A preparation method of enteric-coated sustained-release microcapsules of chlorella protein polypeptide

Abstract

The invention discloses a method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptides, wherein the protein is extracted after enzymatic wall breaking of protein core chlorella, the protein is hydrolyzed into polypeptides by enzymatic hydrolysis, and separated and impurized by alcohol precipitation, and the polypeptide powder is obtained by drying, and then the enteric-coated sustained-release microcapsules of chlorella protein polypeptides are prepared by solidification and secondary cross-linking of sodium alginate-chitosan-sodium tripolyphosphate in an emulsified dispersion system. The chlorella protein polypeptide microcapsules prepared by the microencapsulation technology of the present invention have anti-cancer activity, are not easily dissolved in the gastric fluid environment, are stably released in the intestinal fluid, and achieve the effect of controlled sustained release in the intestine. The preparation process of the present invention is simple and easy to expand production.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of chlorella sustained-release capsules, and in particular to a preparation method of chlorella protein polypeptide enteric-coated sustained-release microcapsules. Background Art

[0002] Chlorella is a ubiquitous spherical unicellular algae belonging to the Chlorophyta, Chlorophyceae, Chlorococcales, Oocystaceae, Chlorella genus, with a diameter of 3 to 8 microns. It is one of the earliest life on Earth, appearing more than 2 billion years ago. It is an efficient photosynthetic plant that grows and reproduces by photosynthesis and is widely distributed. Chlorella is easy to cultivate. It can not only use light energy for autotrophy, but also use organic carbon sources for growth and reproduction under heterotrophic conditions; and it grows and reproduces quickly. It is the only organism on Earth that can grow four times in 20 hours. Chlorella is rich in nutrition, high in protein, low in fat, low in sugar, and low in calories. It is also rich in vitamins and mineral elements, and has high application value. In my country, common species include pyrenoid chlorella, ellipsoid chlorella, and common chlorella. Among them, pyrenoid chlorella has the highest protein content and the highest nutritional value. In 2012, my country's Ministry of Health listed pyrenoid chlorella as a new resource food.

[0003] Bioactive peptides are active molecules composed of several to dozens of amino acid residues, and their sources are very wide, such as milk and dairy products, chicken and eggs, duck meat and duck blood, fish, oysters, grains (rice, wheat, soybeans, buckwheat, barley and corn) and seeds. Modern nutrition research has found that most of the protein ingested by humans is digested and absorbed in the form of low peptides after the enzymes in the digestive tract, and the proportion absorbed in the form of free amino acids is very small. Further experiments have revealed that peptides are digested faster and absorbed more than free amino acids. Bioactive peptides have a variety of physiological functions and can beneficially regulate a series of biomarkers related to health promotion. In recent years, a large number of literatures have shown that bioactive peptides have potential effects on blood pressure and lipid metabolism, in addition to antibacterial, analgesic, antioxidant, anti-inflammatory and other activities.

[0004] Cancer is a disease caused by abnormal proliferation of normal cells, usually manifested as uncontrolled cell growth, which ultimately damages normal cell function. Peptides have significant anti-cancer activity by inducing cancer cell apoptosis, downregulating the PI3K / Akt signaling pathway, inhibiting cancer cell proliferation, and promoting angiogenesis. Compared with common anti-cancer drugs, peptides have a small molecular weight, are easily absorbed, and have strong specificity. In recent years, they have attracted much attention in cancer treatment.

[0005] Protein peptide drugs mainly have two dosage forms: freeze-dried powder injection and solution injection. This type of drug has the disadvantages of short half-life and long-term administration. Most protein peptide drugs are easily degraded by biological enzymes, have poor biological stability, and have low bioavailability after oral administration, which limits their application. Microcapsules refer to the use of natural or synthetic polymer materials to encapsulate solid or liquid drugs to form tiny capsule-like particles. The microencapsulation method has the advantages of improving taste, prolonging the action time of drugs, increasing drug stability, reducing gastric irritation, solidifying liquid drugs to prevent drug loss, facilitating storage and use, being easy to make sustained-release preparations, targeted preparations, and improving bioavailability. In recent years, the microencapsulation method has been widely used in the production of protein peptide drug preparations, and some products have achieved good results in clinical practice.

[0006] Chinese patent CN202110167140.8 provides a pyrenoid chlorella antioxidant peptide and a preparation method thereof, using pyrenoid chlorella as raw material, extracting chlorella protein through pyrenoid chlorella powder wall breaking treatment, preparing chlorella peptides, separating and purifying chlorella peptides, freeze drying, and obtaining pyrenoid chlorella antioxidant peptides. This scheme uses high-pressure homogenization equipment, ultrafiltration equipment and freezing equipment, which has high power consumption and high cost, and is not conducive to promotion and production.

[0007] Chinese patent CN201210318091.4 provides a method for preparing anti-tumor peptides from Chlorella, which first uses a low-temperature ultrahigh-pressure continuous flow cell disruptor to extract Chlorella protein, then hydrolyzes the Chlorella protein with trypsin, and filters it through an ultrafiltration centrifuge tube to obtain Chlorella polypeptides with molecular weights ranging from 0-3KD, 3-5KD, 5-10KD, and greater than 10KD. This method uses a cell disruptor to process Chlorella, which is costly and requires the purchase of additional special equipment, which is not conducive to industrial production; and uses a single enzyme to enzymatically process Chlorella protein, which has few enzymatic hydrolysis points and low conversion yield.

[0008] Chinese patent CN200710008931 provides a phycobiliprotein controlled slow-release microparticle, which is composed of a wall material and a core material made of phycobiliprotein dry powder; the weight ratio of the core material to the wall material is 1:4-8. The weight percentage of each component in the wall material is: sodium alginate 1%-10%, calcium alginate 0.1%-10%, chitosan 0.1%-5%, and the rest is water. This method adds the sodium alginate aqueous solution of phycobiliprotein dropwise into the acetic acid aqueous solution of chitosan-calcium chloride. The droplets of this dripping are relatively large, and the particle size of the prepared microparticles is also relatively large. In addition, special equipment is required for dripping during production, and its production cycle has certain limitations.

