CN112852909B - Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by solid-state fermentation of shrimp heads by mixed strains - Google Patents
Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by solid-state fermentation of shrimp heads by mixed strains Download PDFInfo
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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, preparation of seed liquid, preparation and sterilization of fermentation culture medium, inoculation and fermentation, extraction and separation of peptide, concentration, drying and the like. The beneficial effects of the invention are as follows: the solid state fermentation method is adopted, so that the cost is low, and protease is not required 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 raw material utilization rate are increased, and the heat dissipation in fermentation and the oxygen demand of thalli 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; adopting a process of periodically supplementing sugar, maintaining lower sugar concentration so as to facilitate the thalli to utilize the components on the raw materials and promote the degradation and dissolution of the components on the raw materials; the shrimp head fermented peptide developed by the method disclosed by the invention has high yield and purity and good inhibitory activity.
Description
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 by mixed strains.
Background
Hypertension is one of the most common chronic diseases, mainly represented by the syndrome of increased arterial pressure of the systemic circulation, and is an important cause and inducing factor of various cardiovascular and cerebrovascular diseases. At present, the overall awareness rate, the treatment rate and the control rate of the hypertension diseases in China are obviously lower. The administration of the chemically synthesized antihypertensive drug is a common treatment method, and although the antihypertensive effect is obvious, adverse reactions such as dry cough, rash, angioedema, proteinuria, leukopenia and drug withdrawal syndrome are easy to cause after long-term administration. Therefore, it is very necessary to find a novel, safe and economical functional ingredient for lowering blood pressure as a pharmaceutical or food functional factor for preventing and treating hypertension. The food-source antihypertensive peptide is a peptide segment derived from a food protein sequence, and plays a role in inhibiting or relieving blood pressure rise 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, so that the food source ACE inhibitory peptide becomes an important research direction for non-drug treatment of hypertension.
The shrimp heads are main byproducts for producing shrimp meat or headless shrimp and the like, and account for about one third of the weight of the shrimp. The shrimp heads are removed from fresh shrimps, the histoses are active, the moisture content is high, the shrimps are difficult to store, most of the shrimps are often directly discarded after being processed, or the shrimps are simply processed (such as dried and crushed) to form the feed for cultivation, so that the environment is polluted, and the waste of resources is caused. The shrimp heads contain a large amount of protein nutrient substances, and are good raw materials for obtaining antihypertensive active peptide. The research and development of the shrimp head protein source ACE inhibitory peptide as a functional food factor has 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 disclosed technology for preparing ACE inhibitory peptide by using shrimp head protein, an enzymolysis method is mainly adopted, and few fermentation methods are adopted. Levoqi et al uses chymotrypsin and proline proteinase to perform two-step enzymolysis on shrimp byproducts to prepare ACE inhibitory peptide, and the enzymolysis product ACE inhibitory IC 50 The value was 1.645mg/mL (food industry technology, 2014,35 (10): 181-185). Shi Xushan, etc., wherein shrimp shell powder is used as a raw material, preferably neutral protease is used as an optimal enzyme, the ACE inhibition rate of the polypeptide is 84.04%, and the hydrolysis degree is 26.76% (food science, 2012,33 (11): 131-136). Feng Bing and the like for preparing ACE inhibitory peptide by utilizing alkaline protease to hydrolyze shrimp head protein, and performing gel filtration on the enzymolysis product to obtain IC with highest ACE inhibitory activity component 50 0.79mg/mL (food science, 2012,33 (11): 131-136). Zhu Guoping method for preparing ACE inhibitory peptide by hydrolyzing protein with endogenous protease of shrimp head in autolysis process, ultrafiltering, gel chromatography and ion exchange chromatography 50 0.19mg/mL (aquatic journal 2013,37 (4): 631-640). The enzymolysis method has the advantages of simpler process, mild condition and capability of completing the enzymolysis process in a shorter time. Because of the degree of randomness of the proteolytic process, how to select appropriate reaction conditions to control the proteolytic process is critical to achieving high yields of active peptide. The enzymolysis method can achieve better workFor industrial application, proteases and peptidases with specific cleavage site capability are required, and deep hydrolysis of raw material proteins is also required to achieve a higher degree of hydrolysis, thereby releasing peptides with high ACE activity. To achieve the above object, two or more enzymes are often required for hydrolysis, and high-purity enzymes are required for the process, increasing the production cost.
The invention patent CN200710303512.5 discloses a Chinese penaeus protein antihypertensive peptide, a preparation method and application thereof, wherein bacillus subtilis is used as a strain to prepare an enzyme solution through liquid fermentation, and the enzyme solution is mixed with slurry prepared by pulping Chinese penaeus and subjected to enzymolysis, so that the antihypertensive peptide with six amino acids is separated and purified from the enzymolysis solution, and the antihypertensive peptide has higher Angiotensin Converting Enzyme (ACE) inhibitory activity. In addition, according to the action sites of in vivo enzymes, the three possible enzymolysis products of the hexapeptide are chemically synthesized, and the 12 sequences of the short peptides obtained by screening through an ACE inhibition test have good ACE inhibition activity.
