein the chromatographic conditions are as follows: chromatographic column Znertsil ODS-3 (4.6X 250mm), flow rate 1.0mL/min, detection wavelength 220nm, mobile phase of acetonitrile and ultrapure water, 0-3min, 15% acetonitrile; 3-51min, 15-55% acetonitrile; 51-53min, 55-95% acetonitrile; 53-58min, 95% acetonitrile; 58-59min, 95-15% acetonitrile; 59-60min, 15% acetonitrile. One tube was collected every 2.5min from the mobile phase outlet for 24 tubes. The purification chromatogram is shown in FIG. 3. Each tube was concentrated to a polypeptide concentration of 100. mu.g/mL using a vacuum concentration apparatus, the ACE inhibitory activity thereof was measured, and the molecular weight in each tube was further identified by ESI-MS<The 2kDa polypeptide amino acid sequence, ACE inhibitory activity of partial sequence and identification result are shown in Table 1 (the amino acid sequence is shown in SEQ ID NO:1, and SEQ ID NO:9-SEQ ID NO: 21). Wherein, the sequenceThe small peptide of Ser-Tyr-Pro (the amino acid sequence is shown as SEQ ID NO: 1) has the strongest activity and IC50The yield reaches 52.5 mu g/mL and is the highest, and reaches 0.67 percent. The retention time corresponding to the small peptide Ser-Tyr-Pro is about 8.0min, the purity of the small peptide is 95.0 percent through detection, and the small peptide is the pure product of the ganoderma lucidum mycelium antihypertensive peptide.
TABLE 1 ESI-MS identification table of amino acid sequence of Ganoderma mycelia small molecular peptide and ACE inhibition rate
Example 2: ACE inhibition mechanism analysis of ganoderma lucidum mycelium antihypertensive peptide
Analyzing an inhibition model of ganoderma lucidum mycelium antihypertensive peptide by using a Lineweaver-Burk double reciprocal mapping method, incubating ACE with antihypertensive peptide Ser-Tyr-Pro with different mass concentrations (0, 50 and 100 mu g/mL) and different H H L substrate concentrations (0.8, 1.6, 3.2 and 6.4mmol/L), mixing the ACE with the same reaction conditions as the above-mentioned 'ACE inhibition activity determination', measuring the absorbance value at 228nm, drawing a Lineweaver-Burk double reciprocal diagram, and calculating kinetic parameters, the maximum initial velocity (Vmax) and the Michaelis-Menten constant (Km).
As shown in FIG. 4a, the Lineweaver-Burk reciprocal double curve intersects the y-axis, and Km gradually increases with the increase of the mass concentration of the antihypertensive peptide, while Vmax does not change significantly, indicating that it belongs to a competitive inhibitory peptide. As shown in FIG. 4c, the molecular dynamics simulation analysis shows that the peptide Ser-Tyr-Pro of the ganoderma lucidum mycelium has a salt bond with Lys472 carboxyl of ACE active region through Ser1 amino; hydrogen bonds are formed between Ser1 hydroxyl and Lys472, Tyr2 hydroxyl of ACE active area and Gln242, Pro3 carboxyl and Lys 415; through the ring closure of Tyr2 benzene ring and Pro3, Tyr484 and Phe488 in ACE active area produce hydrophobic effect, thus occupying ACE active area and making it unable to act on substrate and be inactivated.
Example 3: gastrointestinal tract digestion stability analysis of ganoderma lucidum mycelium antihypertensive peptide
Adding 1.0% (w/w) pepsin into Ganoderma mycelia antihypertensive peptide Ser-Tyr-Pro solution (pH of 2, 1mg/mL) and reacting at 37 deg.C for 30 min. After the reaction is finished, the pH value is adjusted to 7.5 by using 1M NaOH, and gastric juice digestive juice of the antihypertensive peptide is obtained. To the digestion solution (pH 7.5), 1.0% (w/w) of pancreatin was added and reacted at 37 ℃ for 90min, and after the reaction was completed, the mixture was left at 95 ℃ for 10min to inactivate the enzyme. Then placing on ice water for 5min to obtain gastrointestinal fluid digestion product of the sample. The digestive stability of gastrointestinal fluid of the antihypertensive peptide is analyzed by RP-HPLC by using a small peptide solution without adding pepsin or pancreatin as an experimental control group, and the ACE inhibitory activity of the antihypertensive peptide is determined. Chromatographic conditions are as follows: the chromatographic column is Znertsil ODS-3 (4.6X 250 mm); the fluidity was water (containing 0.04% trifluoroacetic acid), acetonitrile (0.03% trifluoroacetic acid); eluting for 0-5min with 0% acetonitrile; 5-40min, 0-40% acetonitrile; the temperature is 25 ℃; the detection wavelength is 220 nm; the flow rate was 0.5 mL/min.
As shown in FIG. 5, the ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro can be degraded by pepsin, but not by pancreatin, indicating that the ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro has good stability in intestinal tract.