[0009] The existing patented technologies mainly focus on the extraction technology of Chlorella polypeptides and polypeptide microencapsulation technology, and provide corresponding invention technologies, which mostly use ultrasound and ultrafiltration treatment. The corresponding equipment costs are high, the technical processes are complicated, and it is not conducive to expanding production. Summary of the invention

[0010] Purpose of the invention: In order to overcome the problems existing in the prior art, the present invention proposes a method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptides. The preparation process is simple, the particle size of the emulsified particles is smaller and more uniform, and it is suitable for expanded production.

[0011] Technical solution: In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0012] A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide comprises the following steps:

[0013] (1) taking protein core chlorella powder, adding water and stirring to form a chlorella suspension, and adjusting the pH to 3-7 with an acid solution; then adding a cell wall breaking enzyme, stirring at 30-70° C. for 3-8 hours to perform enzymatic cell wall breaking; cooling to room temperature and centrifuging, taking the supernatant, and concentrating under reduced pressure at 50-80° C. to obtain a chlorella protein extract;

[0014] (2) taking the chlorella protein extract of step (1), adjusting the pH to 5-8 with an alkaline solution; then adding a hydrolase, stirring at 40-80° C. for 3-8 hours to hydrolyze the protein, inactivating the enzyme in the liquid, cooling to room temperature and centrifuging, taking the supernatant, and concentrating under reduced pressure at 50-80° C. to obtain a chlorella polypeptide hydrolyzate;

[0015] (3) adding ethanol to the Chlorella polypeptide hydrolyzate of step (2) while stirring, standing at room temperature for 1-5 hours, precipitating, filtering, concentrating the filtrate under reduced pressure at 50-80° C. and drying to obtain Chlorella polypeptide powder;

[0016] (4) taking the chlorella polypeptide powder of step (3), mixing and dissolving it in a sodium alginate aqueous solution, then adding calcium carbonate powder, stirring evenly to form an aqueous suspension; then adding vegetable oil and an emulsifier, stirring well at 50-80° C., and homogenizing to obtain an emulsion;

[0017] (5) taking the emulsion of step (4), cooling it to room temperature, adding an aqueous solution containing chitosan and acetic acid with stirring, stirring for 1-5 hours to complete solidification, then adding an aqueous solution of sodium tripolyphosphate, and continuing to stir for 1-5 hours for secondary crosslinking to obtain a layered mixed solution;

[0018] (6) Taking the mixed solution of step (5), removing the upper oil phase by stratification and centrifuging, and drying the precipitate in an oven at 50-80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0019] Furthermore, in step (1), the pH of the acid solution in the Chlorella suspension is adjusted to 4-6, the enzymolysis temperature is 40-60°C, and the stirring time is 3-6h; the mass fraction of Chlorella is 1-15%; the acid solution is selected from an aqueous solution of one or more combinations of hydrochloric acid, sulfuric acid, acetic acid, and citric acid; the cell wall-breaking enzyme is selected from one or more combinations of cellulase, hemicellulase, and pectinase, and the mass fraction of the cell wall-breaking enzyme added to the suspension is 0.2-5%.

[0020] Furthermore, in step (1), the mass fraction of Chlorella is 5-12%; the acid solution is selected from an aqueous solution of one or more combinations of hydrochloric acid and citric acid; the wall-breaking enzyme is selected from one or more combinations of cellulase and pectinase, and the concentration of the wall-breaking enzyme added to the suspension is 2-4%.

[0021] Furthermore, the reduced pressure concentration in step (1) specifically comprises: concentrating the Chlorella protein extract to a solid content of 1-10%.

[0022] More preferably, the chlorella protein extract is concentrated to a solid content of 3-8%.

[0023] Furthermore, in the chlorella protein extract described in step (2), the alkaline solution is adjusted to a pH of 6-7, the enzymolysis temperature is 50-60° C., and the stirring time is 4-7 hours; the alkaline solution in step (2) is selected from an aqueous solution of one or more combinations of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; the hydrolase is selected from one or more combinations of papain, bromelain, and neutral protease, and the mass fraction of the hydrolase added to the chlorella protein extract is 0.2-5%.

[0024] More preferably, in step (2), the alkaline solution is selected from an aqueous solution of one or more combinations of sodium hydroxide and sodium carbonate; the hydrolase is selected from one or more combinations of papain and neutral protease, and the concentration of the hydrolase added to the Chlorella protein extract is 1.5-4%.

[0025] Furthermore, the reduced pressure concentration in step (2) specifically comprises: concentrating the Chlorella polypeptide hydrolyzate to a solid content of 5-20%.

[0026] More preferably, the Chlorella polypeptide hydrolyzate is concentrated to a solid content of 10-20%.

[0027] Furthermore, the Chlorella polypeptide hydrolyzate in step (3) is allowed to stand at room temperature for 1-3 hours; and the amount of ethanol added is 1.5-6 times the mass of the hydrolyzate.

[0028] Furthermore, in step (4), after adding the vegetable oil and the emulsifier, the mixture is fully stirred at 70-80° C.; the suspension contains 0.1-2% of chlorella polypeptide powder, 1-10% of sodium alginate, and 1-10% of calcium carbonate by mass.

[0029] More preferably, the suspension contains 0.2-1% of chlorella polypeptide powder, 1-5% of sodium alginate, and 1-5% of calcium carbonate by mass fraction.

[0030] Furthermore, the vegetable oil in step (4) is selected from one or more combinations of soybean oil, corn oil, grape seed oil, olive oil, and sweet almond oil, and the amount used accounts for 40-90% of the total mass of the emulsion; the emulsifier is selected from one or more combinations of Span 40, Span 60, Span 80, lecithin, and glyceryl monostearate, and the amount used accounts for 1-10% of the total mass of the emulsion.

