Hermetia illucens polypeptide-procyanidine nanoparticles and preparation method thereof

Abstract

The invention discloses hermetia illucens polypeptide-procyanidine nanoparticles and a preparation method thereof. The preparation method comprises the following steps: dissolving hermetia illucens polypeptide powder in water to prepare a hermetia illucens polypeptide solution; dissolving procyanidine in water to prepare a procyanidine solution; uniformly mixing and stirring the hermetia illucens polypeptide solution and the procyanidine solution to obtain a hermetia illucens polypeptide-procyanidine nanoparticle solution; wherein the mass ratio of the hermetia illucens polypeptide to the procyanidine is (0.1-0.5): 20; and centrifuging and drying the hermetia illucens polypeptide-procyanidine nanoparticle solution to obtain the hermetia illucens polypeptide-procyanidine nanoparticles. According to the invention, the hermetia illucens polypeptide is taken as a carrier, the procyanidine is loaded to form the nano-particles, the loading rate is high, the bioavailability of the procyanidine is effectively improved, and the preparation method is simple.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to black soldier fly polypeptide-proanthocyanidin nanoparticles and a preparation method thereof. Background Art

[0002] Proanthocyanidins belong to the class of flavanols. Since they contain multiple phenolic hydroxyl groups in their molecular structure, they also belong to the class of polyphenolic compounds. Proanthocyanidins have strong antioxidant capacity, and their monomers are catechin compounds, including catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. However, proanthocyanidins are unstable in the gastrointestinal tract, have low bioavailability, are very sensitive to light, heat, oxygen, humidity, and pH, and have poor stability in neutral or alkaline environments. They are easily diastereoisomerized and oxidized, and are also very unstable in the gastrointestinal environment. It can be seen that if a carrier system can be designed for flavonoids represented by proanthocyanidins, they can be encapsulated in them for gastrointestinal delivery, and intermolecular interactions can be formed between the carriers to resist the degradation of proanthocyanidins by factors such as low pH and enzymes, and can prevent their degradation under acidic conditions in the stomach, and regulate their release in the intestinal absorption site, this is an effective way to improve their bioavailability.

[0003] At present, nanocarrier delivery technology has been widely used in the food, pharmaceutical, medical and cosmetic industries, especially in the encapsulation of bioactive substances. The peptide self-assembly system has great application potential in this field, mainly due to its many advantages, such as: the molecules are composed of natural amino acids, with high biocompatibility and low toxicity; the molecules are highly designable and can self-assemble to form a variety of nano / micro structures with various morphologies and sizes; the self-assembled structure is easy to modify, and specific functional groups and active substances can be easily introduced. However, there is no report in the existing research data on peptides as nanocarriers of proanthocyanidins. In order to solve the problem of proanthocyanidin bioavailability, it is urgent to develop a peptide nano delivery system. Summary of the invention

[0004] The purpose of the present invention is to overcome the above technical deficiencies, provide a black soldier fly polypeptide-proanthocyanidin nanoparticle and a preparation method thereof, and solve the technical problem of low bioavailability of proanthocyanidins in the prior art.

[0005] In order to achieve the above technical purpose, the technical solution provided by the present invention is:

[0006] In a first aspect, the present invention provides a black soldier fly polypeptide-proanthocyanidin nanoparticle, wherein the black soldier fly polypeptide-proanthocyanidin nanoparticle is obtained by uniformly mixing a black soldier fly polypeptide solution and a proanthocyanidin solution, and then centrifuging and drying, wherein the mass ratio of the black soldier fly polypeptide to the proanthocyanidin is (0.1-0.5):20.

[0007] In a second aspect, the present invention provides a method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles, comprising the following steps:

[0008] The black soldier fly polypeptide powder is dissolved in water to prepare a black soldier fly polypeptide solution; the proanthocyanidin is dissolved in water to prepare a proanthocyanidin solution;

[0009] The black soldier fly polypeptide solution and the proanthocyanidin solution are mixed and stirred evenly to obtain a black soldier fly polypeptide-proanthocyanidin nanoparticle solution; wherein the mass ratio of the black soldier fly polypeptide to the proanthocyanidin is (0.1-0.5):20;

[0010] The black soldier fly polypeptide-proanthocyanidin nanoparticle solution is then centrifuged and dried to obtain black soldier fly polypeptide-proanthocyanidin nanoparticles.

[0011] Preferably, the specific preparation steps of the black soldier fly polypeptide solution include: dissolving black soldier fly polypeptide powder in distilled water at a concentration of 10 mg / mL, sequentially subjecting to magnetic stirring, hydration, adjusting pH to weak alkalinity, heating and cooling to obtain a black soldier fly polypeptide solution for use.