Example 4: in vivo and in vitro blood pressure reduction efficacy analysis of antihypertensive peptide Ser-Tyr-Pro
1. Evaluation of in vivo blood pressure lowering efficacy of ganoderma lucidum mycelium antihypertensive peptide
The male spontaneous hypertension rat SHR with 8 weeks and tail arterial systolic pressure over 180mmHg provided by the Hunan Silikscada laboratory animals GmbH is selected. Feeding at 23 deg.C for 12h under light and 12h in dark, feeding food and water freely, and adaptively feeding for 1 week. The SHR was randomly divided into 3 groups of 6 animals, the group 1 was a negative control group, and the same amount of saline was injected, the group 2 was administered by intravenous injection, and the group 3 was administered by intraperitoneal injection. The ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro is administrated once according to the specified dose of 10mg/kg animal body weight, the tail arterial systolic pressure and diastolic pressure 0-8h after SHR administration are measured, and the blood pressure value of each rat is measured for 5 times to obtain an average value.
As shown in FIG. 6, the peptide can be administered intraperitoneally or intravenously to lower both the diastolic and systolic SHR pressure. After 2h of administration, both systolic and diastolic blood pressure decreased significantly, and since the antihypertensive peptide was partially metabolically degraded in the blood, blood pressure increased back to 3h after administration. As shown in fig. 6a, when the drug is administered for 2 hours, the systolic pressure of the group without drug administration is 160.7mmHg, which is better than that of the group administered by intraperitoneal injection, and the systolic pressure is reduced to 122.6mmHg by intravenous injection; in fig. 6b, the diastolic pressure of the group without drug administration is 134.7mmHg, the blood pressure lowering effect of intravenous injection is better than that of intraperitoneal injection, and the diastolic pressure can be lowered to 96.3mmHg.
2. In-vitro blood pressure lowering activity analysis of ganoderma lucidum mycelium blood pressure lowering peptide
Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in RPMI 1640 medium, which also contained 10% fetal bovine serum, 100. mu.g/mL penicillin and 100. mu.g/mL streptomycin. The regulation and control conditions of the antihypertensive peptide Ser-Tyr-Pro on the expression of eNOS and endothelin-1 of HUVEC induced by angiotensin I are analyzed by adopting a microscopic observation and MTS cell viability measuring method.
1) Analysis of regulation of eNOS and endothelin-1 protein expression: mixing HUVEC (1X 10)5Individual cells/mL) were planted in 10cm cell culture dishes containing 5% CO at 37 ℃2The carbon dioxide incubator of (1). After the cells are mature, the supernatant is carefully removed, 1mL hanks liquid is added, the supernatant is removed and repeated for 3 times, 4mL of Gamma-EDTA is added for digestion, and 12mL of RPMI 1640 culture solution is added to stop digestion after the cells shrink and become round. They were divided into 10cm dishes (2 mL/dish) and 5 dishes were made up with fresh medium to 10 mL/dish. When the cells grow to be full of 90%, adding angiotensin I (1.0mg/mL) for induction for 2h, and establishing a hypertension cell model. Respectively adding different concentrations (0, 25, 50 and 100 mu g/mL) of antihypertensive peptide Ser-Tyr-Pro, wherein the control group without angiotensin I and antihypertensive peptide and the induction group without antihypertensive peptide are used, extracting total protein, and detecting the expression levels of eNOS and endothelin-1 protein by using a Wester-blot technology.
As shown in FIGS. 7c, d and e, the antihypertensive peptide Ser-Tyr-Pro from Ganoderma lucidum mycelia up-regulates the eNOS phosphorylation level and inhibits the endothelin-1 protein expression level, compared to the inducible group. Wherein, when the treatment dosage of the antihypertensive peptide is 25 mug/mL, the expression level of phosphorylated eNOS protein is increased by 4.86 times, and the expression level of endothelin-1 protein is reduced by 63.9%.
2) eNOS and endothelin-1 mRNA expression control: mixing HUVEC (1X 10)5Is smallcells/mL) were planted in 10cm cell culture dishes containing 5% CO at 37 deg.C2The carbon dioxide incubator of (1). After the cells are mature, the supernatant is carefully removed, 1mL of hanks solution is added, the supernatant is removed, the process is repeated for 3 times, 4mL of Trypsin-EDTA is added for digestion, and 12mL of RPMI 1640 culture solution is added after the cells shrink and become round to stop digestion. They were divided into 6-well plates (2 mL/well) for a total of 5 wells. When the cells grow to 80%, angiotensin I (1.0mg/mL) is added for induction for 2h, and a hypertension cell model is established. Respectively adding different concentrations (0, 25, 50 and 100 mu g/mL) of antihypertensive peptide Ser-Tyr-Pro, wherein the control group without angiotensin I and antihypertensive peptide and the induction group without antihypertensive peptide are used, extracting total RNA and carrying out reverse transcription to obtain cDNA, and detecting the integrity of the cDNA by agarose electrophoresis; and detecting eNOS and endothelin-1 mRNA expression levels by using RT-qPCR technology.