[0031] More preferably, the vegetable oil in step (4) is selected from one or more combinations of soybean oil and corn oil, and the amount used accounts for 50-80% of the total mass of the emulsion; the emulsifier is selected from one or more combinations of Span 80, lecithin, and glyceryl monostearate, and the amount used accounts for 2-8% of the total mass of the emulsion.

[0032] Furthermore, the aqueous solution of chitosan and acetic acid in step (5) contains 0.5-3% chitosan and 1.5-15% acetic acid, calculated by mass fraction, and the mass ratio of the amount used to the aqueous suspension in step (4) is 1:1; the mass concentration of the sodium tripolyphosphate aqueous solution is 0.08-0.5%, and the mass ratio of the amount used to the aqueous suspension in step (4) is 3:2.

[0033] More preferably, the aqueous solution of chitosan and acetic acid contains 1-2.5% chitosan and 2-8% acetic acid; and the concentration of the aqueous solution of sodium tripolyphosphate is 0.15-0.4%.

[0034] Furthermore, the centrifugation in steps (1), (2) and (6) is carried out at a temperature of 4-8°C, a rotation speed of 5000-10000 r / min, and a centrifugation time of 10-60 min.

[0035] More preferably, the centrifugation is carried out at a temperature of 6-8°C and a rotation speed of 7000-9000 r / min for 20-40 min.

[0036] Beneficial effects: The method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide provided by the present invention comprises the following steps: extracting the protein from the chlorella protein core by enzymatic hydrolysis and breaking the cell wall, then hydrolyzing the protein into polypeptides by enzymatic hydrolysis, separating and removing impurities by alcohol precipitation, and drying to obtain polypeptide powder, and then preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide by solidification and secondary cross-linking of sodium alginate-chitosan-sodium tripolyphosphate in an emulsified dispersion system. Compared with the prior art, the present invention has the following advantages:

[0037] (1) The present invention uses enzymatic hydrolysis to extract Chlorella protein and uses multiple enzymes to prepare Chlorella polypeptides. Compared with single enzyme hydrolysis, the extraction rate is higher and the loss of effective ingredients is smaller. The polypeptide powder is purified by alcohol precipitation process. Compared with the ultrafiltration process which is expensive and requires additional equipment, the process is simple, the cost is low, and it is conducive to expanding production;

[0038] (2) In the current process of preparing microparticles, a physical droplet method is often used to mix a protein sodium alginate solution with a chitosan acetic acid aqueous solution. This droplet addition method forms relatively large microparticles. The difference of the present invention is that the chlorella polypeptide and the sodium alginate solution are emulsified into tiny droplets by an emulsifier, uniformly dispersed in an emulsified system, and then the chitosan acetic acid aqueous solution is added under stirring to solidify and form. The resulting microparticles are smaller and more uniform, and the operation is simpler and easier to produce.

[0039] (3) The present invention uses chitosan-sodium alginate as a carrier material for sustained release of polypeptides, and obtains a microcapsule with a cross-linked network microporous structure through microencapsulation technology. At the same time, a layer of a reinforced membrane with secondary cross-linking of sodium tripolyphosphate and chitosan is coated on the surface of the microcapsule to reduce the swelling rate of the chitosan microcapsule and reduce the dissolution loss of the polypeptide;

[0040] (4) The chlorella protein polypeptide microcapsules prepared by the microencapsulation technology of the present invention can be controlled and sustained-released in the intestine, so that the polypeptide can better exert its effect in the human body, and the preparation process is simple and easy to achieve mass production. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments:

[0042] Examples 1-5 are the preparation of Chlorella protein polypeptide powder.

[0043] Embodiment 1:

[0044] A method for preparing chlorella protein polypeptide powder, the steps are as follows:

[0045] (1) Take protein core chlorella powder, add water and stir to form a chlorella suspension with a mass fraction of 10%, and adjust the pH to 5 with hydrochloric acid solution; then add 1.5% cellulose and 1.5% pectinase by mass fraction, stir at 50° C. for 4 hours, and perform enzymatic hydrolysis and cell wall breaking; after cooling to room temperature, centrifuge at 8000 r / min at 6° C. for 20 minutes, take the supernatant, and concentrate under reduced pressure at 70° C. to obtain a chlorella protein extract with a solid content of 5%;

[0046] (2) The chlorella protein extract obtained in step (1) was adjusted to pH 6 with sodium hydroxide solution; then 1.5% papain and 1% neutral protease were added, stirred at 55° C. for 5 h to hydrolyze the protein, and then the liquid was inactivated, cooled to room temperature, and centrifuged at 8000 r / min for 20 min at 6° C. The supernatant was collected and concentrated under reduced pressure at 70° C. to obtain a chlorella polypeptide hydrolyzate with a solid content of 15%;

[0047] (3) Add ethanol 3 times the mass of the hydrolyzate to the Chlorella polypeptide hydrolyzate in step (2) while stirring, let stand at room temperature for 2 hours to precipitate, filter, and then concentrate the filtrate under reduced pressure at 70° C. and dry to obtain Chlorella polypeptide powder.