[0012] Further preferably, in the specific preparation steps of the black soldier fly polypeptide solution, the magnetic stirring temperature is room temperature, the stirring speed is 550-600 r / min, and the stirring time is 0.5-1.5 h; the hydration temperature is 4-8°C, the hydration time is more than 10 h, and the pH is adjusted to 7.2-7.8 with 0.1 mol / L citric acid; the heating temperature is 75-85°C, and the heating time is 30-45 min; the above parameters are selected to ensure that the black soldier fly polypeptide can be fully dissolved.

[0013] Preferably, the concentration of the proanthocyanidin solution is 0.1 to 0.5 mg / mL.

[0014] Preferably, the proanthocyanidin solution and the black soldier fly polypeptide solution are mixed in a volume ratio of 1:2, stirred at room temperature for 1 to 3 hours, and the stirring speed is 550 to 600 r / min; the centrifugal speed of the black soldier fly polypeptide-proanthocyanidin nanoparticle solution is 4000 to 5000 r / min. The above parameters are selected to ensure that the non-covalent interactions between the black soldier fly polypeptides can be interrupted, the aggregates are degraded, and smaller particles are formed with the proanthocyanidins.

[0015] Preferably, the preparation steps of black soldier fly polypeptide powder include:

[0016] S1, washing the black soldier fly larvae, killing enzymes, and then preparing insect pulp;

[0017] S2, adding alkaline protease to the obtained insect pulp to carry out enzymatic hydrolysis reaction;

[0018] S3, after the enzymatic hydrolysis reaction is completed, the enzyme is inactivated, and centrifugation is performed to obtain a fat layer, an enzymatic hydrolysis liquid layer, and a residue layer, wherein the enzymatic hydrolysis liquid layer is a crude polypeptide product;

[0019] S4. Separate the crude polypeptide product through a membrane and then spray dry it to obtain black soldier fly polypeptide powder.

[0020] Further preferably, in step S1, the method for preparing the insect slurry is specifically as follows: firstly, the black soldier fly larvae are placed under natural conditions for 2 to 3 days, cleaned with a microwave cleaner and then blanched to inactivate the enzyme, the black soldier fly larvae are crushed and homogenized at 9000 to 10000 r / min for 10 to 15 min to obtain the insect slurry.

[0021] Further preferably, in step S2, the amount of alkaline protease added is 0.2-0.6% of the mass of the insect pulp, and the enzymatic hydrolysis reaction conditions are: enzymatic hydrolysis at 45-60° C. for 3-5 hours.

[0022] Further preferably, in step S3, the enzyme is inactivated at high temperature, and the enzyme inactivation parameters are: heating at 80-95°C for 15-20 min; and centrifugation at 9000-11000 r / min for 15-25 min.

[0023] More preferably, in step S4, membrane separation uses a filter membrane of 3000-5000 Da.

[0024] Compared with the prior art, the beneficial effects of the present invention include:

[0025] The present invention uses black soldier fly polypeptide as a carrier to load proanthocyanidins to form nanoparticles. The black soldier fly polypeptide, as a new type of insect-derived bioactive peptide, has the advantages of a polypeptide self-assembly system and is suitable as a carrier for delivering proanthocyanidins. The loading rate is as high as 85-90%, and the bioavailability of proanthocyanidins is effectively improved. The preparation method is simple. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] To avoid redundancy, the measuring method involved in the present invention is described here.

[0028] (1) Determination of particle size and zeta potential: The particle size is measured using a particle size analyzer, where the particle size is the average particle size.

[0029] (2) Determination of polypeptide content: First, separate small peptides by methanol precipitation method, weigh 3 g of each enzymatic hydrolyzed sample into a conical flask, add 30 mL of distilled water, stir magnetically for 30 min, put into a centrifuge tube and centrifuge at 2500 r / min for 10 min, take the supernatant and add the same volume of methanol, mix and let stand for 20 min, centrifuge at 4000 r / min for 20 min, take the supernatant, rotary evaporate and freeze-dry to obtain the polypeptide sample to be tested. The polypeptide content in the polypeptide sample to be tested is determined by the Kjeldahl nitrogen method. This method can remove polysaccharides and eliminate the influence of nitrogen in polysaccharides. The polypeptide content in the sample is determined by the following formula:

[0030]

[0031] Where V1 is the volume of sulfuric acid or hydrochloric acid standard solution consumed by the sample, mL; V2 is the volume of sulfuric acid or hydrochloric acid standard solution consumed by the reagent blank, mL; N is the equivalent concentration of sulfuric acid or hydrochloric acid standard solution; m is the mass of the sample, g; F is the coefficient for converting nitrogen to protein, and the conversion coefficient for meat and fish is selected here, that is, 6.25.

