CN102558296B - Mytilus edulis enzymolysis polypeptide and preparation method and application thereof - Google Patents
Mytilus edulis enzymolysis polypeptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a mytilus edulis enzymolysis polypeptide. The mytilus edulis enzymolysis polypeptide is characterized by containing the following amino acid sequence: Asp Leu Tyr. The mytilus edulis enzymolysis polypeptide is prepared by adopting the following steps of: (1) preparing homogenate from mytilus edulis meat, adding alkaline protease, deactivating the protease, centrifuging, and taking clear solution of the upper layer; (2) performing ultra-filtration on the clear solution, collecting hydrolysate with the molecular weight of below 3K, concentrating, and performing freeze drying; (3) performing chromatographic separation by adopting a DEAE-SepharoseFF ion exchange column; (4) performing chromatographic separation by adopting a Sephadex G-25 gel column; and (5) performing high performance liquid chromatography purification. The invention also discloses application of the mytilus edulis enzymolysis polypeptide prepared by the steps in prostatic cancer resistance. Compared with the prior art, the invention has the advantages that: the mytilus edulis is subjected to enzymolysis and purification by adopting an optimal protease and an optimal technology, a strong cell proliferation inhibiting effect is achieved when the obtained target peptide is applied to prostatic cancer resistant cells, and a feasible research path is provided for resisting prostatic cancer.
Description
Technical field
The present invention relates to a kind of mussel enzymolysis polypeptide, the invention still further relates to the preparation method of this mussel enzymolysis polypeptide and the application on anti-prostate cancer thereof.
Background technology
Polypeptide can be used as neurotransmitter, interleukin-and cell growth factor isoreactivity material in vivo, has the biology growing of maintaining growth, immunomodulatory and metabolic isoreactivity function.Now there are some researches show, the polypeptide that derives from organism has concern antitumor, anti-oxidant, antibiotic, anti-ageing, that hypertension isoreactivity function, especially anti-tumor activity have been subject to various countries researchist.At present, about the existing a large amount of reports of anti-tumor activity of native peptides and synthetic peptide, and there is part of polypeptide to be applied to clinical as antitumor drug; The polypeptide in food proteins source is antibiotic, anti-oxidant for it, antihypertensive active research is a lot of, and less to the relevant report of its anti-tumor activity.
Marine protein is of a great variety, wide material sources, and an important sources as bioactive peptide, has received increasing concern.Mussel is as a halobiontic important component part, be under the jurisdiction of sea mollusk door (Mollusca) Bivalvia (Bivalvia) mussel order (Mytiloida), in northern China, claim Hai Hong, Jiangsu and Zhejiang Provinces is called mussel, and richness originates in marine site around, Shengsi, Zhejiang.Enriched Mussel, containing crude protein, fat and carbohydrate, contains the various trace elements such as calcium, phosphorus, iron, VITAMIN in addition.There are some researches show, that mussel extract has is antitumor, anti-oxidant, anti-freezing, hypotensive, reducing blood-fat, raising immunizing power isoreactivity function, can referenced patent number be ZL200510110096.8 Chinese invention patent " extract of Mytilus crassitesta Lischke, manufacture method and uses thereof " (Granted publication number: CN100467030C), the application of the extract that this patent discloses Mytilus crassitesta Lischke in influenza.Can also referenced patent number be similarly the Chinese invention patent of ZL200510024391.1 " a kind of Trachyostracous mussel extract that improves immunity function " (Granted publication number is CN100486593C); Application number is open " thick-shell mussel fat-soluble extract and its production and use " (publication number: CN101606951A) of Chinese invention patent application of 200910101136.0.But the relevant report of relevant mussel enzymolysis product to prostate cancer cell effect that so far there are no.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of mussel enzymolysis polypeptide for the above-mentioned state of the art.
Another technical problem to be solved by this invention is to provide a kind of preparation method of mussel enzymolysis polypeptide.