[0048] Embodiment 2:

[0049] A method for preparing chlorella protein polypeptide powder, the steps are as follows:

[0050] (1) Take protein core chlorella powder, add water and stir to form a chlorella suspension with a mass fraction of 1%, and adjust the pH to 3 with hydrochloric acid solution; then add 0.1% cellulose and 0.1% pectinase by mass fraction, stir at 30° C. for 3 hours to perform enzymatic hydrolysis and cell wall breaking; after cooling to room temperature, centrifuge at 8000 r / min at 6° C. for 20 minutes, take the supernatant, and concentrate under reduced pressure at 70° C. to obtain a chlorella protein extract with a solid content of 1%;

[0051] (2) The chlorella protein extract obtained in step (1) was adjusted to a pH of 5 with a sodium hydroxide solution; then 0.1% papain and 0.1% neutral protease were added, stirred at 40° C. for 3 h to hydrolyze the protein, and then the liquid was inactivated, cooled to room temperature, and centrifuged at 8000 r / min for 20 minutes at 6° C. The supernatant was collected and concentrated under reduced pressure at 70° C. to obtain a chlorella polypeptide hydrolyzate with a solid content of 5%;

[0052] (3) Add ethanol 1.5 times the mass of the hydrolyzate to the Chlorella polypeptide hydrolyzate of step (2) while stirring, let stand at room temperature for 3 hours to precipitate, filter, and then concentrate the filtrate under reduced pressure at 70° C. and dry to obtain Chlorella polypeptide powder.

[0053] Embodiment 3:

[0054] A method for preparing chlorella protein polypeptide powder, the steps are as follows:

[0055] (1) Take protein core chlorella powder, add water and stir to form a chlorella suspension with a mass fraction of 15%, and adjust the pH to 7 with hydrochloric acid solution; then add 2.5% cellulose and 2.5% pectinase by mass fraction, stir at 70°C for 8 hours, and perform enzymatic hydrolysis and cell wall breaking; after cooling to room temperature, centrifuge at 8000 r / min at 6°C for 20 minutes, take the supernatant, and concentrate under reduced pressure at 70°C to obtain a chlorella protein extract with a solid content of 10%;

[0056] (2) taking the chlorella protein extract of step (1), adjusting the pH to 8 with sodium hydroxide solution; then adding 3% papain and 2% neutral protease by mass fraction, stirring at 80° C. for 8 h to hydrolyze the protein, inactivating the enzyme in the liquid, cooling to room temperature, centrifuging at 8000 r / min at 6° C. for 20 minutes, taking the supernatant, and concentrating under reduced pressure at about 70° C. to obtain a chlorella polypeptide hydrolyzate with a solid content of 20%;

[0057] (3) Add ethanol 6 times the mass of the hydrolyzate to the Chlorella polypeptide hydrolyzate in step (2) while stirring, let stand at room temperature for 5 hours, precipitate, filter, and then concentrate the filtrate at 70° C. under reduced pressure and dry to obtain Chlorella polypeptide powder.

[0058] Embodiment 4:

[0059] A method for preparing chlorella protein polypeptide powder, the steps are as follows:

[0060] (1) Take protein core chlorella powder, add water and stir to form a chlorella suspension with a mass fraction of 5%, and adjust the pH to 4 with hydrochloric acid solution; then add 1% cellulose and 1% pectinase by mass fraction, stir at 40° C. for 3 hours to perform enzymatic hydrolysis and cell wall breaking; after cooling to room temperature, centrifuge at 8000 r / min at 6° C. for 20 minutes, take the supernatant, and concentrate under reduced pressure at 70° C. to obtain a chlorella protein extract with a solid content of 3%;

[0061] (2) taking the chlorella protein extract of step (1), adjusting the pH to 6 with sodium hydroxide solution; then adding 1% papain and 0.5% neutral protease by mass fraction, stirring at 50° C. for 4 hours to hydrolyze the protein, inactivating the enzyme in the liquid, cooling to room temperature, centrifuging at 8000 r / min at 6° C. for 20 minutes, taking the supernatant, and concentrating under reduced pressure at 70° C. to obtain a chlorella polypeptide hydrolyzate with a solid content of 10%;

[0062] (3) Add ethanol twice the mass of the hydrolyzate to the Chlorella polypeptide hydrolyzate of step (2) while stirring, let stand at room temperature for 1 hour to precipitate, filter, and then concentrate the filtrate under reduced pressure at 70° C. and dry to obtain Chlorella polypeptide powder.

[0063] Embodiment 5:

[0064] A method for preparing chlorella protein polypeptide powder, the steps are as follows:

[0065] (1) Take protein core chlorella powder, add water and stir to form a chlorella suspension with a mass fraction of 12%, and adjust the pH to 6 with hydrochloric acid solution; then add 2% cellulose and 2% pectinase by mass fraction, stir at 60° C. for 6 hours to perform enzymatic hydrolysis and cell wall breaking; after cooling to room temperature, centrifuge at 8000 r / min at 6° C. for 20 minutes, take the supernatant, and concentrate under reduced pressure at 70° C. to obtain a chlorella protein extract with a solid content of 5%;

[0066] (2) taking the chlorella protein extract of step (1), adjusting the pH to 7 with sodium hydroxide solution; then adding 2.5% papain and 1.5% neutral protease by mass fraction, stirring at 60° C. for 7 h to hydrolyze the protein, inactivating the enzyme in the liquid, cooling to room temperature, centrifuging at 8000 r / min at 6° C. for 20 minutes, taking the supernatant, and concentrating under reduced pressure at 70° C. to obtain a chlorella polypeptide hydrolyzate with a solid content of 20%;

[0067] (3) Add ethanol in an amount of 4 times the mass of the hydrolyzate to the Chlorella polypeptide hydrolyzate in step (2) while stirring, let stand at room temperature for 3 hours to precipitate, filter, and then concentrate the filtrate under reduced pressure at 70° C. and dry to obtain Chlorella polypeptide powder.