Designing a PCR primer:
endothelin-1 reverse chain: 5 'CAG AAA CTC CAC CCC TGT GT 3' (the sequence is shown in SEQ ID NO: 3),
endothelin-1 positive chain: 5 'TCC TCT GCT GGT TCC TGA CT 3' (SEQ ID NO:4 in sequence)
eNOS reverse-chain: 5 'GGT GGC CCT CGT GGA CTT GC 3' (SEQ ID NO:5 in sequence)
eNOS positive chain: 5 'AGG CCT TCC GAG GCT GG 3' (SEQ ID NO:6 in sequence)
Beta-actin reverse strand: 5 'CAT GTA CGT TGC TAT CCA GGC 3' (SEQ ID NO:7 in sequence)
Beta-actin plus strand: 5 'CTC CTT AAT GTC ACG CAC GAT 3' (the sequence is shown in SEQ ID NO: 8).
As shown in FIGS. 7a and b, the mRNA level regulation of eNOS and endothelin-1 by the peptide Ser-Tyr-Pro of Ganoderma lucidum mycelia was similar to the change trend of the protein expression level. When the antihypertensive peptide therapeutic dose was 25. mu.g/mL, the mRNA level of eNOS was 3.51 times that of the induced group, whereas that of endothelin-1 was decreased by 27.3% compared to the induced group.
SEQUENCE LISTING
<110> technical university of the middle and south forestry
<120> ganoderma lucidum mycelium antihypertensive peptide and preparation method thereof
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Ser Tyr Pro
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<213> Artificial sequence
<400> 20
Thr Cys Gly Pro Leu Gln Val Val
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial sequence
<400> 21
Glu Leu Val Asn Leu Pro Thr Asn
1 5
Claims (5)
1. A ganoderma lucidum strain SCIM 1006 is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 25 days in 2019, and the preservation number is CGMCC No. 18819.
2. A method for producing a peptide with an amino acid sequence of Ser-Tyr-Pro by using the ganoderma lucidum strain as claimed in claim 1, wherein the method is a biological fermentation method; the method comprises performing fermentation production by using the Ganoderma strain SCIM 1006 of claim 1, extracting and purifying the peptide from Ganoderma fermentation broth; the mixed amino acids in the medium used for the fermentation include: 17 of Ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg and Ala; the fermentation is carried out for 6-8 d at the temperature of 20-28 ℃ and the temperature of 100-; the extraction comprises the following steps: collecting Ganoderma lucidum fermentation broth, collecting mycelia, pouring liquid nitrogen, grinding to fine particles, transferring to precooled homogenization buffer, performing tissue homogenization on ice bath, centrifuging to obtain supernatant, collecting ultrafiltrate with molecular weight less than 3kDa from the supernatant, and removing the homogenization buffer from the filtrate to obtain crude product of Ganoderma lucidum mycelia antihypertensive peptide; the homogenate buffer solution is 10mM Tris-HCl buffer solution, has the pH value of 7.0, and contains 2mM EDTA, 1% enzyme inhibitor Roche, 10mM beta-mercaptoethanol and 5-10% glycerol; the purification comprises the following steps: dissolving the crude product of the ganoderma lucidum mycelium antihypertensive peptide in acetonitrile water solution, loading the mixture to a C18 solid phase extraction column, collecting an eluted adsorption part after flowing and eluting, removing acetonitrile, and then loading the sample to RP-HPLC for further purification to obtain the pure product of the ganoderma lucidum mycelium antihypertensive peptide.
3. The application of the peptide with the amino acid sequence of Ser-Tyr-Pro in the preparation of the medicine with the function of reducing blood pressure.
4. The use of claim 3, wherein the medicament is prepared into capsules, tablets, granules, pills, powder or oral liquid which are commonly used in clinic according to the conventional preparation process.
5. A method of modulating eNOS and endothelin-1 expression for the diagnosis and treatment of non-diseases comprising using a peptide having the amino acid sequence Ser-Tyr-Pro.
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Citations (3)
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| WO2009155585A1 (en) * | 2008-06-21 | 2009-12-23 | Blumy Kenneth | N utragenomics |
| CN103865814A (en) * | 2014-03-04 | 2014-06-18 | 昆明理工大学 | High-yield ganoderan engineering strain Rmust and construction method thereof |
| CN110272932A (en) * | 2019-06-10 | 2019-09-24 | 开平健之源保健食品有限公司 | A kind of preparation method of lucid ganoderma spore powder polysaccharide peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009155585A1 (en) * | 2008-06-21 | 2009-12-23 | Blumy Kenneth | N utragenomics |
| CN103865814A (en) * | 2014-03-04 | 2014-06-18 | 昆明理工大学 | High-yield ganoderan engineering strain Rmust and construction method thereof |
| CN110272932A (en) * | 2019-06-10 | 2019-09-24 | 开平健之源保健食品有限公司 | A kind of preparation method of lucid ganoderma spore powder polysaccharide peptide |
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| "食用菌ACE 抑制肽制备及其功能活性研究进展";许新月等;《山东农业科学》;20191130;第51卷(第11期);第157-160,167页 * |
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