CN112852909A

CN112852909A - Method for preparing ACE inhibitory peptide by solid-state fermentation of shrimp heads with mixed strains

Info

Publication number: CN112852909A
Application number: CN202110095953.0A
Authority: CN
Inventors: 朱慧; 李云; 周飞
Original assignee: Hanshan Normal University
Current assignee: Hanshan Normal University
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-05-28
Anticipated expiration: 2041-01-25
Also published as: CN112852909B

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads by mixed strains, which comprises the following steps: pretreatment of shrimp heads, activation of strains and preparation of seed liquid, preparation and sterilization of fermentation media, inoculation and fermentation, extraction and separation of peptides, concentration, drying and the like. The invention has the beneficial effects that: the solid state fermentation method is adopted, the cost is low, and protease does not need to be added; the shrimp heads are prepared into particles with a certain particle size in advance, so that the specific surface area and the utilization rate of raw materials are increased, and the heat dissipation and the oxygen requirement of bacteria in fermentation are facilitated; the mixed fermentation of the two strains is adopted, and the degradation of the raw materials is more complete by utilizing various proteases and peptidases produced by the two strains; a process of adding sugar regularly is adopted, and the lower sugar concentration is maintained to be beneficial to the thallus to utilize the components on the raw material and promote the degradation and dissolution of the components on the raw material; the shrimp head fermented peptide developed by the method has high yield and purity and good inhibitory activity.

Description

Method for preparing ACE inhibitory peptide by solid-state fermentation of shrimp heads with mixed strains

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing ACE inhibitory peptide by solid-state fermentation of shrimp heads with mixed strains.

Background

Hypertension is one of the most common chronic diseases, is mainly characterized by a syndrome of increased systemic arterial pressure, and is an important cause and induction factor of various cardiovascular and cerebrovascular diseases. At present, the overall awareness rate, the treatment rate and the control rate of hypertension in China are also obviously low. The administration of chemically synthesized antihypertensive drugs is a common treatment method, and although the antihypertensive effect is obvious, adverse reactions such as dry cough, rash, angioedema, proteinuria, leukopenia, withdrawal syndrome and the like are easily caused after long-term administration. Therefore, it is very necessary to find a novel, safe and economical functional component for lowering blood pressure as a functional factor of a medicine or food for preventing and treating hypertension. The food-derived antihypertensive peptide is a peptide segment derived from a food protein sequence, and has the effect of inhibiting or relieving the increase of blood pressure mainly by inhibiting the activity of Angiotensin Converting Enzyme (ACE). The food source ACE inhibitory peptide has the advantages of no side effect, and good safety and stability, and becomes a key research direction for non-drug treatment of hypertension.

The shrimp heads are main byproducts of products such as shrimp meat or head-removed shrimps and the like, and account for about one third of the weight of the shrimp bodies. After the shrimp heads are removed from fresh shrimps, the tissue enzyme is active, the water content is higher, the storage is difficult, most of the processed shrimps are usually directly discarded, or the shrimps are simply processed (such as dried and crushed) into feed for cultivation, so that the environment is polluted, and the waste of resources is also caused. The shrimp heads contain a large amount of protein nutrient substances, and are good raw materials for obtaining the antihypertensive active peptide. The research and development of the shrimp head protein source ACE inhibitory peptide as a functional food factor have important social and economic significance for reducing side effects caused by the treatment of synthetic antihypertensive drugs, preventing cardiovascular and cerebrovascular diseases and promoting human health.

The preparation of the food source ACE inhibitory peptide mainly comprises an enzymolysis method and a microbial fermentation method. At present, in the reported or published technology for preparing ACE inhibitory peptide by utilizing shrimp head protein, an enzymolysis method is mainly used, and a fermentation method is rarely adopted. Two-step enzymolysis of shrimp by chymotrypsin and prolease in Zuoqi et alPreparation of ACE inhibitory peptide from by-product, and ACE inhibitory IC of enzymolysis product₅₀The value is 1.645mg/mL (food industry science 2014,35(10): 181-. Asahu and the like take shrimp shell powder as a raw material, preferably neutral protease is taken as an optimal enzyme, the ACE inhibition rate of the prepared polypeptide is 84.04%, and the hydrolysis degree is 26.76% (food science, 2012,33(11): 131-. The method comprises preparing ACE inhibitory peptide by hydrolyzing shrimp head protein with alkaline protease, subjecting enzymolysis product to gel filtration, and obtaining IC with highest ACE inhibitory activity₅₀0.79mg/mL (food science, 2012,33(11): 131-. The method comprises preparing ACE inhibitory peptide from endogenous protease hydrolyzed protein of shrimp head in autolysis process, ultrafiltering, gel chromatography and ion exchange chromatography to obtain hydrolysate, and performing IC₅₀0.19mg/mL (aquatic bulletin, 2013,37(4): 631-640). The enzymolysis method has the advantages of simple process, mild conditions and capability of completing the enzymolysis process in a short time. Because the protease hydrolysis process has a certain degree of randomness, how to select proper reaction conditions to control the enzymolysis process is the key to obtain high-yield active peptides. For better industrial application of the enzymatic hydrolysis method, protease and peptidase with specific enzyme cutting site capability are required to be optimized, and deep hydrolysis of raw material protein is also required to achieve higher hydrolysis degree, so that peptide fragments with high ACE activity are released. To achieve the above purpose, two or more enzymes are often used for hydrolysis, and high-purity enzymes are used in the process, which increases the production cost.