[0032] (3) Determination of proanthocyanidin loading rate: 10 mg of black soldier fly polypeptide-proanthocyanidin nanoparticles were taken and immersed in 10 mL of 0.1 M PBS solution. Ultrasonic stirring was performed to completely decompose the nanoparticles. The proanthocyanidin content was determined by the vanillin sulfate method. The proanthocyanidin loading rate was determined by the following formula:

[0033]

[0034] (4) Bioavailability determination: simulate in vitro gastrointestinal digestion, where the gastric digestion fluid is prepared by accurately weighing 1.0g of pepsin, adding 225mL of 9mg / mL sodium chloride solution to dissolve, adjusting the pH value to 2.0-2.3 with 0.1M HCl, transferring to a 250mL volumetric flask, and making up to volume with 9mg / mL sodium chloride solution. The intestinal digestion fluid is prepared by weighing 562.5mg of trypsin and 562.5mg of pig bile salt, adding 225mL of 9mg / mL sodium chloride solution to dissolve, adjusting the pH to 7-7.4 with 0.1M NaOH, and making up to the scale with 9mg / mL sodium chloride solution. Specific operation steps: take two 50mL centrifuge tubes, add 10mL of 5mg / mL black soldier fly polypeptide-proanthocyanidin nanoparticle solution and 8mL of gastric digestive enzyme, respectively, and shake in a water bath at 37°C and 180r / min for 2h. After gastric digestion, add 0.5M Na2CO3 to the sample to adjust the pH to about 5.3 to stop gastric digestion. Then add 8mL of intestinal digestion fluid, and adjust the pH to 7.0-7.4 with 0.1M NaOH, and shake in a water bath at 37°C and 180r / min for 2h. Centrifuge at 10,000r / min for 10min, collect the supernatant and make it up to 50mL, and store it at -20°C until analysis. The proanthocyanidin content was determined by the vanillin sulfate method, and the bioavailability was determined by the following formula,

[0035]

[0036] A blank group was set up, that is, black soldier fly polypeptide-proanthocyanidin nanoparticles were replaced with proanthocyanidins to prepare a solution with the same concentration, and the bioavailability thereof was determined by the above method to be 4.21% (measured proanthocyanidin amount / (10*5)×100%).

[0037] Example 1

[0038] The black soldier fly larvae were placed under natural conditions for 3 days, washed by a microwave cleaning machine, and then scalded in 100°C hot water for 5 minutes to inactivate enzymes; the black soldier fly larvae obtained after enzyme inactivation were beaten into insect pulp and homogenized at 10000 r / min for 10 minutes to obtain black soldier fly insect pulp.

[0039] To the obtained black soldier fly slurry, 0.4% of the mass of alkaline protease (Angel Enzyme Co., Ltd., AP-20) was added, and enzymolysis was carried out at a temperature of 55°C for 4.5 hours to obtain a black soldier fly slurry hydrolysate, which was placed in a 90°C water bath to inactivate the enzyme for 15 minutes, and then centrifuged at 10000 r / min for 20 minutes to obtain a fat layer, an enzymolysis liquid layer and a residue layer, wherein the enzymolysis liquid layer is a crude polypeptide product, and the crude polypeptide product was separated by a 3500D filter membrane and spray-dried at 125°C to obtain a black soldier fly polypeptide powder, in which the polypeptide content reached 91.03%.

[0040] The black soldier fly polypeptide powder was dissolved in distilled water to a concentration of 10 mg / mL, and magnetically stirred at 600 r / min at room temperature for 1 hour, placed in a refrigerator, hydrated at 4°C for 12 hours, adjusted to pH 7.4 with 0.1 M citric acid, heated at 90°C for 30 minutes, and cooled to obtain a black soldier fly polypeptide solution.

[0041] Proanthocyanidin was dissolved in water to prepare a proanthocyanidin solution with a concentration of 0.5 mg / mL. The proanthocyanidin solution was added to the black soldier fly polypeptide solution under magnetic stirring at 600 r / min. The volume ratio of the proanthocyanidin solution to the black soldier fly polypeptide solution was 1:2. The stirring time was 2 h to obtain a black soldier fly polypeptide-proanthocyanidin nanoparticle solution. The solution was centrifuged at 5000 r / min for 15 min to remove the precipitate. The supernatant was spray dried at 125° C. to obtain black soldier fly polypeptide-proanthocyanidin nanoparticles.