Another technical problem to be solved by this invention is to provide a kind of application of mussel enzymolysis polypeptide.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of mussel enzymolysis polypeptide, is characterized in that comprising following aminoacid sequence:
Asp?Leu?Tyr。
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of mussel enzymolysis polypeptide, is characterized in that adopting following steps preparation:
1. get mussel meat, make homogenate, regulate pH value 9.5～10, solid-liquid ratio is 1: 2～1: 3, adds Sumizyme MP, and enzyme concentration is 1500～2000U/g, and enzymolysis time is 6～8 hours, and hydrolysis temperature is 45～50 ℃, centrifugal after the enzyme that goes out, and gets supernatant liquid;
2. above-mentioned clear liquid is undergone ultrafiltration, collect the hydrolyzed solution below molecular weight 3K, concentrated and lyophilize;
3. DEAE-SepharoseFF ion column exchange column chromatographic separation, lyophilize after product by step in is 2. by DEAE-SepharoseFF ion column exchange column, 0.1～1mol/LNaCL gradient elution, elution speed 1～1.5mL/min, Protein Detection instrument 280nm detects, each elution peak is collected respectively, concentrated postlyophilization, adopt mtt assay to carry out anti-tumor activity experiment each peak component obtaining after dry, further determine the elution peak at target peptide place, Bing Duigai collects at peak in a large number, concentrated and lyophilize;
4. adopt Sephadex G-25 gel column chromatography separated, by step 3. the lyophilize after product with high anti-tumor activity of gained cross Sephadex G-25 gel column, moving phase is distilled water, flow velocity is 1.5～2mL/min, Protein Detection instrument 280nm detects, each elution peak is collected respectively, and concentrated postlyophilization also carries out anti-tumor activity experiment with mtt assay;
5. high-efficient liquid phase chromatogram purification, step is obtained in 4. there is high anti-tumor activity lyophilize after postpartum cross RP-HPLC post, chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile is dense changes to 95%, elution speed 2.5～3.0mL/min, ultraviolet detection wavelength 280nm from 0%.
The application of described mussel enzymolysis polypeptide on anti-prostate cancer.Further, described mussel enzymolysis polypeptide suppresses the application in propagation at anti-prostate cancer DU-145 cell; Described mussel enzymolysis polypeptide suppresses the application in propagation at anti-prostate cancer PC-3 cell.
Compared with prior art, the invention has the advantages that: adopt best proteolytic enzyme and preferred technique to carry out enzymolysis purifying to mussel, the target peptide of gained is applied to on anti-prostate cancer cell, produced very strong cell and suppresses proliferation function, for anti-prostate cancer provides a feasibility study approach; Integrated artistic is simple, and less input, is easy to apply.
Accompanying drawing explanation
Fig. 1 is the zymolyte of the various proteolytic enzyme histogram to DU-145 cell proliferation inhibition rate.
Fig. 2 is the histogram of Sumizyme MP different molecular weight zymolyte to DU-145 cell proliferation inhibition rate.
Fig. 3 is the DEAE Sepharose FF chromatography collection of illustrative plates of the following component of basic protein enzyme molecular weight 3K.
The Sephadex G-25 gel chromatography collection of illustrative plates at Tu4Wei Tu3Zhong peak 2.
The RT-HPLC collection of illustrative plates of Tu5Wei Tu4Zhong peak 2-2.
Fig. 6 is the RT-HPLC collection of illustrative plates of target peptide in Fig. 5.
Fig. 7 is Photomicrograph after normal DU-145 cellular form HE dyeing.
Fig. 8 is the rear Photomicrograph of DU-145 cellular form HE dyeing after mussel polypeptide effect 24h.
Fig. 9 is the rear Photomicrograph of DU-145 cellular form HE dyeing after mussel polypeptide effect 48h.
Figure 10 is Photomicrograph after normal PC-3 cellular form HE dyeing.
Figure 11 is the rear Photomicrograph of PC-3 cellular form HE dyeing after mussel polypeptide effect 24h.
Figure 12 is the rear Photomicrograph of PC-3 cellular form HE dyeing after mussel polypeptide effect 48h.
Embodiment
Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.
1 material
1.1 animals: mussel (Mytilus edulis) is purchased from market, Zhoushan (identifying through professor Zhao Shenglong of Oceanography Institute Of Zhejiang), and after shelling ,-20 ℃ of refrigerations are standby.
1.2 cell strains: Human Prostate Cancer Cells DU-145 and PC-3 are all purchased from Chinese Academy of Sciences's Shanghai cell bank.