In the prior enzymolysis technology, the protease reaction system is carried out in aqueous solution, and the substrate object of the protease reaction is mainly water-soluble protein in materials, but for shrimp head raw materials, the protease reaction system has less soluble components 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 rule, so that the good raw material utilization rate and polypeptide yield are difficult to obtain.
Microbial fermentation is one of the important methods for preparing food source ACE inhibitory active peptides. The key point of the fermentation method for preparing antihypertensive peptide by using marine product protein is food-safe strain with high protease and peptidase activities and capability of releasing specific functional peptide fragments. Wang et al used lactic acid bacteria to ferment shrimp meat to obtain ACE inhibitory peptides (Applied Microbiology and Biotechnology,2008,79 (5): 785-791.). Because lactobacillus growth and fermentation have high requirements on nutrition and weak proteolytic activity, the culture medium is required to contain available carbon sources and growth factors, and the lactobacillus is often used for fermentation, and the protease is often used for hydrolysis of raw materials in advance for fermentation, so that added substances bring difficulty to later separation and purification, and the process is complicated.
Disclosure of Invention
The invention aims to solve the problems that in the prior art, the ACE inhibitory peptide is prepared by taking shrimp heads as raw materials, and a method with high hydrolysis rate of the raw materials and good product inhibitory activity is lacking. At present, the enzymolysis method is mainly adopted, the shrimp heads mainly comprise insoluble solid components, the enzymolysis efficiency is low in a liquid phase system, and the high proteolytic rate is difficult to obtain. The invention adopts a solid state fermentation method, utilizes inoculation of mixed strains, adopts a mode of adding sugar in the fermentation process for fermentation, improves the hydrolysis rate of protein, and obtains the small molecular polypeptide with high inhibition 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 by 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 activation culture on bacillus subtilis and bacillus licheniformis to obtain slant culture;
C. preparing seed liquid: performing expansion culture on the inclined plane culture to obtain seed liquid;
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. inoculating and solid state fermentation: mixing the seed liquid with the fermentation culture medium, starting fermentation, and supplementing a sugar source from the fermentation time of 24-36 hours 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, and then filtering, decoloring, ultrafiltering, eluting to remove impurities to obtain an eluent containing ACE inhibitory peptide;
G. concentrating and drying to obtain the finished product: concentrating and drying the eluent to obtain shrimp hair fermentation polypeptide powder containing ACE inhibitory peptide.
In the step A, after shrimp heads are dried, the shrimp heads are crushed into particles with proper particle size, so that the specific surface area is increased, the shrimp heads 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 maintained, so that the shrimp heads play a role of a filler, and the heat dissipation and air circulation in solid state fermentation are facilitated for oxygen for the growth of the bacteria.
The strain used in the invention is a combination of bacillus subtilis and bacillus licheniformis, namely the strain of bacillus subtilis and bacillus licheniformis with the capability of producing alkaline protease, neutral protease and aminopeptidase can realize the technical scheme. The bacillus growth has low nutrition requirement, the proteolytic enzyme system is rich, and can secrete a plurality of extracellular proteases and aminopeptidases, wherein the bacillus subtilis and the bacillus licheniformis are important production strains for commercial neutral proteases and alkaline proteases. In addition, bacillus subtilis and the like are common strains for producing fermented foods such as fermented soybeans, bean paste, natto and the like, have good safety, and are suitable for preparing food-source ACE inhibitory peptides as fermentation strains. In the existing technology for preparing ACE inhibitory peptide by using whole shrimps, as in the invention patent CN200710303512.5, strains are inoculated in advance into a liquid culture medium containing specific proteins for fermentation and centrifugation to remove thalli so as to prepare enzyme liquid, and the enzyme liquid prepared by fermentation is used for hydrolyzing target proteins (mainly water-soluble proteins and low in utilization rate of insoluble proteins) in raw materials under a liquid phase system, so that a target product is obtained. However, the invention adopts a solid state fermentation method, and the thalli directly grow on a solid raw material matrix, so that protease and aminopeptidase can be directly produced by taking shrimp heads as substrates, and the protease and aminopeptidase can hydrolyze proteins on shrimp heads raw materials on site to produce ACE inhibitory peptides. Compared with the enzymolysis process in a liquid phase system, the bacterial cells grow on the raw materials, which is favorable for further degrading the raw material particles to promote the dissolution of proteins, can effectively degrade insoluble solid proteins and improves the proteolysis rate of the raw materials. Under the continuous existence of the preferred bacillus strain combination, enzyme production and hydrolysis of substrate proteins synchronously occur and mutually promote, the production efficiency of ACE inhibitory peptide is greatly improved, and the cost is saved because the method of pre-adding exogenous enzyme hydrolysis (such as wang and the like (Applied Microbiology and Biotechnology,2008,79 (5): 785-791)) is not needed.