[0042] After testing, the particle size of the black soldier fly polypeptide-proanthocyanidin nanoparticles in Example 1 was 90.78 nm, the Zeta potential was -27.27 mV, the proanthocyanidin loading rate was 86.13%, and the proanthocyanidin bioavailability was increased from 4.21% to 66.80%.

[0043] Comparative Example 1

[0044] The only difference from Example 1 is that a conventional polypeptide (source is soybean peptide, Jiangsu Peptide Bioengineering Co., Ltd., <3000D) is used to replace the black soldier fly polypeptide powder in Example 1, and its molecular weight is close to that of the black soldier fly polypeptide in Example 1. The other steps and conditions are the same as those in Example 1.

[0045] In comparative example 1, the particle size of soybean peptide-proanthocyanidin nanoparticles was 160.23 nm, the Zeta potential was -18.97 mV, the proanthocyanidin loading rate was 72.83%, and the proanthocyanidin bioavailability was 50.22%. It can be seen that black soldier fly polypeptide can form nanoparticles with proanthocyanidins that are smaller in size, more stable, and have higher loading rate and bioavailability.

[0046] Comparative Example 2

[0047] The only difference from Example 1 is that the concentration of the proanthocyanidin solution is 2 mg / mL, and the other steps and conditions are the same as those in Example 1.

[0048] In Comparative Example 2, the particle size of the black soldier fly polypeptide-proanthocyanidin nanoparticles was 51.95 nm, the Zeta potential was -26.90 mV, the proanthocyanidin loading rate was 55.78%, and the proanthocyanidin bioavailability was 46.83%.

[0049] Example 2

[0050] The only difference from Example 1 is that the concentration of the proanthocyanidin solution is 0.3 mg / mL, and the other steps and conditions are the same as those in Example 1.

[0051] The particle size of the black soldier fly polypeptide-proanthocyanidin nanoparticles in Example 2 is 81.56 nm, the Zeta potential is -29.67 mV, the proanthocyanidin loading rate is 83.42%, and the proanthocyanidin bioavailability is 62.15%.

[0052] Comparative Example 3

[0053] The only difference from Example 1 is that the hydration time is 6 hours, and the other steps and conditions are the same as those in Example 1.

[0054] In Comparative Example 3, the particle size of the black soldier fly polypeptide-proanthocyanidin nanoparticles was 357.13 nm, the Zeta potential was -16.07 mV, the proanthocyanidin loading rate was 13.22%, and the proanthocyanidin bioavailability was 15.10%.

[0055] Example 3

[0056] The only difference from Example 1 is that the hydration time is 10 h, and the other steps and conditions are the same as those in Example 1.

[0057] The particle size of the black soldier fly polypeptide-proanthocyanidin nanoparticles in Example 3 is 119.80 nm, the Zeta potential is -13.33 mV, the proanthocyanidin loading rate is 58.36%, and the proanthocyanidin bioavailability is 44.79%.

[0058] The test results of the above embodiments 1-3 and comparative examples 1-3 are statistically analyzed, and the results are shown in the following Table 1.

[0059] Table 1 Test results of Examples 1-3 and Comparative Examples 1-3

[0060]

[0061]

[0062] It can be seen from Example 1 and Comparative Example 1 that the effect of using soybean peptide is not as good as that of black soldier fly polypeptide; the black soldier fly polypeptide used in the present invention can form nanoparticles with proanthocyanidins with smaller particle size, greater stability, higher loading rate and bioavailability.

[0063] It can be seen from Examples 1-3 and Comparative Examples 2-3 that when the concentration of the proanthocyanidin solution is too high and the hydration time is too short, the black soldier fly polypeptide-proanthocyanidin nanoparticles will be more unstable, the proanthocyanidin loading rate will be lower, and the proanthocyanidin bioavailability will be lower. Therefore, the present invention preferably has a proanthocyanidin solution concentration of 0.3 to 0.5 mg / mL and a hydration time of 10 to 12 h, more preferably 0.5 mg / mL and 12 h.