1.3 main agents: stomach en-, trypsinase, papoid, Sumizyme MP, dextran gel SephadexG-25 and DEAE SepharoseFF anion-exchange column are all purchased from Heng Xin bio tech ltd, Asia-Pacific, Beijing; Foetal calf serum is purchased from Hangzhou folium ilicis chinensis biotechnology company limited; F12 powder culture medium and MTT are all purchased from U.S. SIGMA company; Dimethyl sulfoxide (DMSO) is purchased from U.S. AMRESCO company; Acetonitrile is purchased from Ningbo Hai Shuhenglong experiment equipment company limited; All the other reagent are analytical pure.
1.4 key instruments: BSA124S type electronic balance (Germany, Sartorius AG company); DS-1 type high-speed tissue mashing machine (Shanghai Sample Model Factory); CF16RXII high speed freezing centrifuge (HITACHI company of Hitachi); Automatic Fraction Collector (BSZ-40-LCD), Protein Detection instrument (HD-21-88) (Shanghai Qi Te Analytical Instrument Co., Ltd); SSW type Microcomputerized electric thermostatic bath (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); Yi Lite P1201 type high performance liquid chromatograph (Dalian Yilite Analytical Instrument Co., Ltd); MSC300 ultrafiltration cup (ultra-filtration membrane: 3KDa and 5KDa) (Shanghai rub fast science equipment company limited).ZHJH-C1209C type Bechtop (Shanghai Zhi Cheng analytical instrument Manufacturing Co., Ltd); Forma 3111 type CO
2incubator (U.S. Thermo company); Inverted microscope (Japanese OLYMPUS company); Microplate reader (U.S. Bio-Rad company).
2 methods
2.1 enzymolysis process flow processs: select Sumizyme MP, trypsinase, stomach en-and 4 kinds of proteolytic enzyme of papoid respectively mussel meat to be carried out to enzymolysis, the enzymatic hydrolysis condition of various proteolytic enzyme is in Table 1, and enzymolysis process is as follows successively:
Mussel is cleaned; Shell and get meat; Homogenate; 0.5mol/LNaOH and 0.1mol/LHCl solution are adjusted pH value; Add enzymic hydrolysis; Enzyme (90 ℃, heating 15min) goes out; Centrifugal (5000r/min, 20min); Get supernatant liquor; Lyophilize; Mtt assay anti-tumor activity rough determination.
The enzymatic hydrolysis condition of several proteolytic enzyme of table 1
2.2 cell cultures: human prostata cancer DU-145 and PC-3 cell (former purchased from cell bank ，You Zhe laboratory, Chinese Academy of Sciences Shanghai go down to posterity preservation), with the F12 perfect medium containing 10% calf serum in 37 ℃, 5%CO
2in incubator, be cultured to logarithmic phase.
The mensuration of 2.3 cell proliferation inhibition rates: adopt mtt assay to detect.Preparation PBS solution (pH value is 7.2) and certain density enzymolysis polypeptide solution.The prostate cancer cell of taking the logarithm vegetative period is made suspension, is seeded to 96 orifice plate ，Mei hole 200 μ L, in 5%CO
2, 37 ℃ of adherent 4h, then add respectively enzymolysis polypeptide solution, and each concentration is established 3 parallel holes, establishes not dosing control group simultaneously, puts 5% CO
2, in 37 ℃ of incubators, hatch, cultivate and finish to add 180 μ LMTT and 20 μ L PBS to continue to cultivate 4h, inhale the liquid of abandoning 96 orifice plates, add the DMSO of 150 μ L, fully mix.Put enzyme-linked immunosorbent assay instrument and survey absorbancy at 490nm, calculate cell inhibitory effect index (IR), by following formula, calculate.
IR=[(control group A value-medicine group A value)/control group A value] * 100%
The initial gross separation of 2.4 anti-tumor activity peptides: get the supernatant liquor that protease hydrolysis mussel meat obtains, carry out ultrafiltration with the ultra-filtration membrane that molecular weight cut-off is 10K, 5K and 3K respectively, obtain molecular weight and be that 10K is above, 5K-10K, 3K-5K and the hydrolyzed solution below 3K.After lyophilize, adopt mtt assay to measure respectively the proliferation inhibition rate of each component to prostate cancer cell, tentatively determine and have compared with the molecular weight ranges of powerful antitumor activity polypeptide.Hydrolyzed solution to this molecular weight ranges is collected in a large number through ultrafiltration, and concentrated and lyophilize, does further separation and purification.