The invention discloses CN200710303512.5, a method for preparing the peptide for reducing blood pressure of protein of the Chinese Acete chinensis and an application thereof, which adopts bacillus subtilis as a strain to prepare enzyme liquid through liquid fermentation, then the enzyme liquid is mixed with slurry prepared by pulping the Chinese Acete chinensis to carry out enzymolysis, the peptide for reducing blood pressure with six amino acids is separated and purified from the enzymolysis liquid, and the peptide has higher Angiotensin Converting Enzyme (ACE) inhibition activity. In addition, the three possible enzymolysis products of the hexapeptide are chemically synthesized according to the action sites of enzymes in vivo, and the short peptides with 12 sequences are obtained by screening through an ACE inhibition test and have good ACE inhibitory activity.

In the existing enzymolysis technology, a protease reaction system is carried out in an aqueous solution, a substrate object of the protease reaction is mainly water-soluble protein in a material, and for a shrimp head raw material, the soluble component is less and mainly consists of insoluble solid components, wherein insoluble hard protein (such as keratin) or hydrophobic protein is difficult to utilize by adopting an enzymolysis method, so that the raw material utilization rate and the polypeptide yield are difficult to obtain.

The microbial fermentation method is one of the important methods for preparing the food-source ACE inhibitory active peptide. The key of the fermentation method for preparing the antihypertensive peptide by using the marine product protein is a food safe strain with high protease and peptidase activities and the capability of releasing specific functional peptide fragments. Wang et al fermented Acetes chinensis meat with lactic acid bacteria to obtain ACE inhibitory peptide (Applied Microbiology and Biotechnology,2008,79(5): 785-. Because the growth and fermentation of the lactic acid bacteria have high nutritional requirements and weak proteolytic activity, the culture substrate is required to contain available carbon sources and necessary growth factors, the raw materials are required to be hydrolyzed by protease in advance and then fermented when the lactic acid bacteria are used for fermentation, and the added substances bring difficulties for later separation and purification, so that the process is complicated.

Disclosure of Invention

The invention aims to solve the problems that in the prior art, a method for preparing ACE inhibitory peptide by taking shrimp heads as raw materials is lack of a method with high raw material hydrolysis rate and good product inhibitory activity. At present, the enzymolysis method is mainly used, and the shrimp heads mainly comprise insoluble solid components, so that the enzymolysis efficiency in a liquid phase system is low, and the high proteolysis rate is difficult to obtain. The invention adopts a solid state fermentation method, utilizes inoculated mixed strains, and adopts a mode of adding sugar in the fermentation process for fermentation, thereby improving the hydrolysis rate of protein and obtaining the micromolecule polypeptide with high inhibitory activity.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains comprises the following steps:

A. pretreatment of raw materials: drying and crushing fresh shrimp heads to obtain crushed shrimp head powder;

B. strain activation: respectively carrying out solid slant activated culture on bacillus subtilis and bacillus licheniformis to obtain slant cultures;

C. preparing a seed solution: carrying out amplification culture on the slant culture to obtain a seed solution;

D. preparing a fermentation medium, mixing materials and sterilizing: mixing the crushed shrimp head powder with a culture solution and sterilizing to obtain a fermentation culture medium;

E. inoculation and solid state fermentation: mixing the seed liquid with the fermentation medium, starting fermentation, and supplementing a sugar source from 24-36 hours of fermentation time until the fermentation is finished to obtain a fermentation material;

F. extraction and isolation of peptides: mixing the fermentation material with an acid agent, stirring, filtering, decoloring, ultrafiltering, eluting and removing impurities to obtain an eluent containing ACE inhibitory peptide;

G. concentrating and drying to obtain a finished product: and concentrating and drying the eluent to obtain the shrimp head fermented polypeptide powder containing the ACE inhibitory peptide.

In the step A, the dried shrimp heads are crushed into particles with proper particle size, so that the specific surface area is increased, the bacteria can be fully contacted with protein in the raw materials to facilitate enzymolysis and release of small molecular peptides, and in addition, the particle size with a certain size is kept to play a role of a filling agent, so that the requirements of heat dissipation and air circulation for oxygen for bacterial growth in solid state fermentation are facilitated.

The strain used by the invention is the combination of bacillus subtilis and bacillus licheniformis, namely the strains of bacillus subtilis and bacillus licheniformis with the capability of producing alkaline protease, neutral protease and aminopeptidase can realize the technical scheme. The bacillus has low nutrient requirement for growth and rich proteolytic enzyme system, and can secrete a plurality of extracellular proteases and aminopeptidases, wherein bacillus subtilis and bacillus licheniformis are important production strains of commercial neutral proteases and alkaline proteases. In addition, the bacillus subtilis and the like are common strains for producing fermented foods such as fermented soybeans, soybean paste, natto and the like, have good safety and are suitable for being used as a fermentation strain to prepare the food source ACE inhibitory peptide. In the prior art of preparing ACE inhibitory peptide by using whole shrimps, for example, in patent CN200710303512.5, a liquid culture medium containing specific protein is inoculated with a strain in advance for fermentation, bacteria are removed by centrifugation to prepare an enzyme solution, and the enzyme solution prepared by fermentation is used for hydrolyzing target protein (mainly water-soluble protein, and the utilization rate of insoluble protein is low) in a raw material in a liquid phase system, so that a target product is obtained. However, the invention adopts a solid state fermentation method, the thallus directly grows on a solid raw material substrate, protease and aminopeptidase can be directly generated by taking shrimp heads as substrates, and the protease and the aminopeptidase can hydrolyze proteins on the shrimp head raw materials in situ to generate ACE inhibitory peptide. Compared with the enzymolysis process in a liquid phase system, the thalli grow on the raw materials, which is beneficial to further degrading raw material particles to promote the dissolution of protein, and can also effectively degrade insoluble solid protein, thereby improving the proteolysis rate of the raw materials. In the continuous presence of the preferred combination of Bacillus strains, enzyme production and hydrolysis of substrate proteins occur simultaneously and promote each other, the efficiency of ACE inhibitory peptides production is greatly enhanced, and costs are saved by not requiring the prior addition of exogenous enzymatic hydrolysis (e.g., the method of wang et al (Applied Microbiology and Biotechnology,2008,79(5): 785-)).