[0068] Examples 6-10 are methods for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide using the chlorella polypeptide powder prepared in Example 1, as follows:

[0069] Embodiment 6:

[0070] A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide, comprising the following steps:

[0071] (1) The chlorella polypeptide powder prepared in Example 1 was mixed and dissolved in a sodium alginate aqueous solution, and then calcium carbonate powder was added and stirred to form an aqueous suspension, wherein the aqueous suspension contained 0.4% chlorella polypeptide powder, 2% sodium alginate, and 2% calcium carbonate by weight; then soybean oil (60% by weight of the total mass of the emulsion), 3% Span 80, and 2% glyceryl monostearate were added, and the mixture was stirred at 75° C. to obtain an emulsion;

[0072] (2) taking the emulsion of step (1), cooling it to room temperature, adding with stirring an aqueous solution containing 1% chitosan and 3.5% acetic acid by mass, the mass ratio of which to the aqueous suspension in step (1) is 1:1, stirring for 2 h to complete solidification, then adding 0.15% sodium tripolyphosphate aqueous solution, the mass ratio of which to the aqueous suspension in step (1) is 3:2, and stirring is continued for 1 h for secondary crosslinking to obtain a layered mixed solution;

[0073] (3) The mixed solution of step (2) is taken, the upper oil phase is removed by stratification, and then centrifuged, and the precipitate is dried in an oven at 80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0074] Embodiment 7:

[0075] A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide, comprising the following steps:

[0076] (1) The chlorella polypeptide powder prepared in Example 1 is mixed and dissolved in a sodium alginate aqueous solution, and then calcium carbonate powder is added and stirred to form an aqueous suspension, wherein the aqueous suspension contains 0.1% chlorella polypeptide powder, 1% sodium alginate, and 1% calcium carbonate by weight; then 40% soybean oil, 0.6% Span 80, and 0.4% glyceryl monostearate are added to the mixture, and the mixture is stirred at 50° C. to obtain an emulsion;

[0077] (2) taking the emulsion of step (1), cooling it to room temperature, adding with stirring an aqueous solution containing 0.5% chitosan and 1.5% acetic acid by mass, the mass ratio of which to the aqueous suspension in step (1) is 1:1, stirring for 1 hour to complete solidification, then adding 0.08% sodium tripolyphosphate aqueous solution, the mass ratio of which to the aqueous suspension in step (1) is 3:2, and stirring is continued for 1 hour for secondary crosslinking to obtain a layered mixed solution;

[0078] (3) The mixed solution of step (2) is taken, the upper oil phase is removed by stratification, and then centrifuged, and the precipitate is dried in an oven at 80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0079] Embodiment 8:

[0080] A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide, comprising the following steps:

[0081] (1) The chlorella polypeptide powder prepared in Example 1 is mixed and dissolved in a sodium alginate aqueous solution, and then calcium carbonate powder is added and stirred to form an aqueous suspension, wherein the aqueous suspension contains 2% chlorella polypeptide powder, 10% sodium alginate, and 10% calcium carbonate by weight; then 90% soybean oil, 6% Span 80, and 4% glyceryl monostearate are added, and the mixture is stirred at 80° C. to obtain an emulsion;

[0082] (2) taking the emulsion of step (1), cooling it to room temperature, adding an aqueous solution containing 3% chitosan and 15% acetic acid by weight, with the weight ratio of 1:1 to the aqueous suspension in step (1), stirring for 5 hours to complete solidification, then adding 0.5% sodium tripolyphosphate aqueous solution, with the weight ratio of 3:2 to the aqueous suspension in step (1), continuing stirring for 5 hours to perform secondary crosslinking, and obtaining a layered mixed solution;

[0083] (3) The mixed solution of step (2) is taken, most of the oil layer is removed by stratification, and then centrifuged, and the precipitate is dried in an oven at 80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0084] Embodiment 9:

[0085] A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide, comprising the following steps:

[0086] (1) The chlorella polypeptide powder prepared in Example 1 is mixed and dissolved in a sodium alginate aqueous solution, and then calcium carbonate powder is added and stirred to form an aqueous suspension, wherein the aqueous suspension contains 0.2% chlorella polypeptide powder, 1% sodium alginate, and 1% calcium carbonate by weight; then 50% soybean oil, 1.2% Span 80, and 0.8% glyceryl monostearate are added to the mixture, and the mixture is stirred at 70° C. to obtain an emulsion;

[0087] (2) taking the emulsion of step (1), cooling it to room temperature, adding with stirring an aqueous solution containing 1% chitosan and 2% acetic acid by mass, the mass ratio of which to the aqueous suspension in step (1) is 1:1, stirring for 1 hour to complete solidification, then adding 0.15% sodium tripolyphosphate aqueous solution, the mass ratio of which to the aqueous suspension in step (1) is 3:2, and stirring is continued for 1 hour for secondary crosslinking to obtain a layered mixed solution;

[0088] (3) The mixed solution of step (2) is taken, the upper oil phase is removed by stratification, and then centrifuged, and the precipitate is dried in an oven at 80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0089] Embodiment 10:

[0090] The preparation method of chlorella protein polypeptide enteric-coated sustained-release microcapsules comprises the following steps:

[0091] (1) The chlorella polypeptide powder prepared in Example 1 is mixed and dissolved in a sodium alginate aqueous solution, and then calcium carbonate powder is added and stirred to form an aqueous suspension, wherein the aqueous suspension contains 1% chlorella polypeptide powder, 5% sodium alginate, and 5% calcium carbonate by mass; then 80% soybean oil, 4.8% Span 80, and 3.2% glyceryl monostearate are added, and the mixture is fully stirred at 80° C. and homogenized to obtain an emulsion;

[0092] (2) taking the emulsion of step (1), cooling it to room temperature, adding an aqueous solution containing 2.5% chitosan and 8% acetic acid by mass in a ratio of 1:1 to the aqueous suspension in step (1), stirring for 3 h to complete solidification, then adding 0.4% sodium tripolyphosphate aqueous solution in a ratio of 3:2 to the aqueous suspension in step (1), continuing stirring for 3 h to perform secondary crosslinking, and obtaining a layered mixed solution;

[0093] (3) The mixed solution of step (2) is taken, the upper oil phase is removed by stratification, and then centrifuged, and the precipitate is dried in an oven at 80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

[0094] Comparative Example 1:

[0095] The Chlorella polypeptide was prepared by the preparation method of the Chlorella anti-tumor polypeptide of patent CN201210318091.4, and then prepared into a microcapsule form according to the microcapsule preparation method of Example 1.

