CN115501346A

CN115501346A - Tea residue protein-epsilon-polylysine nano material and anthocyanin nano compound and preparation method thereof

Info

Publication number: CN115501346A
Application number: CN202211189178.6A
Authority: CN
Inventors: 陈琪; 方康志; 夏雨琴; 王宇晴; 高学玲; 宛晓春
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2022-12-23
Anticipated expiration: 2042-09-28
Also published as: CN115501346B

Abstract

The invention belongs to the technical field of nanoparticle preparation, and particularly relates to a tea residue protein-epsilon-polylysine nano material, an anthocyanin nano compound and a preparation method thereof. The invention provides a tea-leaf protein-epsilon-polylysine nano material, which comprises tea-leaf protein nano particles and epsilon-polylysine; the tea residue protein nano-particles and the epsilon-polylysine form a reticular compound by a polyelectrolyte compounding method. The results of the examples show that the tea residue protein-epsilon-polylysine nano material provided by the invention has the advantages that the thermal stability of anthocyanin is improved by 15%, the light stability is improved by 14%, and the accurate slow control of anthocyanin gastrointestinal tract is realized.

Description

Tea residue protein-epsilon-polylysine nano material and anthocyanin nano compound and preparation method thereof

Technical Field

The invention belongs to the technical field of nanoparticle preparation, and particularly relates to a tea residue protein-epsilon-polylysine nano material, an anthocyanin nano compound and a preparation method thereof.

Background

The tea residue contains 20-30% of tea protein, wherein the tea protein mainly comprises glutelin and prolamin. The tea residue protein belongs to vegetable protein, is rich in amino acid composition, does not contain cholesterol, and is very suitable for eating. Researches find that the tea protein has better functions such as blood pressure reduction, blood sugar reduction, radiation protection and the like, so the tea protein has higher development and utilization values. Tea residue protein is mainly applied to the preparation of health Food, feed, antioxidant and microcapsule, just as the prior art (Ren, z., chen, z., zhang, y., zhao, t., ye, x., gao, x., (2019) Functional properties and structural profiles of water-insoluble proteins from the same types of oils of tea wastes, lwt-Food Science and Technology,110, 324-331) adopts tea residue protein nanoparticles as the stabilizer of Pickering emulsion to maintain the oxidation stability of oil and fat, and the application effect is good. At present, the tea residue protein is not reported as a nano delivery carrier of anthocyanin. Anthocyanin is an extremely unstable and poorly bioavailable active substance and is particularly sensitive to the environment. Anthocyanin is easily affected by environmental factors such as temperature, oxygen, enzyme, light and ascorbic acid to cause the structure to be damaged, and the absorption rate of anthocyanin in gastrointestinal tract is very low due to the easy damage of the structure, so that the problem how to solve the stability of anthocyanin and improve the long-term absorption of anthocyanin in gastrointestinal tract is the problem to be solved at present.

The nano-delivery carrier (nano-delivery) refers to a drug (bioactive substance) delivery system with the average particle size of 10-1000 nm prepared by a mechanical or chemical method, and small-molecule active substances are embedded or loaded in the particles, so that the purposes of protection, slow release and targeted delivery are achieved.

In the prior art, the nano delivery carrier of anthocyanin is mainly soybean protein, beta-lactoglobulin, chitosan, sodium alginate and the like, just as in the prior art (Chen, Z., wang, C., gao, X., chen, Y., santhanam, R.K. (2019) Interaction chromatography of pretreated so protein isolate with anthocyanin-3-O-glucose and the present effects on the stability of black soy protein prepared by inorganic nanoparticles Chemistry,271, 266-273) discloses that the anthocyanin shows good thermal and oxidative stability by using the soybean protein modified by heating as a wall material and the anthocyanin as a core material. Meanwhile, the stability of anthocyanin is improved to a certain extent by the nano materials such as soybean protein, beta-lactoglobulin, chitosan, sodium alginate and the like, but the improvement range of the heat stability of anthocyanin is limited after the anthocyanin is embedded by the soybean protein, the beta-lactoglobulin, the chitosan and the sodium alginate, and the substances such as the soybean protein, the beta-lactoglobulin, the chitosan and the sodium alginate are difficult to reduce the cost by waste utilization.