In the step E, a mode of adding sugar regularly is adopted, the reduced sugar concentration is maintained all the time in the fermentation process, so that the components on the shrimp head raw material can be favorably utilized by the thalli as a carbon source, the degradation and dissolution of the components on the raw material are promoted, the utilization rate of the raw material and the fermentation yield of the active peptide can be effectively improved, and the economic benefit is improved.

Preferably, the method comprises the following steps:

A. pretreatment of raw materials: drying the fresh shrimp heads in an oven at the temperature of 60-70 ℃ for 18-20 hours until the weight is constant, crushing the fresh shrimp heads by a crusher, and sieving the dried fresh shrimp heads by a standard sieve with the mesh of 8-80 to obtain crushed shrimp head powder, wherein the particle size of the crushed shrimp head powder is 0.2-2.5 mm;

B. strain activation: respectively inoculating the bacillus subtilis and the bacillus licheniformis to a slant culture medium, and culturing at 37-40 ℃ for 18-24 hours to respectively obtain a bacillus subtilis slant culture and a bacillus licheniformis slant culture;

C. preparing a seed solution: subjecting the Bacillus subtilis slant culture and the Bacillus licheniformis slantRespectively inoculating the culture to a seed liquid culture medium, performing shake culture at 37-40 deg.C and 200-300 rpm for 18-30 hr, adding appropriate amount of sterile seed liquid culture medium, diluting, and adjusting the thallus concentration to 1-50 × 10⁷CFU/mL to obtain a bacillus subtilis seed solution and a bacillus licheniformis seed solution, and mixing the bacillus subtilis seed solution and the bacillus licheniformis seed solution to obtain the seed solution;

D. preparing a fermentation medium, mixing materials and sterilizing: mixing the crushed shrimp head powder with 30-80 mL of the culture solution per 100g, uniformly mixing, adjusting the pH to 7.0-8.0 by using 0.5-0.6 mol/L sodium hydroxide solution, and sterilizing at 121-125 ℃ for 15-20 min to obtain the fermentation culture medium;

E. inoculation and solid state fermentation: mixing the seed solution with the fermentation medium, inoculating 5-20 mL of the seed solution per 100g of shrimp head meal, fermenting at 28-40 ℃ and relative humidity of 60-100%, starting from 24-36 hours of fermentation time, adding 10-50 mL of sterilized 20-25 g/L glucose solution per 100g of shrimp head meal per 12-24 hours, and fermenting for 4-7 days to obtain the fermentation material;

F. extraction and isolation of peptides: mixing the fermentation material with 200-500 mL of hydrochloric acid solution with the concentration of 0.05-0.08 mol/L per 100g, stirring at 150-200 rpm, extracting for 1-2 hours at 10-30 ℃, filtering the extract by filter cloth of 200-300 meshes to obtain a primary filtrate, adding active carbon according to 0.3-0.5% of the volume of the primary filtrate, adsorbing for 30-60 minutes at 40-45 ℃, filtering again, performing ultrafiltration by using a 5kDa ceramic membrane, retaining components with the molecular weight of less than 5kDa, performing secondary ultrafiltration by using a 1kDa ceramic membrane, retaining components with the molecular weight of less than 1kDa, combining and collecting filtrate with the molecular weight of less than 1kDa, loading the filtrate on a cation exchange column, and cleaning the column by using deionized water to remove non-peptide impurities after small peptides are completely adsorbed on the column, then eluting the polypeptide adsorbed on the cation exchange column by using 1-2 mol/L ammonia water, and collecting the eluate containing ACE inhibitory peptide;

G. concentrating and drying to obtain a finished product: and carrying out low-temperature vacuum concentration and freeze drying on the eluent to obtain the shrimp head fermented polypeptide powder containing ACE inhibitory peptide.

And E, mixing 5-20 mL of seed liquid per 100g of shrimp head powder contained in the fermentation medium, mixing the shrimp head powder with a small amount of the fermentation medium and the seed liquid to meet the requirement of thallus growth, and keeping the main body of the fermentation material in the step F to be solid, so that the fermentation cost can be effectively reduced.

Preferably, in the step C, the bacillus subtilis seed solution and the bacillus licheniformis seed solution are mixed according to the volume ratio of 1: 0.5-3 to obtain the seed solution.

Preferably, the bacillus subtilis is bacillus subtilis CMCC63501, and the bacillus licheniformis is bacillus licheniformis AS 1.269.

The bacillus comprises bacillus subtilis and bacillus licheniformis, the two strains can produce alkaline protease, neutral protease and aminopeptidase, and under the combined action of the alkaline protease, the neutral protease and the aminopeptidase, peptide fragments which are difficult to release by a general enzymolysis method can be obtained, and the bacillus subtilis strain CMCC63501 and the bacillus licheniformis strain AS1.269 are preferably used in the method.