2.5 DEAE-SepharoseFF ion exchange column chromatographies are separated: the strongest part of the activity that above-mentioned steps is obtained is crossed DEAE-SepharoseFF ion exchange column, use respectively PBS (PH is 7.4), 0.1mol/LNaCL, 0.3mol/LNaCL, 0.5mol/LNaCL and 1mol/LNaCL solution to carry out stepwise elution, elution speed is 1ml/min, Protein Detection instrument 280nm detects, collect respectively each elution peak, and lyophilize.Adopt mtt assay to carry out anti-tumor activity experiment each peak component obtaining after dry, further determine that the elution peak ，Bing Duigai peak at target peptide place is collected in a large number, concentrated and lyophilize, as being further purified specimen in use.
2.6 Sephadex G-25 gel column chromatographies are separated: the strongest component of activity being obtained by above-mentioned steps is crossed Sephadex G-25 gel column (80cm * 2.6cm), moving phase is distilled water, flow velocity is 2mL/min, Protein Detection instrument 280nm detects, each elution peak is collected respectively to concentrated postlyophilization.Adopt mtt assay to carry out anti-tumor activity experiment each peak component obtaining after dry, determine that the elution peak ，Bing Duigai peak at target peptide place is collected in a large number, concentrated and lyophilize, as high performance liquid phase specimen in use.
2.7 high performance liquid chromatography (HPLC) purifying and purity detecting: purifying chromatographic condition: chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile concentration changes to 95% with 0%; Elution speed 3.0mL/m in; Ultraviolet detection wavelength 220nm.Purity detecting chromatographic condition: chromatographic column is Hypersil BDS C18 (250mm * 4.6mm, 5 μ m); Column temperature is room temperature; Gradient elution: 0～20min, acetonitrile concentration changes to 95% by 0%; Elution speed 0.8mL/m in; Ultraviolet detection wavelength 220nm.
The aminoacid sequence of 2.8 target peptide detects.By detection, obtain this mussel enzymolysis polypeptide, comprise following aminoacid sequence: Asp Leu Tyr.
2.9 cellular fories are observed: adopt HE dyeing process.DU-145 and PC-3 cell are seeded on six orifice plates with cover glass, after cell climbing sheet 24h, add the target peptide of 20mg/ml, cultivate respectively after 24h and 48h, cover glass is taken out to 95% alcohol fixation 15min.PBS washes 2 times, and Hematorylin dyes, and tap water is fully washed, and redye in Yihong, and tap water embathes, the dehydration of alcohol gradient, and dimethylbenzene is transparent, neutral tree mounting, optical microphotograph Microscopic observation is also taken the photograph sheet.
3 results
Determining of 3.1 proteolytic enzyme kinds:
4 kinds of proteolytic enzyme all have certain inhibited proliferation to the resulting zymolyte of mussel meat enzymolysis to DU-145 cell, wherein Sumizyme MP zymolyte is the strongest to the proliferation inhibition activity of DU-145 cell, concentration is 20mg/ml, and after effect 48h, inhibiting rate is 28.4% (seeing Fig. 1).4 components that hydrolysis by novo liquid obtains after ultra-filtration membrane ultrafiltration are 20mg/ml in concentration, and after effect 48h, the following component of 3K is maximum to the proliferation inhibition rate of DU-145 cell, is 30.3% (seeing Fig. 2).