Therefore, intensive research is needed to solve the technical problem of low improvement of thermal stability after anthocyanin embedding.

Disclosure of Invention

The invention aims to provide a tea residue protein-epsilon-polylysine nano material, which is used as a carrier for embedding anthocyanin and improves the thermal stability and the light stability of the anthocyanin.

In order to achieve the technical effects, the invention provides the following technical scheme:

the invention provides a tea leaf protein-epsilon-polylysine nano-material, which comprises tea leaf protein nano-particles and epsilon-polylysine; the tea-leaf protein nanoparticles and epsilon-polylysine form a network complex.

The invention provides a tea residue protein-epsilon-polylysine-anthocyanin nano compound, which adopts the technical scheme that tea residue protein nano particles and epsilon-polylysine are used as wall materials, and anthocyanin is used as a core material.

The invention provides a preparation method of the tea residue protein-epsilon-polylysine-anthocyanin nano compound, which comprises the following steps: mixing the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution with the epsilon-polylysine nano solution, and homogenizing to form a tea residue protein-epsilon-polylysine-anthocyanin nano compound;

the pH value of the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution is 2-6 after the mixed solution and the epsilon-polylysine nano solution are mixed;

the mass concentration of the tea leaf protein in the tea leaf protein nano solution is 0.02-0.125 mg/mL; the mass concentration of anthocyanin in the anthocyanin nano solution is 0.2-1.25 mg/mL; the mass concentration of the epsilon-polylysine in the epsilon-polylysine nano solution is 0.02-0.125 mg/mL.

Preferably, the mixed solution of the tea leaf protein nano solution and the anthocyanin nano solution is obtained by adding the anthocyanin nano solution into the tea leaf protein nano solution and mixing;

the volume ratio of the tea residue protein nano solution to the epsilon-polylysine nano solution to the anthocyanin nano solution is (1-5) to 1.

Preferably, the tea residue protein nano solution comprises tea residue protein nano particles and a buffer solution; the mass volume ratio of the tea-leaf protein nanoparticles to the buffer solution is (2-10) mg: (80-100) mL; the buffer solution comprises PBS buffer solution, and the concentration of the PBS buffer solution is 0.01-0.06M.

Preferably, the preparation method of the tea residue protein nano solution comprises the following steps: and mixing and homogenizing the tea leaf protein nanoparticles and the buffer solution to obtain the tea leaf protein nano solution.

Preferably, the epsilon-polylysine nano solution comprises epsilon-polylysine nano particles and a buffer;

the mass-volume ratio of the epsilon-polylysine nano particles to the buffer solution is (2-10) mg: (80-100) mL;

the buffer solution comprises PBS buffer solution, and the concentration of the PBS buffer solution is 0.01-0.06M.

Preferably, the solvent of the anthocyanin nano solution is 0.01M PBS buffer solution, and the mass-volume ratio of the anthocyanin to the solvent is (20-100) mg, (80-100) mL.

Preferably, the particle size of the tea residue protein-epsilon-polylysine-anthocyanin nano compound is 100-200 nm.

Preferably, the homogenizing comprises ultrasonic homogenizing, wherein the ultrasonic homogenizing time is 5-20 min, the power is 200-400W, and the frequency is 25Hz.