[0064] The invention discloses a black soldier fly polypeptide-proanthocyanidin nanoparticle and a preparation method thereof, wherein the method comprises: washing black soldier fly larvae, killing enzymes, and preparing insect pulp; adding alkaline protease to the enzymatic hydrolyzate to obtain black soldier fly polypeptide; magnetically stirring the hydrated black soldier fly polypeptide solution and the proanthocyanidin solution according to a certain volume ratio, then centrifuging to remove precipitation, and spray drying the supernatant to obtain black soldier fly polypeptide-proanthocyanidin nanoparticles. The method provided by the invention constructs a proanthocyanidin nano-delivery system with black soldier fly polypeptide as a carrier, with a loading rate of 58.36-90%, and the preparation method is simple, and the bioavailability of proanthocyanidin is improved, with a bioavailability of 44.79-66.80%.

[0065] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A black soldier fly polypeptide-proanthocyanidin nanoparticle, characterized in that: The black soldier fly polypeptide-proanthocyanidin nanoparticles are obtained by mixing a black soldier fly polypeptide solution and a proanthocyanidin solution, stirring them uniformly, and then centrifuging and drying them, wherein the mass ratio of the black soldier fly polypeptide to the proanthocyanidin is (0.1-0.5):

20.

2. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 1, characterized in that: The following steps are involved: The black soldier fly polypeptide powder is dissolved in water to prepare a black soldier fly polypeptide solution; the proanthocyanidin is dissolved in water to prepare a proanthocyanidin solution; The black soldier fly polypeptide solution and the proanthocyanidin solution are mixed and stirred evenly to obtain a black soldier fly polypeptide-proanthocyanidin nanoparticle solution; The mass ratio of black soldier fly polypeptide to proanthocyanidins is (0.1-0.5):20; The black soldier fly polypeptide-proanthocyanidin nanoparticle solution is then centrifuged and dried to obtain black soldier fly polypeptide-proanthocyanidin nanoparticles.

3. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 2, characterized in that: The specific preparation steps of the black soldier fly polypeptide solution include: dissolving black soldier fly polypeptide powder in distilled water at a concentration of 10 mg / mL, sequentially subjecting the mixture to magnetic stirring, hydration, adjusting pH to weak alkalinity, heating and cooling to obtain a black soldier fly polypeptide solution for use.

4. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 3, characterized in that: In the specific preparation steps of the black soldier fly polypeptide solution, the magnetic stirring temperature is room temperature, the stirring speed is 550-600 r / min, and the stirring time is 0.5-1.5 h; the hydration temperature is 4-8°C, the hydration time is more than 10 h, and the pH is adjusted to 7.2-7.8 with 0.1 mol / L citric acid; the heating temperature is 75-85°C, and the heating time is 30-45 min.

5. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 2, characterized in that: The concentration of the proanthocyanidin solution is 0.1-0.5 mg / mL.

6. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 2, characterized in that: The proanthocyanidin solution and the black soldier fly polypeptide solution are mixed in a volume ratio of 1:2, stirred at room temperature for 1 to 3 hours, and the stirring speed is 550 to 600 r / min; the centrifugal speed of the black soldier fly polypeptide-proanthocyanidin nanoparticle solution is 4000 to 5000 r / min.

7. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 2, characterized in that: The preparation steps of black soldier fly polypeptide powder include: S1, washing the black soldier fly larvae, killing enzymes, and then preparing insect pulp; S2, adding alkaline protease to the obtained insect pulp to carry out enzymatic hydrolysis reaction; S3, after the enzymatic hydrolysis reaction is completed, the enzyme is inactivated, and centrifugation is performed to obtain a fat layer, an enzymatic hydrolysis liquid layer, and a residue layer, wherein the enzymatic hydrolysis liquid layer is a crude polypeptide product; S4. Separate the crude polypeptide product through a membrane and then spray dry it to obtain black soldier fly polypeptide powder.

8. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 7, characterized in that: In step S1, the method for preparing insect slurry is specifically as follows: firstly, the black soldier fly larvae are placed under natural conditions for 2 to 3 days, cleaned with a microwave cleaner and then blanched to inactivate enzymes, the black soldier fly larvae are crushed and homogenized at 9000 to 10000 r / min for 10 to 15 minutes to obtain insect slurry.

9. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 7, characterized in that: In step S2, the amount of alkaline protease added is 0.2-0.6% of the mass of the insect pulp, and the enzymatic hydrolysis reaction conditions are: enzymatic hydrolysis at 45-60° C. for 3-5 hours.

10. The method for preparing black soldier fly polypeptide-proanthocyanidin nanoparticles according to claim 7, characterized in that: In step S3, the enzyme is inactivated at high temperature, and the parameters for inactivation are: heating at 80-95° C. for 15-20 min; Centrifugation is carried out at 9000-11000 r / min for 15-25 min; In step S4, membrane separation uses a filter membrane of 3000 to 5000 Da.

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