[0096] (1) Chlorella protein was extracted using a low-temperature ultra-high pressure continuous flow cell disruptor: Chlorella powder and pure water were mixed at a mass ratio of 1:20 and stirred for 50 minutes to obtain a mixed solution. The above mixed solution was used to extract Chlorella protein at a temperature of 6°C and a pressure of 100 MPa, and the crude extract was used as the next extraction solution for re-extraction. The number of extractions was 3 times, and the time for each extraction was 20 minutes. Finally, the obtained solution was centrifuged at a temperature of 4°C and a speed of 6000r / min for 30 minutes, the precipitate was removed to obtain the protein supernatant, and then vacuum freeze-dried to obtain Chlorella protein powder.

[0097] (2) Based on the chlorella protein powder obtained in step (1), a 2% chlorella protein solution is prepared, and trypsin is added for hydrolysis. The experimental conditions are temperature 40°C, pH=6, and the ratio of enzyme to chlorella protein aqueous solution is 3%. After hydrolysis for 5 hours, the enzyme is inactivated in 100°C water for 10 minutes. After cooling to room temperature, the hydrolyzate is centrifuged at 8000r / min for 25 minutes, and the supernatant is taken. Then, it is filtered through an ultrafiltration centrifuge tube with a molecular weight cutoff of 10KD at 8000g and 4°C. The filtrate is then filtered through 3KD and 5KD ultrafiltration centrifuge tubes (also at 8000g and 4°C). Chlorella polypeptide hydrolyzate with a molecular weight range of 0-3KD, 3-5KD, 5-10KD, and >10KD can be obtained.

[0098] (3) The Chlorella polypeptide hydrolyzate was dried by rotary evaporation to obtain Chlorella polypeptide powder, which was then prepared into microcapsules according to the method of Example 6.

[0099] Comparative Example 2: Chlorella polypeptide controlled-release microparticles were prepared according to the phycobiliprotein controlled-release microparticles and preparation method thereof disclosed in patent CN200710008931.

[0100] (1) Preparation of chitosan: The crude chitosan product was mixed with 1% acetic acid solution at a ratio of 1 g:10 ml for 30 minutes, the upper layer of liquid was centrifuged and filtered, the filtrate was neutralized with NaOH solution to pH = 8, filtered, the filter cake was rinsed in distilled water until neutral, dried and ground to obtain chitosan powder;

[0101] (2) preparing an acetic acid aqueous solution of chitosan-calcium chloride: adding calcium chloride, acetic acid and water to the chitosan powder prepared in step (1) to prepare a corresponding aqueous solution, wherein the aqueous solution comprises: 5% chitosan content, 10% calcium chloride content, 15% acetic acid content, and the remaining component is water;

[0102] (3) preparing a sodium alginate aqueous solution of chlorella polypeptide: taking the chlorella polypeptide powder prepared in Example 1 and sodium alginate, mixing them in a weight ratio of 1:7, and then adding water to make the sodium alginate content in the solution reach 10%;

[0103] (4) Preparation of Chlorella polypeptide controlled sustained-release microparticles: At 20°C, in the dark, add the sodium alginate aqueous solution of Chlorella polypeptide dropwise into the acetic acid aqueous solution of chitosan-calcium chloride using a syringe or dropper, wherein the volume of the acetic acid aqueous solution of chitosan-calcium chloride should be more than 50 times the volume of each drop. After the dropwise addition, let it stand for 30 minutes, separate the microparticles by centrifugation or filtration, wash with water, and dry in a vacuum at 35°C to obtain Chlorella polypeptide controlled sustained-release microparticles.

[0104] Anticancer activity test:

[0105] The MTT colorimetric method was used to evaluate the tumor inhibition activity of Chlorella peptides, with the inhibition rate of liver cancer cells HepG2 as an indicator. The principle is that succinate dehydrogenase in living cells can reduce MTT to blue-purple product formazan crystals that are insoluble in water and deposited in cells, while dead cells do not have this function. After formazan is dissolved by dimethyl sulfoxide (DMSO), its absorbance OD value can be measured at a wavelength of 490nm using an enzyme marker. Within a certain range of cell numbers, the amount of formazan crystals formed is proportional to the number of living cells, so the number of living cells can be inferred based on the optical density OD value.

[0106] 1. Reagent preparation

[0107] (1) PBS buffer: Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 mL distilled water. Adjust the pH of the solution to 7.4 with HCl. Add water to make up to 1 L. Sterilize with high-pressure steam for 20 min and store at room temperature.

[0108] (2) MTT (tetraquinone) solution: Take an appropriate amount of MTT and dissolve it in phosphate buffered saline (PBS) at pH 7.4 to make a 5 mg / mL solution. Place it on a magnetic stirrer and stir for 30 min to completely dissolve it. Filter it with a 0.22 μm filter membrane for sterilization. Store it at 4°C away from light for two weeks. It can be stored at -20°C for a long time.

[0109] (3) Sample solution: Take a certain amount of the Chlorella polypeptide enteric-coated sustained-release microcapsules prepared in Example 6 and Comparative Example 1, accurately weigh them, dissolve them in DMEM, sterilize them with a 0.22 μm filter membrane, store them at -4°C in the dark for later use, and dilute them with fresh DMEM to a working solution when needed.

[0110] (4) Complete medium for cells: Under sterile conditions, add 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin to the basal medium and store at 4°C until use.