That is, it is understood that the method of the present invention is not limited to the strains of CMCC63501 and Bacillus licheniformis is not limited to AS1.269, i.e., strains of Bacillus subtilis and Bacillus licheniformis have the ability to produce alkaline protease, neutral protease and aminopeptidase.

Preferably, in step B, the slant medium comprises: 5-6 g/L of glucose, 10-12 g/L of tryptone, 5-6 g/L of sodium chloride, 2-3 g/L of dipotassium hydrogen phosphate and 15-18 g/L of agar; adjusting the pH value to 7.0-7.2, and sterilizing for 15-20 minutes at 121-125 ℃.

Preferably, in step C, the seed liquid culture medium comprises: 10-20 g/L of glucose, 5-10 g/L of tryptone, 2-5 g/L of yeast powder, 1-5 g/L of sodium chloride, 2-3 g/L of dipotassium hydrogen phosphate, 7.0-7.5 of pH and 15-20 minutes of sterilization at 121-125 ℃.

Preferably, in step D, the culture solution comprises a carbon source, a nitrogen source, inorganic salts, and a surfactant; the concentration of the carbon source is 5-60 g/L, the concentration of the nitrogen source is 5-100 g/L, the concentration of the inorganic salt is 1-5 g/L, and the concentration of the surfactant is 0.1-1 g/L.

Preferably, the concentration of the carbon source is 10-30 g/L.

Preferably, the carbon source comprises one or more of glucose, mannitol and starch; the nitrogen source comprises one or more of yeast extract powder and ammonium chloride; the inorganic salt comprises one or more of dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate and calcium chloride; the surfactant comprises one or more of Tween 80 and surfactant.

Dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate, calcium chloride and the like can be used as inorganic salt to promote fermentation and supplement trace elements such as potassium, magnesium, zinc, iron, calcium and the like.

Preferably, in step F, the cation exchange column is Dowex 50WX 2.

Screening comparison through different cation exchange columns shows that Dowex 50WX2 can separate ACE inhibitory peptide more efficiently.

Compared with the prior art, the implementation of the invention has the following beneficial effects:

1. the invention adopts a fermentation method, the microorganism has the characteristics of fast growth and reproduction, simple growth condition, rich proteolytic enzyme systems and the like, and compared with the enzymolysis method, the fermentation method has low cost, does not need to add protease with high cost, and can hydrolyze and utilize a plurality of types of proteins. The polypeptide fragments which cannot be produced by the enzymolysis method can be obtained by the combined action of various proteases and peptidases produced by microorganisms in the fermentation process.

2. Aiming at the property that the shrimp head raw material is insoluble particles, the method is designed to be carried out by adopting a solid state fermentation mode. The dried shrimp heads are crushed to form solid particles with shells, the particles with proper particle size increase the specific surface area, the bacteria can be fully contacted with protein in the raw materials, so that small molecular peptides can be conveniently released by enzymolysis, particles with certain sizes are maintained, the function of a filling agent is realized, and the requirements of heat dissipation and air circulation for oxygen for bacteria growth in solid state fermentation are facilitated. In the solid state fermentation mode, the thallus can be adsorbed and adhered on the surface of the solid material to degrade insoluble protein, so that the defect that an enzymolysis method cannot hydrolyze insoluble protein is avoided, and the hydrolysis degree of the raw material is increased.

3. The invention adopts bacillus as a fermentation strain, and designs two strains to carry out mixed fermentation according to a proportion. The bacillus has the capability of producing various proteases and peptidases, and the combined action of the various proteases and the peptidases produced by the various enzymes of the two strains is beneficial to obtaining a peptide segment which is difficult to release by a general enzymolysis method.

4. In the solid-state fermentation process, a mode of adding sugar regularly is adopted, the reduced sugar concentration is maintained all the time in the fermentation process, so that the components on the shrimp head raw materials are favorably utilized by thalli as a carbon source, the degradation and dissolution of the components on the raw materials are promoted, the utilization rate of the raw materials and the fermentation yield of active peptides can be effectively improved, and the economic benefit is improved.

5. The invention provides two implementation modes of a triangular flask system and a fermentation tank system, has practicability and can be selectively implemented according to specific actual conditions.

6. The shrimp head fermented peptide developed by the method described by the invention has high yield and purity and good inhibitory activity (IC)₅₀Value less than 0.089mg/mL), and has good flavor and color

Can be added into nutritional food and special food as functional factor.

Drawings

FIG. 1 is a graph comparing the effect of solid state fermentation and enzymatic processes of the present application on the ACE inhibitory activity of raw material proteolysis and products;

FIG. 2 is a graph comparing the effects of the present application using single and mixed species;

FIG. 3 is a graph comparing the effect of sugar addition during fermentation according to the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings. Wherein, the Bacillus subtilis CMCC63501 and the Bacillus licheniformis AS1.269 are purchased from public depositary institutions according to the preservation numbers.

Example 1

In the embodiment, the ACE inhibitory peptide is prepared by performing solid state fermentation on shrimp heads by using mixed strains of Bacillus subtilis CMCC63501 and Bacillus licheniformis AS1.269 in a triangular flask system.