In the step E, the method of periodically supplementing sugar is adopted, and the reduced sugar concentration is maintained all the time in the fermentation process, so that the thalli can utilize the components on the shrimp head raw material as a carbon source, the degradation and dissolution of the components on the raw material are promoted, the raw material utilization rate 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 60-70 ℃ for 18-20 hours to constant weight, crushing by a crusher, and sieving by a 8-80 mesh standard sieve 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 for 18-24 hours at 37-40 ℃ to obtain a bacillus subtilis slant culture and a bacillus licheniformis slant culture respectively;
C. preparing seed liquid: inoculating the bacillus subtilis slant culture and the bacillus licheniformis slant culture into seed liquid culture medium respectively, shake culturing at 37-40deg.C and 200-300 rpm for 18-30 hr, adding appropriate amount of sterile seed liquid culture medium for dilution, and adjusting thallus concentration to 1-50X10 7 CFU/mL is used for obtaining a bacillus subtilis seed solution and a bacillus licheniformis seed solution, and the bacillus subtilis seed solution and the bacillus licheniformis seed solution are mixed 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, regulating the pH to 7.0-8.0 by using 0.5-0.6 mol/L sodium hydroxide solution after uniformly mixing, and sterilizing for 15-20 min at 121-125 ℃ to obtain the fermentation culture medium;
E. inoculating and solid state fermentation: mixing the seed liquid with the fermentation medium, inoculating 5-20 mL of the seed liquid to each 100g of shrimp head powder, fermenting at 28-40 ℃ under the condition of 60-100% relative humidity, starting from 24-36 hours of fermentation time, adding 10-50 mL of sterilized 20-25 g/L glucose solution to each 100g of shrimp head powder every 12-24 hours, and ending the fermentation until 4-7 days to obtain the fermentation material;
F. extraction and isolation of peptides: mixing the fermented 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 at 10-30 ℃ for 1-2 hours, filtering the extracting solution through 200-300 meshes of filter cloth to obtain primary filtrate, adding activated carbon according to 0.3-0.5% of the volume of the primary filtrate, adsorbing for 30-60 minutes at 40-45 ℃, filtering again, ultrafiltering by adopting a 5kDa ceramic membrane, retaining components with the molecular weight of less than 5kDa, ultrafiltering for the second time by adopting 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, washing by adopting deionized water after the small peptides are completely adsorbed on the column, eluting the polypeptides adsorbed on the cation exchange column by using 1-2 mol/L of ammonia water, and collecting eluent containing ACE inhibitory peptides;
G. concentrating and drying to obtain the finished product: concentrating the eluent in vacuum at low temperature, and freeze-drying to obtain shrimp hair ferment polypeptide powder containing ACE inhibitory peptide.
The shrimp head meal in the step E is mixed with 5-20 mL of seed liquid according to the mass of each 100g of the shrimp head meal, and the shrimp head meal is only mixed with a small amount of the fermentation medium and the seed liquid so as to provide the requirement of bacterial growth, and the fermentation material main body in the step F is still 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 AS1.269.
The bacillus comprises bacillus subtilis and bacillus licheniformis, and the two strains can produce alkaline protease, neutral protease and aminopeptidase, under the combined action of the alkaline protease, neutral protease and aminopeptidase, peptide fragments which are difficult to release by a general enzymolysis method are obtained, and the bacillus subtilis strain CMCC63501 and the bacillus licheniformis strain AS1.269 are used in the method.
That is, it is to be understood that the strain of Bacillus subtilis used in the method of the present invention is not limited to CMCC63501 and Bacillus licheniformis strain, and is not limited to AS1.269, i.e., the strains of Bacillus subtilis and Bacillus licheniformis having the ability to produce alkaline protease, neutral protease and aminopeptidase can achieve the technical scheme.
Preferably, in step B, the slant culture medium comprises: glucose 5-6 g/L, tryptone 10-12 g/L, sodium chloride 5-6 g/L, dipotassium hydrogen phosphate 2-3 g/L, agar 15-18 g/L; adjusting the pH value to 7.0-7.2, sterilizing for 15-20 minutes at the temperature of 121-125 ℃.
Preferably, in step C, the seed solution medium includes: 10-20 g/L glucose, 5-10 g/L tryptone, 2-5 g/L yeast powder, 1-5 g/L sodium chloride, 2-3 g/L dipotassium hydrogen phosphate, pH of 7.0-7.5 and sterilizing at 121-125 ℃ for 15-20 minutes.
Preferably, in the step D, the culture solution includes a carbon source, a nitrogen source, an inorganic salt, 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 to 30g/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 salts to promote fermentation, and can also supplement microelements such as potassium, magnesium, zinc, iron, calcium and the like.
Preferably, in step F, the cation exchange column is Dowex 50WX2.
Comparison of screening by different cation exchange columns found that Dowex 50WX2 allows ACE inhibiting peptides to be separated more efficiently.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
1. the invention adopts a fermentation method, and the microorganism has the characteristics of quick growth and reproduction, simple growth condition, rich proteolytic enzyme system 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 more protein types. 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 solid state fermentation mode is adopted. The dried shrimp heads are crushed to form solid particles with shells, the particles with proper particle size increase the specific surface area, so that the particles can fully contact with protein in the raw materials to facilitate enzymolysis and release of small molecular peptides, and meanwhile, the particles with a certain size are kept to play a role of a filler, thereby being beneficial to the heat dissipation and air circulation in solid fermentation for oxygen for the growth of the bacteria. In the solid state fermentation mode, the thalli can be adsorbed and adhered on the surface of a solid material to degrade insoluble proteins, so that the defect that an enzymolysis method cannot hydrolyze insoluble proteins is avoided, and the hydrolysis degree of raw materials is increased.