[0111] 2. MTT assay

[0112] (1) Take cells in the logarithmic growth phase, digest them with 0.25% trypsin, add complete culture medium to neutralize the trypsin, and repeatedly pipette to prepare a single-cell suspension. Avoid excessive bubbles during pipetting. After counting on a hemocytometer, adjust the concentration of the cell suspension to 3×10 3 / 100μL, and inoculated into 96-well plates. Incubate in a 37°C, 5% CO2 incubator for 24h, remove the original culture medium, add 100μL of fresh high-glucose DMEM complete culture medium without calf serum, and then add the sample solution to the final concentration. The culture medium well without sample solution is used as a negative control;

[0113] (2) After culturing for 24 h, the culture medium was discarded after centrifugation and the cells were washed twice with PBS;

[0114] (3) Add 20 μL of culture medium containing 5 mg / mL MTT, continue culturing for 4 h, then aspirate, add 100 μL DMSO, and shake for 10 min to fully dissolve the blue-purple crystals;

[0115] (4) The optical density was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay, and the cell inhibition rate was calculated using the following formula. The cell survival rate was plotted against the dose, and the IC50 value was calculated using SPSS Probit regression analysis. (A positive inhibition rate indicates a killing effect on the cells, and a negative inhibition rate indicates a promoting effect on the cells).

[0116] Cell inhibition rate (%) = (1-test group OD 490 Value / control group OD 490 value)×100%

[0117] Cell survival rate (%) = (OD 490 / Control group OD 490 )×100%

[0118] concentration OD value Cell inhibition rate% Cell survival rate% Comparison 0.4325±0.0185 0 100 Example 6 (0.1 mg / mL) 0.3824±0.0099 11.59 88.41 Example 6 (0.2 mg / mL) 0.3487±0.0101 19.38 80.62 Example 6 (0.3 mg / mL) 0.2983±0.0124 31.03 68.97 Example 6 (0.4 mg / mL) 0.2301±0.0113 46.80 53.20 Comparative Example 1 (0.1 mg / mL) 0.3964±0.0107 8.35 91.65 Comparative Example 1 (0.2 mg / mL) 0.3702±0.0134 14.41 85.59 Comparative Example 1 (0.3 mg / mL) 0.3161±0.0112 26.91 73.09 Comparative Example 1 (0.4 mg / mL) 0.2521±0.0125 41.71 58.29

[0119] The data show that the enteric-coated sustained-release microcapsules of Chlorella polypeptide prepared by the present invention exhibit an inhibitory effect on tumor cells HepG2, and the cell survival rate decreases with the increase of drug concentration, with an obvious dose-effect relationship. The IC50 value of the enteric-coated sustained-release microcapsules of Chlorella polypeptide is calculated to be 453 μg / mL. Compared with Comparative Example 1, the Chlorella polypeptide prepared by this method has a stronger inhibitory ability on tumor cells HepG2 and a higher anti-tumor activity.

[0120] Drug loading, embedding rate determination and in vitro release experiment:

[0121] 1. Reagent preparation

[0122] (1) Artificial gastric juice: (prepared according to the Chinese Pharmacopoeia 2005 edition) Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, and dilute with water to 1000 mL.

[0123] (2) Artificial intestinal fluid: (prepared according to the Chinese Pharmacopoeia 2005 edition) Take 6.8 g of potassium dihydrogen phosphate and add about 300 ml of water to dissolve; take 10 g of trypsin and add 300 ml of water to dissolve. After mixing the two liquids, adjust the pH value to 6.8 with 0.1 mol / L sodium hydroxide solution and dilute with water to 1000 mL.

[0124] 2. Standard curve drawing

[0125] Accurately weigh 25 mg of the Chlorella polypeptide powder prepared in Example 1 and Comparative Example 2, dissolve in simulated gastric juice / intestinal juice (prepared according to the Chinese Pharmacopoeia 2005 edition), and dilute to a 100 mL volumetric flask. After shaking, accurately pipette 0, 2, 4, 6, 8, 10, and 12 mL respectively, dissolve in simulated gastric juice / intestinal juice in a 50 mL volumetric flask, dilute to the scale, and measure the light absorption value (A) at 214 nm.

[0126] 3. Determination of drug loading and embedding rate

[0127] The fresh microcapsules prepared in Example 6 and Comparative Example 2 were centrifuged at 4°C, 15000 r / min for 30 min, and the supernatant was taken to determine the concentration of free Chlorella polypeptide and calculate the embedding rate. The fresh microcapsules were washed with distilled water and dried for calculation of drug loading.

[0128] Drug loading = weight of drug in microspheres / weight of microspheres × 100%;

[0129] Embedding rate = weight of drug in microspheres / total amount of drug added × 100%.

[0130] The drug loading was calculated to be 12.6% and the encapsulation efficiency was 70.9%.

[0131] 4. Simulate release

[0132] (1) Release in simulated gastric fluid environment 500 mg of dried Chlorella polypeptide enteric-coated sustained-release microcapsules (from Example 6) were accurately weighed into a 100 mL conical flask, and 100 mL of freshly prepared artificial gastric fluid was added. The mixture was reacted in a constant temperature water bath oscillator at 37°C ± 1°C and 60 rpm. 1 mL of simulated gastric fluid was taken out every 2 h, and 1 mL of simulated gastric fluid was added at the same time. The polypeptide content in the simulated gastric fluid was measured and calculated, the cumulative release amount was calculated, and the release curve was plotted.

[0133] Release rate (%) = total peptide amount released in gastric fluid simulation / (weight of added microcapsules × drug loading)

[0134] (2) Release in simulated intestinal fluid environment 500 mg of Chlorella polypeptide enteric-coated sustained-release microcapsules (from Example 6) were added to 100 mL of simulated intestinal fluid, and the measurement method was the same as that in simulated gastric fluid.