The separation of 3.2 anti-PCa bioactive peptides
The following component of 3K, after DEAE-Sepharose FF ion exchange column wash-out, obtains 4 peak components (seeing Fig. 3) altogether, and wherein peak 2 anti-tumor activities are the strongest, and when concentration is 20mg/ml, after DU-145 cytosis 48h, inhibiting rate is 41.2%.Peak 2 obtains 3 peak components (seeing Fig. 4) after Sephadex G-25 gel column wash-out, wherein the anti-tumor activity of peak 2-2 is the strongest, in concentration, be 20mg/ml, after DU-145 cytosis 48h, inhibiting rate is purifying and the purity testing of the anti-PCa bioactive peptide of 46.5%3.3: peak 2-2 is through Zorbax SB C
18after purifying, finally obtain 1 polypeptide, be this and test purified target peptide, see Fig. 5.This peptide being crossed to Hypersil BDS C18 chromatographic column and detect its purity, occur simple spike when retention time is about 15min, see Fig. 6, illustrate that this tests resulting target peptide purity higher, is one-component.
3.4 target peptide are to DU-145 and the effect of PC-3 cell inhibitory effect: target peptide is arranged to 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml and five concentration groups of 25mg/ml, respectively after mensuration effect 24h, 48h and 72h, the impact on DU-145 and PC-3 cell proliferation.Result shows, target peptide is stronger than PC-3 cell to the proliferation inhibition activity of DU-145 cell, and the proliferation inhibition activity of two kinds of cells is all presented to timeliness and dose-effect relationship (table 2).
The impact (x ± s, n=3) of table 2 mussel enzymolysis polypeptide on human prostata cancer DU-145 and PC-3 cell proliferation
3.4 morphological observation: HE dyes as shown in Fig. 7 to Figure 14, normal cell kytoplasm dyeing homogeneous, and cell fission is obvious, and form is full, and nucleus is not of uniform size, and kernel number is many.After target peptide effect 24h, tenuigenin is concentrated, and karyon starts pyknosis and diminishes, and intercellular space increases.After effect 48h, above-mentioned change aggravation, intercellular space is strengthened obviously, and cell outline is fuzzy, and cell space sharply dwindles, and there is cavity in parts of fine kytoplasm, kernel reduced number, apoptotic body forms, chromatin color burn.Morphological observation demonstration, proving again target peptide has time-effect relationship to the effect of DU-145 and PC-3 cell; Apoptotic body forms, and illustrates that the antitumor action of target peptide may be realized by cell death inducing.
In the last few years, from natural product, found the focus that antitumor drug novel, efficient, low toxicity is various countries' research always.Wherein, anti-tumor activity peptide is because the features such as its multifunctionality, hypersensitivity and high stability also receive increasing concern.First this experiment is chosen several proteolytic enzyme mussel protein is hydrolyzed, and relatively the anti-tumor activity of each zymolyte, finds that its anti-tumor activity of different zymolytes is not identical yet, and this should be relevant with the composition structure of zymolyte.Can infer accordingly, while utilizing enzyme solution to obtain anti-tumor activity peptide, the selection of enzyme is most important, and under the prerequisite of conditions permit, the multiselect of should trying one's best carries out screening active ingredients with several proteolytic enzyme.Secondly, in separation and purification, obtain in the process of target peptide, the component of different molecular weight and different elution peaks detected, its anti-tumor activity also has very large difference, and the strongest active part is the small molecules amount polypeptide below 3K, and in polypeptide within the scope of this, what play active function is mainly also the polypeptide fraction at certain peak, and along with the carrying out of separation and purification, the anti-tumor activity of polypeptide also strengthens gradually, to the inhibiting rate of tumour cell, 28.4% before by separation and purification is increased to 47.6% for it.Therefore,, when preparing anti-tumor activity peptide, carrying out separation and purification, to obtain sterling be also very necessary.Finally, according to existing result of study, same bioactive peptide is not identical to the active function of different germline cancer cells yet, this is tested resulting active polypeptide and screens for prostate cancer DU-145 cell, although this target peptide also has proliferation inhibition activity to PC-3 cell, activity is starkly lower than the former, so, when carrying out anti-tumor activity peptide screening, the selection of cancer cells germline plays a key effect for the target peptide finally obtaining.
Originally studies confirm that utilization enzyme solution can extract anti-tumor activity peptide from mussel protein, this bioactive peptide has significant proliferation inhibition activity to prostate cancer cell, this experiment is laid a good foundation for the research and development of food proteins source antitumor drug and healthcare products, still for its antitumor mechanism and toxicity thereof, needs further to study.