3. The invention adopts bacillus as a fermentation strain, and designs two strains to perform mixed fermentation according to a proportion. The bacillus strain 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 peptide fragments which are difficult to release by a general enzymolysis method.
4. In the solid state fermentation process, the method adopts a mode of periodically supplementing sugar, and the reduced sugar concentration is maintained all the time in the fermentation process, so that the thallus can utilize the components on the shrimp head raw material as a carbon source, the degradation and dissolution of the components on the raw material are promoted, the raw material utilization rate and the fermentation yield of the active peptide can be effectively improved, and the economic benefit is improved.
5. The invention provides two embodiments of a triangular bottle system and a fermentation tank system, which have practicability and can be selectively implemented according to specific practical conditions.
6. The shrimp head fermented peptide developed by the method has high yield and purity, and good inhibitory activity (IC) 50 A value of less than 0.089 mg/mL), and has good flavor and color, and can be used as
Is added into nutritional food and special food for functional factors.
Drawings
FIG. 1 is a graph showing the comparison of the effect of solid state fermentation and enzymolysis methods of the present application on ACE inhibitory activity of raw material protein hydrolysates and products;
FIG. 2 is a graph showing the comparison of the effects of the application using a single strain and a mixed strain;
FIG. 3 is a graph comparing the effect of feed sugar addition during fermentation of the present application.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Among them, bacillus subtilis (Bacillus subtilis) CMCC63501 and Bacillus licheniformis (Bacillus licheniformis) AS1.269, purchased from public depository according to accession number.
Example 1
In the embodiment, ACE inhibitory peptide is prepared by solid state fermentation of shrimp heads in a triangular flask system by using a mixed strain of bacillus subtilis (Bacillus subtilis) CMCC63501 and bacillus licheniformis (Bacillus licheniformis) AS1.269.
The specific implementation steps are as follows:
(1) Preparation for raw material treatment
Fresh shrimp heads (by-products after processing shrimp meat) are dried in an oven at 60 ℃ for 18 hours to constant weight, the dried shrimp heads are crushed by a crusher and then pass through a 10-mesh standard sieve, and the particle size of the crushed particles is about 2 mm.
(2) Activation of strains
The strain was inoculated on a slant for 24 hours based on 37℃for strain activation. The slant culture medium comprises: glucose 5g/L, tryptone 10g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 2g/L and agar 15g/L; adjusting pH to 7.2, sterilizing at 121deg.C for 20min
(3) Seed liquid preparation
Inoculating bacillus subtilis and bacillus licheniformis inclined plane strains into a seed liquid culture medium respectively, wherein the seed liquid culture medium comprises the following components: 10g/L glucose, 5g/L tryptone, 2g/L yeast powder, 1g/L sodium chloride, 2g/L dipotassium hydrogen phosphate, pH7.5 and sterilizing at 121 ℃ for 20 minutes. Shaking culture at 37deg.C and 200rpm for 24 hr, adding appropriate amount of sterile seed solution culture medium to dilute the cultured bacterial solution to adjust bacterial concentration to 1×10 7 CFU/mL。
(4) Mixing and sterilizing
Fermentation medium: glucose 10g/L and mannitol 5g/L; yeast extract powder 0.5g/L and ammonium chloride 0.5g/L; dipotassium phosphate 2g/L, magnesium sulfate 0.2g/L, ferrous sulfate 0.05g/L, calcium chloride 0.1g/L, tween 80 0.5g/L, and surfactant (surfactant) 0.02g/L. 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. The shrimp head powder 50g and the culture solution 35mL are filled into a 250mL triangular flask, stirred and fully mixed, the pH value is regulated to 7.5 by 0.5mol/L sodium hydroxide solution after the mixture is evenly mixed, and the mixture is sterilized for 15min at 121 ℃.
(5) Inoculation and fermentation
The bacillus subtilis and bacillus licheniformis seed liquid are mixed according to the proportion of 1:2, and 10mL of mixed seed liquid is inoculated per 100g of shrimp head powder. Fermenting in an incubator with the temperature of 28 ℃ and the relative humidity of 60%, adding 50mL of sterilized 20g/L glucose solution into 100g of shrimp head powder every 24 hours from 24 hours of fermentation time, and fully stirring and uniformly mixing to facilitate oxygen supply and heat generation of fermentation; fermentation was completed by day 6.
(6) Extraction and separation of peptides
300mL of 0.05mol/L hydrochloric acid solution is added into each 100g of fermentation material, the mixture is stirred at 100rpm and extracted for 1 hour at 10 ℃, the extract is filtered by a 200-mesh filter cloth to obtain primary filtrate, activated carbon is added according to 0.3 percent of the volume of the primary filtrate, and the primary filtrate is filtered again after being adsorbed for 30 minutes at 40 ℃ to obtain supernatant extracted by peptide. Ultrafiltering the supernatant with 5kDa ceramic membrane to retain the component with molecular weight less than 5kDa, ultrafiltering with 1kDa ceramic membrane for the second time to retain the component with molecular weight less than 1kDa, and mixing the filtrates. The filtrate was applied to a cation exchange column (Dowex 50WX 2), the column was washed with deionized water to remove non-peptide impurities, then the polypeptide adsorbed on the column was eluted with 1mol/L aqueous ammonia, and the eluate was collected.