The specific implementation steps are as follows:

(1) preparation for raw material treatment

Drying fresh shrimp heads (by-products after processing shrimp meat) in a 60 ℃ oven for 18 hours to constant weight, crushing the dried shrimp heads by a crusher, and sieving the crushed shrimp heads by a standard sieve of 10 meshes, wherein the particle size of the crushed particles is about 2 mm.

(2) Activation of the Strain

The strain is inoculated on a slant culture medium and cultured for 24 hours at 37 ℃ for strain activation. The slant culture medium comprises: 5g/L of glucose, 10g/L of tryptone, 5g/L of sodium chloride, 2g/L of dipotassium hydrogen phosphate and 15g/L of agar; adjusting pH to 7.2, sterilizing at 121 deg.C for 20min

(3) Seed liquid preparation

Respectively inoculating slant strains of bacillus subtilis and bacillus licheniformis to a seed liquid culture medium, wherein the seed liquid culture medium comprises: 10g/L of glucose, 5g/L of tryptone, 2g/L of yeast powder, 1g/L of sodium chloride, 2g/L of dipotassium hydrogen phosphate, pH7.5 and sterilization at 121 ℃ for 20 minutes. Shake culturing at 37 deg.C and 200rpm for 24 hr, adding appropriate amount of sterile seed liquid culture medium into the cultured bacterial liquid, and diluting to adjust the bacterial concentration to 1 × 10⁷CFU/mL。

(4) Mixing and sterilizing

Fermentation medium: 10g/L of glucose and 5g/L of mannitol; 0.5g/L of yeast extract powder and 0.5g/L of ammonium chloride; 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate, 0.05g/L of ferrous sulfate, 0.1g/L of calcium chloride, 800.5 g/L of Tween and 0.02g/L of surfactant (Surfactin). The components are weighed and dissolved in deionized water to prepare a culture solution, and the culture solution is mixed according to the crushed shrimp head powder. 50g of shrimp head powder and 35mL of culture solution are filled into a 250mL triangular flask, stirred and mixed fully, the pH value is adjusted to 7.5 by 0.5mol/L sodium hydroxide solution after uniform mixing, and the mixture is sterilized for 15min at 121 ℃.

(5) Inoculation and fermentation

After the bacillus subtilis and the bacillus licheniformis seed liquid are mixed according to the proportion of 1:2, 10mL of mixed seed liquid is inoculated to every 100g of shrimp head powder. Fermenting in an incubator with the temperature of 28 ℃ and the relative humidity of 60 percent, adding 50mL of sterilized 20g/L glucose solution into 100g of shrimp head powder every 24 hours from the 24 th hour of fermentation time, and fully stirring and uniformly mixing so as to facilitate oxygen supply and release of fermentation heat; fermentation was completed by day 6.

(6) Peptide extraction and isolation

Adding 300mL of 0.05mol/L hydrochloric acid solution into every 100g of fermentation material, uniformly mixing, stirring at 100rpm, extracting at 10 ℃ for 1 hour, filtering the extracting solution through 200-mesh filter cloth to obtain an initial filtrate, adding activated carbon according to 0.3% of the volume of the initial filtrate, adsorbing at 40 ℃ for 30 minutes, and filtering again to obtain a supernatant of peptide extraction. And (3) carrying out ultrafiltration on the supernate by adopting a 5kDa ceramic membrane, reserving components with the molecular weight of less than 5kDa, carrying out secondary ultrafiltration by adopting a 1kDa ceramic membrane, reserving components with the molecular weight of less than 1kDa, and combining and collecting filtrates with the molecular weight of less than 1 kDa. The filtrate was applied to a cation exchange column (Dowex 50WX2), the column was washed with deionized water to remove non-peptide impurities, and then the polypeptide adsorbed on the column was eluted with 1mol/L ammonia water to collect the eluate.

(7) Drying to obtain final product

And (3) carrying out low-temperature vacuum concentration and freeze drying on the peptide solution obtained by ultrafiltration separation to obtain the shrimp head fermented peptide powder.

The performance of the shrimp head active peptide powder obtained by the method described in the embodiment is tested by adopting the following detection method:

1. the ACE inhibitory activity is determined by a High Performance Liquid Chromatography (HPLC) method:

ACE enzyme was dissolved in buffer solution containing 50 mmol/L4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and 300mmol/L NaCl to a final concentration of 0.1U/mL, and substrate equacy-histidyl-leucine (HHL) was dissolved in buffer solution containing 50mmol/L HEPES and 300mmol/L NaCl to a final concentration of 5 mmol/L. The total volume of the reaction system is 100 mu L, 40 mu L of reaction buffer solution (50mmol/L HEPES and 300mmol/L NaCl) and 40 mu L of sample to be testedMixing, keeping the temperature at 37 ℃ for 5min, then sequentially adding 10 mu L of 5mmol/L substrate HHL and 10 mu L of 0.1U/mL ACE, uniformly mixing, reacting at 37 ℃ for 30min, adding 250 mu L of 1mol/L HCl to terminate the reaction, and determining HA generated by the enzyme reaction by an HPLC method. Distilled water was used as a blank instead of the sample. HPLC detection conditions: mobile phase composition: 0.2% (v/v) TFA was dissolved in an aqueous solution containing 23% (v/v) acetonitrile. The flow rate is 1mL/min, the sample loading amount is 10 muL, the detection wavelength is 228nm, and the detection column temperature is 25 ℃. When the method is used for detection at 228nm, HA of a reaction product and HHL of an unreacted substrate both have absorption peaks, and the two absorption peaks can be well separated under the condition of the chromatographic analysis. IC (integrated circuit)₅₀Values are defined as the concentration of inhibitor required to inhibit 50% of ACE enzyme activity under the assay conditions.