The invention has the beneficial effects that: the invention provides a tea-leaf protein-epsilon-polylysine nano material, which comprises tea-leaf protein nano particles and epsilon-polylysine; the tea residue protein nano-particles and the epsilon-polylysine form a reticular compound by a polyelectrolyte compounding method. According to the invention, tea residue protein can carry negative charges above the isoelectric point pH =3, and epsilon-polylysine can carry positive charges below the isoelectric point pH = 9.74; tea residue protein with negative charges and epsilon-polylysine with positive charges carry out polyelectrolyte complex reaction to realize that the epsilon-polylysine and the tea residue protein are complexed and crosslinked to generate nano particles, and the nano particles can realize effective embedding of anthocyanin under the conditions of certain concentration and pH value. The tea residue raw material has wide sources, and the invention extracts tea residue protein from the tea residue waste and develops products, thereby greatly improving the residual value of the tea residue and being beneficial to the comprehensive utilization and sustainable development of the bio-based material. The results of the examples show that the tea residue protein-epsilon-polylysine nano material provided by the invention has the advantages that after the anthocyanin is embedded, the heat stability of the anthocyanin is improved by 15%, the light stability is improved by 14%, and the precise and slow control of the gastrointestinal tract of the anthocyanin is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.

FIG. 1 is the amino acid composition of tea residue protein prepared in example 1;

FIG. 2-1 is a scanning electron micrograph of TP-PLL prepared in comparative example 2, in which A1, A2, A3 are magnified electron micrographs of TP-PLL nanocomposites at 8000-fold, 15000-fold, and 40000-fold, respectively;

FIG. 2-2 is a scanning electron micrograph of ACNs-TP-PLL prepared in example 1, wherein B1, B2, B3 are magnified electron micrographs at 8000 times, 15000 times, 40000 times, respectively, of ACNs-TP-PLL nanocomposites;

FIGS. 2 to 3 are scanning electron micrographs of ACNs-TP (tea residue protein anthocyanin nanocomposites) prepared in comparative example 1, wherein C1, C2, and C3 are magnified electron micrographs of the ACNs-TP nanocomposites at 8000 times, 15000 times, and 40000 times, respectively;

FIGS. 2 to 4 are scanning electron micrographs of ACNs (anthocyanin nanoparticles) prepared in comparative example 3, wherein D1, D2 and D3 are magnified electron micrographs of ACNs nanoparticles at 8000 times, 15000 times and 40000 times, respectively;

FIGS. 2 to 5 are magnified electron micrographs at 15000 times of the TP-PLL of comparative example 2, the ACNs-TP-PLL of example 1, the ACNs-TP of comparative example 1 and the ACNs of comparative example 3;

FIG. 3 is a Fourier infrared spectrum of the tea residue protein TP prepared in example 1, the ACNs-TP-PLL (tea residue protein-epsilon-polylysine-anthocyanin nanocomposite) prepared in example 1, the ACNs prepared in comparative example 3, the ACNs-TP prepared in comparative example 1, and the TP-PLL prepared in comparative example 2;

FIG. 4 is a UV spectrum of ACNs-TP-PLL prepared in example 1, ACNs prepared in comparative example 3, and ACNs-TP prepared in comparative example 1;

fig. 5 is a graph showing the retention of ACNs in simulated gastric fluid and simulated intestinal fluid for ACNs-TP-PLL prepared in example 1 and ACNs prepared in comparative example 3, where a is simulated gastric fluid and B is simulated intestinal fluid.

FIG. 6-1 shows the retention of ACNs-TP-PLL of example 3 and ACNs of comparative example 5 under different treatments; wherein A is different continuous heating time, B is different illumination time, C is different pH value, and D is different K ⁺ The strength of the ion concentration;

FIG. 6-2 shows different pH values and different K ⁺ The particle size of ACNs-TP-PLL under the condition of ionic strength, wherein A is the particle size of ACNs-TP-PLL under the condition of different pH values, and B is different K ⁺ The particle size of ACNs-TP-PLL under the condition of ionic strength;

FIG. 7 is a graph showing anthocyanin retention rates of ACNs-TP-PLL prepared in example 1, ACNs prepared in comparative example 3, ACNs-TP prepared in comparative example 1, ACNs-PLL and ACNs-SPi.

Detailed Description

The invention provides a tea-leaf protein-epsilon-polylysine nano material, which comprises tea-leaf protein nano particles and epsilon-polylysine; the tea residue protein nanoparticles and epsilon-polylysine form a network complex.