(7) Drying to obtain the final product
And (3) carrying out low-temperature vacuum concentration and freeze drying on the peptide solution obtained by ultrafiltration separation to obtain shrimp hair ferment peptide powder.
The shrimp head bioactive peptide powder obtained by the method described in the embodiment is subjected to performance test by adopting the following detection method:
1. ACE inhibition activity was measured by High Performance Liquid Chromatography (HPLC):
ACE enzyme was dissolved in buffer containing 50 mmol/L4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and 300mmol/L NaCl to a final concentration of 0.1U/mL, and substrate hippocampal-histidyl-leucine (HIL) was dissolved in buffer containing 50mmol/L HEPES and 300mmol/L NaCl to a final concentration of 5mmol/L. After the reaction buffer solution (50 mmol/L HEPES and 300mmol/L NaCl) and 40. Mu.L sample to be tested were mixed together in a total volume of 100. Mu.L, the mixture was incubated at 37℃for 5 minutes, then 10. Mu.L of 5mmol/L substrate HHT and 10. Mu.L of 0.1U/mL ACE were sequentially added, reacted at 37℃for 30 minutes after the mixture was homogenized, and the reaction was terminated by adding 250. Mu.L of 1mol/L HCl, and HA produced by the enzymatic reaction was measured by HPLC. Distilled water was used instead of the sample as a blank. 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 was 1mL/min, the loading amount was 10. Mu.L, the detection wavelength was 228nm, and the detection column temperature was 25 ℃. When 228nm detection is adopted, the reaction product HA and the unreacted substrate HIL both have absorption peaks, and the two absorption peaks can be well separated under the condition of the intrinsic chromatographic. IC (integrated circuit) 50 The value is defined as the concentration of inhibitor required to inhibit 50% of ace enzyme activity under the assay conditions.
2. The degree of hydrolysis DH% was determined using the o-phthalaldehyde (OPA) method:
the Degree of Hydrolysis (DH) the alpha-amino content released by hydrolysis was determined according to the o-phthalaldehyde (OPA) method (Nielsen P M. Journal of Food Science,2010,66 (5): 642-646.). The alpha-amino content is expressed as serine concentration corresponding to the standard curve. The percentage of alpha-amino content measured as an enzymatic sample to the alpha-amino content measured after complete acid hydrolysis of the substrate protein represents the degree of hydrolysis DH%.
3. The molecular weight distribution of the active peptide is determined by adopting a high-efficiency size exclusion chromatography injection method:
chromatographic column TSKgel G2000 SWXL 300 mm. Times.7.8 mm gel column, mobile phase composition: acetonitrile: water: trifluoroacetic acid (volume ratio) =40:59.95:0.05, detection wavelength: 220nm, flow rate 0.5mL/min, column temperature: 30 ℃, sample injection volume: 10 mu L. The freeze-dried sample was prepared as a 0.2mg/mL solution, filtered through a 0.22 μm filter and loaded, and analyzed under the chromatographic conditions described above. And calculating the chromatograms of the samples and the data thereof according to the 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 assay
Protease activity was measured using Fu Lin Fenfa, reference GB/T23527-2009, and alkaline protease and neutral protein were measured at pH10.0 and pH7.0, respectively. Aminopeptidase assay L-leucine-4-nitroaniline is used as a substrate, and the amount of enzyme consumed for hydrolyzing the substrate to generate 1 mu g of p-nitroaniline in unit time under the assay condition is defined as an enzyme activity unit.
5. The protein content was determined by Kjeldahl method.
The peptide content was determined by the method of GB/T22492-2008 appendix B.
The shrimp head active peptide powder obtained by the method described in the embodiment has the following indexes:
8.63g of active peptide powder can be obtained per 100g of dry shrimp head powder, the protein content (N×6.25 on dry basis) of the prepared active peptide powder is 95.7%, the peptide content (on dry basis) is 93.85%, IC 50 The value reaches 0.054mg/mL, which is higher than the activity reported in the literature for preparing ACE inhibitory peptide by hydrolyzing shrimp heads by an enzymolysis method (food science, 2012,33 (11): 131-136).
Example 2
In this example, the effects of raw material proteolysis and ACE inhibitory activity of the product were compared using the solid state fermentation method described in the present invention and using the enzymatic hydrolysis method, respectively.
The solid state fermentation method was the same as described in example 1, sampling was performed at 5 days of fermentation, and performance test was performed by the same test method as in example 1.