2. Degree of hydrolysis DH% was determined by the ortho-phthalaldehyde (OPA) method:

degree of Hydrolysis (DH) the content of alpha-amino groups released by hydrolysis was determined according to the o-phthalaldehyde (OPA) method (Nielsen P. journal of Food Science,2010,66(5): 642-646.). The alpha-amino content is expressed in terms of serine concentration corresponding to the standard curve. The degree of hydrolysis DH% was expressed as the percentage of the alpha-amino group content determined on the enzymatically hydrolyzed sample to the alpha-amino group content determined after complete acid hydrolysis of the substrate protein.

3. The molecular weight distribution of the active peptide is determined by adopting a high-efficiency size exclusion chromatography injection method:

TSKgel G2000 SWXL 300mm × 7.8mm gel column, mobile phase composition: acetonitrile, water, trifluoroacetic acid (volume ratio) 40:59.95:0.05, detection wavelength: 220nm, flow rate of 0.5mL/min, column temperature: 30 ℃, injection volume: 10 μ L. The freeze-dried samples were prepared as 0.2mg/mL solutions, passed through a 0.22 μm filter and analyzed by sample injection under the chromatographic conditions described above. And (4) calculating the chromatogram and the data of the sample according to a relative molecular mass correction curve equation, and calculating the sum of the relative percentages of the polypeptides in each relative molecular mass range by adopting a peak area normalization method.

4. Protease and aminopeptidase Activity assays

The protease activity is determined by adopting a Folin phenol method, and referring to GB/T23527-2009, alkaline protease and neutral protein are respectively determined under the conditions of pH10.0 and pH7.0. The aminopeptidase measurement uses L-leucine-4-nitroaniline as a substrate, and the enzyme quantity consumed by hydrolyzing the substrate to generate 1 mu g of p-nitroaniline in unit time under the measurement condition is defined as one enzyme activity unit.

5. The protein content is determined by a Kjeldahl method.

The peptide content is determined by the method of GB/T22492-2008 appendix B.

Through detection, the shrimp head active peptide powder obtained by the method described in the embodiment has the following indexes:

the active peptide powder can be obtained at an amount of 8.63g per 100g of dried shrimp head powder, and has protein content (calculated on dry basis, N × 6.25) of 95.7%, peptide content (calculated on dry basis) of 93.85%, and IC₅₀The value reaches 0.054mg/mL, which is higher than the activity of preparing ACE inhibitory peptide by hydrolyzing shrimp heads by an enzymolysis method reported in the literature (food science, 2012,33(11): 131-.

Example 2

This example compares the effect of raw material proteolysis and the ACE inhibitory activity of the product using solid state fermentation and enzymatic hydrolysis methods, respectively, as described herein.

The solid state fermentation method was the same as that described in example 1, and samples were taken at 5 days of fermentation and the performance test was conducted by the same test method as that of example 1.

An enzymolysis method comprises the following steps: adding 20g of shrimp head dry powder into 200mL of distilled water, fully mixing to prepare 0.1g/mL of suspension, adding protease according to the ratio of 3000U/g of enzyme to substrate, carrying out enzyme reaction under the optimal condition of each enzyme (alkaline protease, trypsin and flavourzyme), dropwise adding 1mol/L of HCl or NaOH during the enzyme reaction to maintain the pH value at the optimal condition, and sampling and measuring after 6 hours of enzymolysis. The bacillus subtilis zymoprotein liquid is prepared by the following method: the strain CMCC63501 is inoculated to a seed liquid culture medium and is subjected to shake cultivation for 24 hours at 37 ℃ and 200 rpm. Inoculating the seed solution into a liquid fermentation enzyme production culture medium according to the inoculation amount of 2%, performing shake culture at 37 ℃ and 200rpm for 36 hours, then centrifuging at 6000rpm for 10min, and collecting the supernatant, namely the zymoproteinase. Liquid fermentation enzyme production culture medium: 15g/L of glucose, 30g/L of soybean meal and CaCl₂ 2g/L，K₂HPO₄2g/L, Tween 801 g/L, NaCl 2gL, sterilizing at 115 deg.C for 15min, pH 8.0. And mixing the enzyme solution obtained by fermentation with 0.2g/mL shrimp head powder suspension in equal volume, performing enzymolysis for 6 hours at the temperature of 40 ℃ at the pH of 7.5, sampling, and performing performance test by adopting the detection method in the same example 1.

The comparative results are shown in FIG. 1. As can be seen from FIG. 1, the highest proteolysis rate achieved by the solid-state fermentation method is up to 60.7%, and the proteolysis rates obtained by various enzymatic hydrolysis methods using different enzymes are between 18% and 24% (similar to the proteolysis rate of shrimp heads hydrolyzed by enzymatic hydrolysis method reported in the literature (food science, 2012,33(11): 131-. In addition, the solid-state fermentation method also obtains more micromolecule polypeptides with the molecular weight less than 1KDa, which shows that the method can more effectively utilize the solid shrimp head meal raw material to release the micromolecule polypeptides. However, the existing research and report show that the ACE inhibitory peptides with good activity are all small-molecule peptides with the molecular weight less than 1 KDa.

Example 3

This example illustrates the use of single and mixed species fermentation, respectively, for comparison.