Claims (3)
1. a mussel enzymolysis polypeptide, is characterized in that comprising following aminoacid sequence:
Asp Leu Tyr; And mussel enzymolysis polypeptide makes as follows
1. get mussel meat, make homogenate, regulate pH value 9.5～10, solid-liquid ratio is 1: 2～1: 3, adds Sumizyme MP, and enzyme concentration is 1500～2000U/g, and enzymolysis time is 6～8 hours, and hydrolysis temperature is 45～50 ℃, centrifugal after the enzyme that goes out, and gets supernatant liquid;
2. by above-mentioned clear liquid process ultrafiltration, collect the hydrolyzed solution below molecular weight 3K, concentrated and lyophilize;
3. DEAE-SepharoseFF ion column exchange column chromatographic separation, lyophilize after product by step in is 2. by DEAE-SepharoseFF ion column exchange column, 0.1～1mol/LNaCL gradient elution, elution speed 1～1.5mL/min, Protein Detection instrument 280nm detects, each elution peak is collected respectively, concentrated postlyophilization, adopt mtt assay to carry out anti-tumor activity experiment each peak component obtaining after dry, further determine the elution peak at target peptide place, Bing Duigai collects at peak in a large number, concentrated and lyophilize;
4. adopt Sephadex G-25 gel column chromatography separated, by step 3. the lyophilize after product with high anti-tumor activity of gained cross Sephadex G-25 gel column, moving phase is distilled water, flow velocity is 1.5～2mL/min, Protein Detection instrument 280nm detects, each elution peak is collected respectively, and concentrated postlyophilization also carries out anti-tumor activity experiment with mtt assay;
5. high-efficient liquid phase chromatogram purification, what step was obtained in 4. has after high anti-tumor activity material lyophilize, and through RP-HPLC post, chromatographic column is Zorbax SB C
18(250mm * 9.4mm, 5 μ m); Column temperature is room temperature; Moving phase is acetonitrile and water, gradient elution: 0～20min, and acetonitrile is dense changes to 95%, elution speed 2.5～3.0mL/min, ultraviolet detection wavelength 220nm from 0%.
2. the application of mussel enzymolysis polypeptide claimed in claim 1 in anti-prostate cancer DU-145 cell proliferation.
3. the application of mussel enzymolysis polypeptide claimed in claim 1 in anti-prostate cancer PC-3 cell proliferation.
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| CN103805662A (en) * | 2012-11-15 | 2014-05-21 | 浙江海洋学院 | Preparation method and application of sinonovacula constricta enzymolysis polypeptide |
| CN103204906A (en) * | 2013-01-29 | 2013-07-17 | 浙江海洋学院 | Mussel meat protein antioxidative peptide and preparation method thereof |
| CN103204904B (en) * | 2013-02-01 | 2015-07-22 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
| CN103992387B (en) * | 2014-05-29 | 2016-08-31 | 浙江海洋学院 | A kind of mussel boiling liquid bioactive peptide and its preparation method and application |
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| CN106047975A (en) * | 2016-08-22 | 2016-10-26 | 得利斯集团有限公司 | Mussel protein peptide extraction method |
| CN106854671A (en) * | 2016-10-26 | 2017-06-16 | 浙江海洋大学 | A kind of preparation method of clam antineoplastic extract |
| CN106676155B (en) * | 2017-01-10 | 2021-07-06 | 大连工业大学 | Preparation method of mussel polypeptide with antithrombotic activity |
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| CN107955068A (en) * | 2017-09-29 | 2018-04-24 | 浙江海洋大学 | A kind of Trachyostracous mussel closed shell creatase demodulates fat pentapeptide |
| CN109206483B (en) * | 2018-10-18 | 2021-07-27 | 大连深蓝肽科技研发有限公司 | ACE (angiotensin converting enzyme) inhibition and anti-tumor active peptide from mussels |
| CN110724175B (en) * | 2019-10-14 | 2021-10-22 | 浙江海洋大学 | Preparation method for extracting antibacterial peptide from Mytilus edulis processing leftovers by utilizing ultrasonic homogenization |
| CN115246871B (en) * | 2021-12-27 | 2024-02-27 | 浙江海洋大学 | Preparation method of thick-shell mussel immunocompetent hexapeptide |
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