And (3) enzymolysis: 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 enzyme-substrate ratio of 3000U/g, carrying out enzyme reaction under the optimal conditions (alkaline protease, trypsin and flavourzyme) of each enzyme, dropwise adding 1mol/L of HCl or NaOH during the period to maintain the pH at the optimal conditions, carrying out enzymolysis for 6 hours, and then sampling and measuring. The bacillus subtilis fermentation protease liquid is prepared by the following method: strain CMCC63501 was inoculated into a seed liquid medium and shake-cultured at 37 ℃ and 200rpm for 24 hours. Inoculating the seed solution into a liquid fermentation enzyme production culture medium according to the inoculum size of 2 percent, carrying out shake culture for 36 hours at 37 ℃ and 200rpm, centrifuging for 10 minutes at 6000rpm, and collecting supernatant to obtain the fermentation protease. Liquid fermentation enzyme-producing culture medium: 15g/L glucose, 30g/L soybean meal and CaCl 2 2g/L，K 2 HPO 4 2g/L Tween 80 1g/L, naCl 2g/L, pH 8.0, and sterilizing at 115deg.C for 15min. The enzyme solution obtained by fermentation was mixed with 0.2g/mL of shrimp head powder suspension in equal volume, and subjected to enzymolysis at pH7.5 and 40℃for 6 hours to sample, and the performance test was carried out by the same test method as in example 1.
The comparison result is shown in FIG. 1. As can be seen from FIG. 1, the highest protein hydrolysis rate is obtained by the solid state fermentation method, which reaches 60.7%, and the protein hydrolysis rates obtained by the enzymolysis methods using different enzymes are between 18 and 24% (which is similar to the protein hydrolysis rate reported in the literature (food science, 2012,33 (11): 131-136) for hydrolyzing shrimp heads by the enzymolysis method). In addition, the solid state fermentation method also obtains more small molecular polypeptides with the molecular weight smaller than 1kDa, which indicates that the method can more effectively utilize the solid state shrimp head powder raw material to release the small molecular polypeptides. The prior researches and reports show that ACE inhibitory peptides with good activity are small molecular peptides with molecular weight smaller than 1 kDa.
Example 3
This example illustrates the use of single and mixed species fermentation, respectively, for comparison.
After pretreatment of the raw materials, stirring and sterilization, B.subilis and B.lichenifermis which are used independently are added respectively, and the two strains are combined and inoculated according to different proportions of 1:0.5, 1:1, 1:2 and 1:3, are fermented according to the fermentation conditions described in the example 1, are sampled after fermentation for 6 days, and are subjected to performance test by adopting the detection method of the example 1.
The comparison result is shown in FIG. 2. As can be seen from FIG. 2, when a single strain is adopted, three indexes of the degree of proteolysis, ACE inhibitory activity and small molecule polypeptide content less than 1kDa are lower than those of mixed strain fermentation. From the different ratio results of the two strains, the protein hydrolysis degree and ACE inhibition activity obtained by the four experimental ratios are not significantly different, but the small molecular polypeptide smaller than 1kDa obtained by the two ratios of 1:1 and 1:2 is higher in proportion, which is more beneficial to release the small molecular polypeptide.
Example 4
This example illustrates the effect of feeding sugar during fermentation.
Raw material pretreatment, material mixing sterilization, seed liquid preparation and inoculation are the same as those in example 1, glucose is not added in the fermentation process after inoculation, the method 1 (32 mL of 50g/L glucose is added once on day 2), the method 2 (20 mL of 40g/L glucose is added twice on days 3 and 5), the method 3 (20 mL of 20g/L glucose is added on days 2,3, 4 and 5), and the fermentation is carried out for 6 days, and the performance test is carried out by adopting the detection method of example 1.
The comparison result is shown in FIG. 3. As can be seen from fig. 3, the addition of sugar during fermentation can significantly increase the degree of proteolysis, ACE inhibitory activity and the content of small molecule polypeptides less than 1 KDa. Of the three modes of sugar supplementation, the effect of one-time addition (mode 1) is the worst, and the three indexes are lower than those of modes 2 and 3. By adopting 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, which shows that the mode can lead the hydrolysis efficiency of the raw materials to be higher and the yield to be better.
Example 5
This example illustrates the effect of surfactant addition on enzyme production.
The contents and combinations of different surfactants were added to the fermentation medium, and the other conditions and operations were the same as described in example 1, and after mixing and sterilizing the materials, solid state fermentation was performed, and after 3 days of fermentation, samples were taken to determine the activities of alkaline protease, neutral protease and aminopeptidase, and the results were shown in table 1.
As can be seen from Table 1, the addition of the surfactant significantly improved the yields of protease and aminopeptidase, and the combination of the two surfactants was more advantageous for enzyme production than the addition of one surfactant alone, with the two proteases being increased by more than 40% and the aminopeptidase being increased by 56.8%, respectively
TABLE 1 Effect of different surfactant additions on bacterial Strain enzyme production
Example 6
This example illustrates the preparation of ACE inhibitory peptides in a solid state fermenter (horizontal rotary shaft mechanical stirred tank fermentor 30L) system using a mixture of Bacillus subtilis (Bacillus subtilis) CMCC63501 and Bacillus licheniformis (Bacillus licheniformis) AS1.269 strain for solid state fermentation of shrimp heads.
The specific implementation steps are as follows:
(1) Preparation for raw material treatment
Fresh shrimp heads (by-products after processing shrimp meat) are dried in an oven at 70 ℃ for 20 hours to constant weight, the dried shrimp heads are crushed by a crusher and then pass through an 80-target standard sieve, and the particle size of the crushed particles is about 0.2 mm.