After pretreatment of raw materials, stirring and sterilization, B.subtilis and B.licheniformis which are used independently and two strains which are combined and inoculated according to different proportions of 1:0.5, 1:1, 1:2 and 1:3 are added respectively, fermentation is carried out according to the fermentation conditions described in the example 1, samples are taken after 6 days of fermentation, and the performance test is carried out by adopting the detection method of the example 1.

The comparative results are shown in FIG. 2. As can be seen from FIG. 2, when a single strain is adopted, three indexes of the degree of proteolysis, the ACE inhibitory activity and the content of small molecular polypeptide smaller than 1KDa are all lower than those of mixed strain fermentation. As can be seen from the results of different ratios of the two strains, the proteolysis degree and the ACE inhibitory activity obtained by the four experimental ratios are not obviously different, but the proportion of the micromolecule polypeptide which is less than 1KDa and is obtained by the two ratios of 1:1 and 1:2 is higher, so that the micromolecule polypeptide is more favorably released.

Example 4

This example illustrates the investigation of the effect of the feeding addition of sugar on the fermentation process.

The raw material pretreatment, material mixing and sterilization, seed solution preparation and inoculation are the same as those in example 1, glucose is not added in the fermentation process after inoculation, the mode 1 (32 mL of 50g/L glucose is added once on day 2), the mode 2 (20 mL of 40g/L glucose is added twice on days 3 and 5) and the mode 3 (20 mL of 20g/L glucose is added respectively on days 2,3, 4 and 5) are adopted respectively, samples are taken after 6 days of fermentation, and the performance test is carried out by adopting the detection method in example 1.

The comparative results are shown in FIG. 3. As can be seen from FIG. 3, the addition of sugar during the fermentation process can significantly improve the degree of proteolysis, ACE inhibitory activity and the content of small molecular polypeptides smaller than 1 KDa. Of the three modes of adding sugar, the effect of one-time addition (mode 1) is the worst, and the three indexes are all lower than the modes 2 and 3. By means of the mode 3, namely adding a small amount of sugar every day from the 2 nd day of fermentation, better hydrolysis degree and more small molecular polypeptides can be obtained, and the mode is shown to enable the hydrolysis efficiency of raw materials to be higher and the yield to be better.

Example 5

This example illustrates the investigation of the effect of surfactant addition on enzyme production.

The contents and combinations of different surfactants were added to the fermentation medium, the other conditions and operations were the same as those described in example 1, solid state fermentation was performed after separately mixing and sterilizing, and after 3 days of fermentation, samples were taken to determine the activities of alkaline protease, neutral protease and aminopeptidase, and the results are shown in Table 1.

As can be seen from Table 1, the addition of the surfactant can significantly improve the yield of the protease and the aminopeptidase, and the combination of the two surfactants is more favorable for producing the enzyme than the single addition of the surfactant, the two proteases are respectively improved by more than 40 percent, and the aminopeptidase is improved by 56.8 percent

TABLE 1 Effect of different surfactant additions on enzyme production by the strains

Example 6

This example illustrates the preparation of ACE inhibitory peptides using solid state fermentation shrimp heads of a mixed strain of Bacillus subtilis CMCC63501 and Bacillus licheniformis AS1.269 in a solid state fermentor (horizontal rotary shaft mechanically agitated fermentor 30L) system.

The specific implementation steps are as follows:

(1) preparation for raw material treatment

Drying fresh shrimp heads (by-products after processing shrimp meat) in a 70 ℃ oven for 20 hours to constant weight, crushing the dried shrimp heads by a crusher, and sieving the crushed shrimp heads by a standard sieve of 80 meshes to obtain granules with the particle size of about 0.2 mm.

(2) Activation of the Strain

The strain is inoculated on a slant culture medium and cultured for 18 hours at 40 ℃ for strain activation. The slant culture medium comprises: 6g/L of glucose, 12g/L of tryptone, 6g/L of sodium chloride, 3g/L of dipotassium hydrogen phosphate and 18g/L of agar; adjusting pH to 7.0, and sterilizing at 125 deg.C for 15 min.

(3) Seed liquid preparation

Respectively inoculating slant strains of bacillus subtilis and bacillus licheniformis to a seed liquid culture medium, wherein the seed liquid culture medium comprises: 20g/L of glucose, 10g/L of tryptone, 5g/L of yeast powder, 5g/L of sodium chloride, 3g/L of dipotassium hydrogen phosphate, pH7.0 and sterilizing for 15 minutes at 125 ℃. Shake culturing at 40 deg.C and 300rpm for 18 hr, adding appropriate amount of sterile seed liquid culture medium into the cultured bacterial liquid, and diluting to adjust the bacterial concentration to 5 × 10⁸CFU/mL。

(4) Mixing and sterilizing

Fermentation medium: 8g/L of glucose, 2g/L of mannitol and 5g/L of starch; 0.5g/L of yeast extract powder and 0.5g/L of ammonium chloride; 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate, 0.05g/L of ferrous sulfate and 0.1g/L of calcium chloride; tween 800.5 g/L, and Surfactin (Surfactin)0.02 g/L. The components are weighed and dissolved in deionized water to prepare culture solution, the culture solution is mixed according to the crushed shrimp head powder, 80mL of culture solution is adopted for every 100g of shrimp head powder, and the pH value is adjusted to 7.0 by 0.5mol/L sodium hydroxide solution after uniform mixing. 30L solid state fermenter with 35% weight, and sterilizing in situ at 121 deg.C for 15 min.