The tea residue protein nanoparticles are not particularly limited in source, and can be prepared from any tea residue protein nanoparticles. The grain size of the tea residue protein is preferably in a nanometer level.

In the present invention, the preparation method of the tea residue protein nanoparticles preferably comprises the steps of: carrying out alkali extraction on the tea residue powder to obtain tea residue powder supernatant, and carrying out acid precipitation on the tea residue powder supernatant to obtain crude tea residue powder protein; and drying the crude tea residue powder protein to obtain tea residue protein nanoparticles.

The tea residue powder is preferably prepared by the following steps: the tea residue is extracted by boiling water and filtered to obtain tea residue, and the tea residue is dried and crushed to obtain tea residue powder. In the present invention, the ratio of the mass of the tea leaves to the mass of the boiling water in the boiling water extraction is preferably (1 to 5) g: (30 to 60) mL, more preferably (1 to 5) g: (40-55) mL; more preferably 1g:50mL. The time for the boiling water leaching is preferably 5 to 20min, more preferably 10 to 18min, and even more preferably 15min. The boiling water leaching and filtering are preferably repeated, the tea residue is obtained by particularly leaching and filtering with boiling water, the tea residue is further leached and filtered with boiling water to obtain the tea residue, the process is preferably repeated for 2-4 times, more preferably for 3 times, the process is preferably carried out for 3 times, the extraction rate of tea residue protein can be improved, and the loss in the tea residue protein extraction process is reduced. When the boiling water leaching is repeated, the volume of the boiling water added every time is the same.

After the tea leaves are leached by boiling water, the invention preferably filters the feed liquid obtained by leaching to obtain the tea leaves. The present invention does not require any particular means of filtration, as will be appreciated by those skilled in the art. In the invention, the filtering mode is preferably to filter the leaching liquor by using cotton cloth with the thickness of 550 mu m, and the filtering mode can remove the residual polyphenols in the tea leaves.

After the tea leaves are obtained, the tea leaves are preferably dried and crushed to obtain tea leaf powder. In the present invention, the temperature for drying is preferably 60 to 80 ℃, more preferably 62 to 70 ℃, and still more preferably 65 ℃. The invention is preferably dried until the tea leaves can be smashed into powder; in the embodiment of the present invention, the drying time is preferably 72 hours. The crushing mode is not particularly limited, and the conventional mode can be adopted. After the tea residue powder is obtained, the invention preferably utilizes a 180 mu m screen when the crushed tea residue powder is screened.

Obtaining the sieved tea powder, preferably performing alkali extraction on the tea powder to obtain the supernatant of the tea powder. In the present invention, the alkali extraction lye preferably comprises a NaOH solution; the mass volume ratio of the tea residue powder to the NaOH solution is preferably 1g (20-50) mL, more preferably 1g (35-50) mL, and more preferably 1g (50mL); the alkali extraction time is preferably 80-100 min, more preferably 85-95 min, and even more preferably 90min; the temperature of the alkali extraction is preferably 80 to 100 ℃, more preferably 86 to 93 ℃, and still more preferably 90 ℃.

After the alkali extraction, the invention preferably centrifuges the alkali extraction solution to obtain the supernatant of the tea residue powder. The rotating speed of the centrifugation is preferably 4500-8500 rpm, more preferably 6000-8300 rpm, and more preferably 8000rpm; the time for centrifugation is preferably 10 to 30min, more preferably 13 to 20min, and still more preferably 15min.

After the supernatant of the tea residue powder is obtained, the invention preferably carries out acid precipitation on the supernatant of the tea residue powder to obtain crude protein of the tea residue powder. According to the acid precipitation method, the pH value of the supernatant of the tea residue powder is preferably adjusted by using an HCl solution until a large amount of crude protein precipitates of the tea residue powder are obtained. The concentration of the HCl solution of the present invention is preferably 0.06 to 0.1M, more preferably 0.08 to 0.1M, and still more preferably 0.1M. The pH value of the adjusted tea residue powder supernatant is preferably 2.0-3.4, more preferably 2.5-3.2, and even more preferably 3.0.