[0135] The experimental results are:

[0136]

[0137] In the simulation experiment, the initial release of the Chlorella polypeptide microcapsules may come from the polypeptide molecules or fine particles adsorbed on the surface of the microcapsules, and the later stable release is the diffusion of the polypeptide components embedded in the microcapsules. The data show that the release rate of the Chlorella polypeptide enteric-coated sustained-release microcapsules prepared by the present invention in simulated intestinal fluid is much greater than the release rate in simulated gastric fluid. This is the result of the pH-responsive swelling of the microcapsules, that is, the microcapsules have good response behaviors of shrinking at low pH values ​​and swelling at high pH values; and the release amount of the microcapsules in each time period is relatively stable, and the cumulative release amount continues to increase with time. The above results show that the microcapsules prepared by the present invention can protect the Chlorella polypeptide in gastric fluid and continue to release in intestinal fluid, so as to play a better role.

[0138] Comparison between Example 6 prepared by the present invention and Comparative Example 2 shows that the sustained-release effect of the Chlorella polypeptide sustained-release microparticles prepared by the method provided by the patent in artificial intestinal fluid is not as good as that of the present patent, and the release time is close to the maximum release rate at about 8h. This method does not have a good embedding effect on the Chlorella polypeptide. At the same time, the performance of Comparative Example 2 in artificial gastric fluid is also unsatisfactory.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide, characterized in that The steps include: (1) taking protein core chlorella powder, adding water and stirring to form a chlorella suspension, and adjusting the pH to 3-7 with an acid solution; then adding a cell wall breaking enzyme, stirring at 30-70° C. for 3-8 hours to perform enzymatic cell wall breaking; cooling to room temperature and centrifuging, taking the supernatant, and concentrating under reduced pressure at 50-80° C. to obtain a chlorella protein extract; the cell wall breaking enzyme is cellulase and pectinase, and the mass fraction of the cell wall breaking enzyme added to the suspension is 0.2-5%; (2) taking the chlorella protein extract of step (1), adjusting the pH to 5-8 with an alkaline solution; then adding a hydrolase, stirring at 40-80° C. for 3-8 hours to hydrolyze the protein, inactivating the enzyme in the liquid, cooling to room temperature and centrifuging, taking the supernatant, and concentrating under reduced pressure at 50-80° C. to obtain a chlorella polypeptide hydrolyzate; the hydrolase is papain and neutral protease, and the mass fraction of the hydrolase added to the chlorella protein extract is 0.2-5%; (3) adding ethanol to the Chlorella polypeptide hydrolyzate of step (2) while stirring, standing at room temperature for 1-5 hours, precipitating, filtering, concentrating the filtrate under reduced pressure at 50-80° C. and drying to obtain Chlorella polypeptide powder; (4) taking the chlorella polypeptide powder obtained in step (3), mixing and dissolving it in a sodium alginate aqueous solution, and then adding calcium carbonate powder, stirring evenly to form an aqueous suspension; then adding vegetable oil and an emulsifier, stirring thoroughly at 50-80° C., and homogenizing to obtain an emulsion; the aqueous suspension contains 0.1-2% chlorella polypeptide powder, 1-10% sodium alginate, and 1-10% calcium carbonate by mass fraction; the amount of the vegetable oil accounts for 40-90% of the total mass of the emulsion; and the amount of the emulsifier accounts for 1-8% of the total mass of the emulsion; (5) taking the emulsion of step (4), cooling it to room temperature, adding an aqueous solution containing chitosan and acetic acid, stirring it, stirring it for 1-5 hours to complete solidification, then adding an aqueous solution of sodium tripolyphosphate, stirring it for 1-5 hours to perform secondary crosslinking, and obtaining a layered mixed solution; the aqueous solution of chitosan and acetic acid contains 0.5-3% chitosan and 1.5-15% acetic acid by mass fraction, and the mass ratio of the amount used to the aqueous suspension in step (4) is 1:1; the mass concentration of the aqueous solution of sodium tripolyphosphate is 0.08-0.5%, and the mass ratio of the amount used to the aqueous suspension in step (4) is 3:2; (6) Taking the mixed solution from step (5), removing the upper oil phase by layers and centrifuging, and drying the precipitate in an oven at 50-80° C. to obtain enteric-coated sustained-release microcapsules of Chlorella polypeptide.

2. The method for preparing the enteric-coated sustained-release microcapsules of Chlorella protein polypeptide according to claim 1, characterized in that: The mass fraction of Chlorella in the Chlorella suspension in step (1) is 1-15%; the acid solution is selected from an aqueous solution of one or more combinations of hydrochloric acid, sulfuric acid, acetic acid and citric acid.

3. The method for preparing the enteric-coated sustained-release microcapsules of Chlorella protein polypeptide according to claim 1, characterized in that: The reduced pressure concentration in step (1) specifically comprises: concentrating the Chlorella protein extract to a solid content of 1-10%.

4. The method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide according to claim 1, characterized in that: The alkaline solution in step (2) is selected from an aqueous solution of one or more combinations of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate.

5. The method for preparing enteric-coated sustained-release microcapsules of Chlorella protein polypeptide according to claim 1, characterized in that: The reduced pressure concentration in step (2) specifically comprises: concentrating the Chlorella polypeptide hydrolyzate to a solid content of 5-20%.

6. The method for preparing enteric-coated sustained-release microcapsules of Chlorella protein polypeptide according to claim 1, characterized in that: The amount of ethanol added in step (3) is 1.5-6 times the mass of the hydrolyzate.

7. The method for preparing enteric-coated sustained-release microcapsules of Chlorella protein polypeptide according to claim 1, characterized in that: In step (4), the vegetable oil is selected from one or more combinations of soybean oil, corn oil, grape seed oil, olive oil, and sweet almond oil; and the emulsifier is selected from one or more combinations of Span 40, Span 60, Span 80, lecithin, and glyceryl monostearate.

8. The method for preparing enteric-coated sustained-release microcapsules of chlorella protein polypeptide according to claim 1, characterized in that: The centrifugation in steps (1), (2) and (6) is carried out at a temperature of 4 to 8°C and a rotation speed of 5000 to 10000 r / min, and the centrifugation time is 10 to 60 min.

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