(2) Activation of strains
The strain was inoculated on a slant for 18 hours based on 40℃for strain activation. The slant culture medium comprises: glucose 6g/L, tryptone 12g/L, sodium chloride 6g/L, dipotassium hydrogen phosphate 3g/L and agar 18g/L; the pH was adjusted to 7.0 and sterilized at 125℃for 15 minutes.
(3) Seed liquid preparation
Inoculating bacillus subtilis and bacillus licheniformis inclined plane strains into a seed liquid culture medium respectively, wherein the seed liquid culture medium comprises the following components: glucose 20g/L, pancreatic protein10g/L peptone, 5g/L yeast powder, 5g/L sodium chloride, 3g/L dipotassium hydrogen phosphate, pH7.0 and sterilizing at 125 ℃ for 15 minutes. Shaking culture at 40deg.C and 300rpm for 18 hr, adding appropriate amount of sterile seed solution culture medium to dilute the cultured bacterial solution to adjust bacterial concentration to 5×10 8 CFU/mL。
(4) Mixing and sterilizing
Fermentation medium: glucose 8g/L, mannitol 2g/L and starch 5g/L; yeast extract powder 0.5g/L and ammonium chloride 0.5g/L; dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.2g/L, ferrous sulfate 0.05g/L and calcium chloride 0.1g/L; tween 80.5 g/L and surfactant (Surfactin) 0.02g/L. The components are weighed and dissolved in deionized water to prepare a culture solution, the culture solution is mixed according to crushed shrimp head powder, 80mL of the culture solution is adopted for each 100g of shrimp head powder to be mixed, and the pH value is regulated to 7.0 by 0.5mol/L sodium hydroxide solution after the uniform mixing. The 30L solid state fermenter was charged at 35% and sterilized in situ at 121℃for 15min.
(5) Inoculation and fermentation
The bacillus subtilis and bacillus licheniformis seed liquid are mixed according to the proportion of 1:1, and each 100g of shrimp head powder is inoculated with 20mL of mixed seed liquid. The conditions of the fermentation tank after inoculation are controlled as follows: the temperature is 40 ℃, the relative humidity is 95%, the stirring rotating 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 according to 100g of shrimp head powder every 12 hours from the fermentation time of 36 hours, and the fermentation is finished until 5 days.
(6) Extraction and separation of peptides
Adding 500mL of 0.08mol/L hydrochloric acid solution into every 100g of fermentation material, uniformly mixing, stirring at 200rpm, extracting at 30 ℃ for 2 hours, filtering the extracting solution by 300-mesh filter cloth to obtain primary filtrate, adding activated carbon according to 0.5% of the volume of the primary filtrate, adsorbing at 45 ℃ for 60 minutes, and filtering again to obtain supernatant extracted by peptide. Ultrafiltering the supernatant with 5kDa ceramic membrane to retain the component with molecular weight less than 5kDa, ultrafiltering with 1kDa ceramic membrane for the second time to retain the component with molecular weight less than 1kDa, and mixing the filtrates. The filtrate was applied to a cation exchange column (Dowex 50WX 2), the column was washed with deionized water to remove non-peptide impurities, then the polypeptide adsorbed on the column was eluted with 1mol/L aqueous ammonia, and the eluate was collected.
(7) Drying to obtain the final product
And (3) carrying out low-temperature vacuum concentration and freeze drying on the peptide solution obtained by ultrafiltration separation to obtain shrimp hair ferment peptide powder.
The performance test was carried out by using the same detection method as in example 1, and the shrimp head peptide powder obtained by the method described in this example had the following index:
12.05g of active peptide powder can be obtained per 100g of dry shrimp head powder, the protein content (N×6.25 on dry basis) of the prepared active peptide powder is 92.2%, the peptide content (on dry basis) is 89.6%, IC 50 The value reaches 0.089mg/mL.
It can be seen that the method of the present invention can obtain ACE inhibitory peptides with high yield and purity and good inhibitory activity, both in the pilot plant of example 1 and in the scale-up production of the fermenter of example 6. Compared with the traditional enzymolysis method, the method does not need to add substrate enzyme in advance, thereby greatly saving the cost in the large-scale production. Under the condition that the strain exists, shrimp heads are used as raw materials, enzyme production and substrate proteolysis synchronously occur and are mutually promoted, the proteolysis rate is greatly improved, and more and purer ACE inhibitory peptides are obtained.
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent changes may be made in the claims herein without departing from the scope of the invention.