(5) Inoculation and fermentation

After the bacillus subtilis and the bacillus licheniformis seed liquid are mixed according to the proportion of 1:1, 20mL of mixed seed liquid is inoculated to every 100g of shrimp head powder. The conditions of the fermentation tank after inoculation are controlled as follows: the temperature is 40 ℃, the relative humidity is 95%, the stirring speed is 5rpm, the aeration flow is 0.2-0.3 VVM, the tank pressure is 0.01-0.03 MPa, 10mL of sterilized 25g/L glucose solution is added into 100g of shrimp head powder every 12 hours from the beginning of 36 hours of fermentation time, and the fermentation is finished for 5 days.

(6) Peptide extraction and isolation

Adding 500mL of 0.08mol/L hydrochloric acid solution into every 100g of fermentation material, uniformly mixing, extracting for 2 hours in a container at the temperature of 30 ℃ under the condition of stirring at 200rpm, filtering the extracting solution through 300-mesh filter cloth to obtain primary filtrate, adding activated carbon according to 0.5% of the volume of the primary filtrate, adsorbing for 60 minutes at 45 ℃, and filtering again to obtain the supernatant of peptide extraction. And (3) ultrafiltering the supernatant by using a 5kDa ceramic membrane, reserving components with the molecular weight of less than 5kDa, then carrying out second ultrafiltration by using a 1kDa ceramic membrane, reserving components with the molecular weight of less than 1kDa, merging and collecting filtrate. The filtrate was applied to a cation exchange column (Dowex 50WX2), the column was washed with deionized water to remove non-peptide impurities, and then the polypeptide adsorbed on the column was eluted with 1mol/L ammonia water to collect the eluate.

(7) Drying to obtain final product

The performance test is carried out by adopting the detection method in the example 1, and the shrimp head peptide powder obtained by the method described in the example has the following indexes:

12.05g of active peptide powder can be obtained per 100g of dried shrimp head powder, and the prepared active peptide powder has protein content (calculated by dry basis, N × 6.25) 92.2%, peptide content (calculated by dry basis) 89.6%, and IC₅₀The value reached 0.089 mg/mL.

It can be seen that both in the pilot plant of example 1 and in the fermenter scale up of example 6, the process of the present invention provides ACE inhibitory peptides with good inhibitory activity in high yield and purity. Compared with the traditional enzymolysis method, the method has the advantage that the substrate does not need to be added in advance to produce the enzyme, so that the cost is greatly saved in large-scale production. In the presence of the strain, the shrimp heads are used as raw materials, the enzyme production and the substrate proteolysis are synchronously generated and mutually promoted, the proteolysis rate is greatly improved, and more and purer ACE inhibitory peptides are obtained.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.

Claims

1. A method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains is characterized by comprising the following steps:

E. inoculation and solid state fermentation: mixing the seed liquid with the fermentation medium, starting solid state fermentation, and supplementing a sugar source from the fermentation time of 24-36 hours until the fermentation is finished to obtain a fermentation material;

2. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 1, comprising the following steps:

C. preparing a seed solution: respectively inoculating the bacillus subtilis slant culture and the bacillus licheniformis slant culture to a seed liquid culture medium, performing shake culture for 18-30 hours at 37-40 ℃ and 200-300 rpm, then adding a proper amount of sterile seed liquid culture medium for dilution, and adjusting the concentration of thalli to 1-50 × 10⁷CFU/mL to obtain a bacillus subtilis seed solution and a bacillus licheniformis seed solution, and mixing the bacillus subtilis seed solution and the bacillus licheniformis seed solution to obtain the seed solution;

3. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 2, wherein in step C, the bacillus subtilis seed solution and the bacillus licheniformis seed solution are mixed according to a volume ratio of 1: 0.5-3 to obtain the seed solution.

4. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains AS claimed in claim 3, wherein the Bacillus subtilis strain is Bacillus subtilis CMCC63501 and the Bacillus licheniformis strain is Bacillus licheniformis AS 1.269.

5. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 2, wherein in step B, the slant culture medium comprises: 5-6 g/L of glucose, 10-12 g/L of tryptone, 5-6 g/L of sodium chloride, 2-3 g/L of dipotassium hydrogen phosphate and 15-18 g/L of agar; adjusting the pH value to 7.0-7.2, and sterilizing for 15-20 minutes at 121-125 ℃.

6. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 2, wherein in step C, the seed liquid culture medium comprises: 10-20 g/L of glucose, 5-10 g/L of tryptone, 2-5 g/L of yeast powder, 1-5 g/L of sodium chloride, 2-3 g/L of dipotassium hydrogen phosphate, 7.0-7.5 of pH and 15-20 minutes of sterilization at 121-125 ℃.

7. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 2, wherein in step D, the culture solution comprises carbon source, nitrogen source, inorganic salt and surfactant; the concentration of the carbon source is 5-60 g/L, the concentration of the nitrogen source is 5-100 g/L, the concentration of the inorganic salt is 1-5 g/L, and the concentration of the surfactant is 0.1-1 g/L.

8. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 7, wherein the concentration of the carbon source is 10-30 g/L.

9. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 7, wherein the carbon source comprises one or more of glucose, mannitol, starch; the nitrogen source comprises one or more of yeast extract powder and ammonium chloride; the inorganic salt comprises one or more of dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate and calcium chloride; the surfactant comprises one or more of Tween 80 and surfactant.

10. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains as claimed in claim 2, wherein in step F, the cation exchange column is Dowex 50WX 2.