Claims (9)
1. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads by mixed strains is characterized by comprising 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 activation culture on bacillus subtilis and bacillus licheniformis to obtain slant culture;
C. preparing seed liquid: performing expansion culture on the slant culture, and adjusting the concentration of the bacterial cells to be 1-50 multiplied by 10 7 CFU/mL, obtain bacillus subtilis seed respectivelyThe method comprises the steps of mixing liquid and bacillus licheniformis seed liquid according to the volume ratio of 1:0.5-3 to obtain seed liquid;
D. preparing a fermentation medium, mixing materials and sterilizing: mixing the crushed shrimp head powder with 30-80 mL of 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 a fermentation culture medium;
E. inoculating and solid state fermentation: mixing the seed liquid with the fermentation medium, inoculating 5-20 mL of the seed liquid to each 100g of shrimp head powder, performing solid-state fermentation at 28-40 ℃ under the condition of 60-100% relative humidity, starting from 24-36 hours of fermentation time, adding 10-50 mL of sterilized 20-25 g/L glucose solution to each 100g of shrimp head powder every 12-24 hours, and ending the fermentation until 4-7 days to obtain a fermentation material;
F. extraction and isolation of peptides: mixing the fermented material with an acid agent, stirring, filtering, decolorizing, ultrafiltering, wherein the ultrafiltration step is to perform ultrafiltration by adopting a 5kDa ceramic membrane, reserving components with molecular weight less than 5kDa, performing secondary ultrafiltration by adopting a 1kDa ceramic membrane, reserving components with molecular weight less than 1kDa, combining and collecting filtrate with molecular weight less than 1kDa, loading the filtrate, and eluting to remove impurities to obtain eluent containing ACE inhibitory peptide;
G. concentrating and drying to obtain the finished product: concentrating and drying the eluent to obtain shrimp hair fermentation polypeptide powder containing ACE inhibitory peptide.
2. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads by mixed strains according to claim 1, comprising the steps of:
A. pretreatment of raw materials: drying the fresh shrimp heads in an oven at 60-70 ℃ for 18-20 hours to constant weight, crushing by a crusher, and sieving by a standard sieve with 8-80 meshes 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 for 18-24 hours at 37-40 ℃ to respectively obtain a bacillus subtilis slant culture and a bacillus licheniformis slant culture;
C. preparing seed liquid: respectively inoculating the bacillus subtilis inclined plane culture and the bacillus licheniformis inclined plane culture into a seed solution culture medium, performing shake cultivation for 18-30 hours at the temperature of 37-40 ℃ and the speed of 200-300 rpm, then adding a proper amount of sterile seed solution culture medium for dilution to respectively obtain the bacillus subtilis seed solution and the 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 each 100g of crushed shrimp head powder with 30-80 mL of the culture solution, regulating the pH to 7.0-8.0 by using 0.5-0.6 mol/L sodium hydroxide solution after uniformly mixing, and sterilizing at 121-125 ℃ for 15-20 min to obtain the fermentation culture medium;
E. inoculating and solid state fermentation: mixing the seed liquid with the fermentation medium, inoculating 5-20 mL of the seed liquid to each 100g of shrimp head powder, fermenting at 28-40 ℃ under the condition of 60-100% relative humidity, starting from 24-36 hours of fermentation time, adding 10-50 mL of sterilized 20-25 g/L glucose solution to each 100g of shrimp head powder every 12-24 hours, and ending the fermentation until 4-7 days to obtain the fermentation material;
F. extraction and isolation of peptides: mixing 100g of the fermentation material with 200-500 mL of hydrochloric acid solution with the concentration of 0.05-0.08 mol/L, stirring at 150-200 rpm, extracting at 10-30 ℃ for 1-2 hours, filtering the extracting solution through 200-300 meshes of filter cloth to obtain primary filtrate, adding activated carbon accounting for 0.3-0.5% of the volume of the primary filtrate, adsorbing for 30-60 minutes at 40-45 ℃, filtering again, ultrafiltering by adopting a 5kDa ceramic membrane, retaining components with the molecular weight of less than 5kDa, ultrafiltering for the second time by adopting 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, washing by adopting deionized water after the small peptides are completely adsorbed on the column, eluting the polypeptides adsorbed on the cation exchange column by using 1-2 mol/L of ammonia water, and collecting eluent containing ACE inhibitory peptides;
G. concentrating and drying to obtain the finished product: concentrating the eluent in vacuum at low temperature, and freeze-drying to obtain shrimp hair ferment polypeptide powder containing ACE inhibitory peptide.
3. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed bacteria according to claim 1, wherein the bacillus subtilis strain is bacillus subtilis cmCC63501 and the bacillus licheniformis strain is bacillus licheniformis AS1.269.
4. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed bacteria according to claim 2, wherein in the 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; and adjusting the pH to 7.0-7.2, and sterilizing for 15-20 minutes at the temperature of 121-125 ℃.
5. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed bacteria according to claim 2, wherein in the step C, the seed liquid 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, pH of 7.0-7.5 and sterilization at 121-125 ℃ for 15-20 minutes.
6. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 2, wherein in the step D, the culture solution comprises a carbon source, a nitrogen source, inorganic salt 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.
7. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads by mixed strains according to claim 6, wherein the concentration of the carbon source is 10-30 g/L.
8. The method for preparing ACE inhibitory peptide by solid state fermentation of shrimp heads with mixed strains according to claim 6, wherein 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.
9. The method for producing ACE inhibitory peptides by solid state fermentation of shrimp heads with mixed bacteria according to claim 2, wherein in step F, the cation exchange column is Dowex 50WX2.
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| CN106119173A (en) * | 2016-08-22 | 2016-11-16 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide without bitterness and preparation method and application |
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| CN106119173A (en) * | 2016-08-22 | 2016-11-16 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide without bitterness and preparation method and application |
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