After acid precipitation, the invention preferably centrifuges the supernatant of the tea residue powder obtained by precipitation to obtain crude protein of the tea residue powder. The rotation speed of the centrifugation is preferably 3000-5000 rpm, more preferably 4000-4800 rpm, and more preferably 4500rpm; the time for the centrifugation is preferably 10 to 30min, more preferably 20 to 30min, and still more preferably 30min. The invention preferably uses pure water to wash the crude protein of the tea residue powder until the pH value of the crude protein is neutral.

After washing with pure water, the invention preferably dries the crude tea residue powder protein to obtain tea residue protein nanoparticles. The drying mode of the invention is preferably freeze drying, the temperature of the freeze drying is preferably-50 ℃, and the time of the freeze drying is preferably 72h.

The invention can effectively improve the added value of tea leaves by deeply processing the byproduct of tea dregs to extract tea dreg protein nanoparticles as the wall material of an anthocyanin embedding material, and simultaneously, the tea dreg protein is richer in amino acid content relative to other protein tea dreg proteins, contains various proteins required by human bodies, and is safer and more reliable as the wall material of the embedding material of anthocyanin relative to polysaccharide. The invention extracts the tea residue protein and the epsilon-polylysine to prepare the nano material by combination.

The source of the epsilon-polylysine is not particularly limited, and the epsilon-polylysine can be prepared by adopting a conventional commercial product. The particle size of the epsilon-polylysine is preferably nano-grade.

The source of the anthocyanin is not particularly limited, and the conventional commercial product is adopted. The particle size of the anthocyanin is preferably nano-grade.

The invention provides a preparation method of the tea residue protein-epsilon-polylysine-anthocyanin nano compound in the technical scheme, which comprises the following steps: mixing the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution with the epsilon-polylysine nano solution, and homogenizing to form a tea residue protein-epsilon-polylysine-anthocyanin nano compound;

the mass concentration of the tea-leaf protein in the tea-leaf protein nano solution is 0.02-0.125 mg/mL, and more preferably 0.08mg/mL; the mass concentration of anthocyanin in the anthocyanin nano solution is 0.2-1.25 mg/mL, and more preferably 0.6mg/mL; the mass concentration of the epsilon-polylysine in the epsilon-polylysine nano solution is 0.02-0.125 mg/mL, and more preferably 0.08mg/mL.

Under the condition of a certain pH value and within a certain concentration range, the tea leaf protein nanoparticles and the epsilon-polylysine can be crosslinked to form a net structure, and the tea leaf protein nanoparticles and the epsilon-polylysine can generate flocculation and precipitation when the pH value and the concentration range are exceeded.

In the present invention, the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution is preferably obtained by adding the anthocyanin nano solution to the tea residue protein nano solution and mixing.

In the present invention, the tea residue protein nano solution preferably comprises tea residue protein nanoparticles and a buffer solution; the mass-volume ratio of the tea-residue protein nanoparticles to the buffer solution is preferably (2-10) mg: (80 to 100) mL, more preferably (5 to 9) mg: (90-100) mL, more preferably 8mg:100mL. The buffer solution of the present invention preferably includes a phosphate buffer solution, and the concentration of the phosphate buffer solution is preferably 0.01M to 0.06M, more preferably 0.01M to 0.03M, and still more preferably 0.01M. The PBS buffer solution is selected because the PBS buffer solution can keep the pH of the system to be relatively stable, meanwhile, the PBS buffer solution can better dissolve and protect solute compared with distilled water, has better salt balance effect, and is more stable and reliable in the result of adjusting the pH by the PBS compared with the distilled water.

In the present invention, the preparation method of the tea residue protein nano solution preferably comprises: and mixing and homogenizing the tea leaf protein nanoparticles and the buffer solution to obtain the tea leaf protein nano solution.

The mixing mode is not particularly limited, and the uniform mixing can be ensured by adopting a conventional mode.

In the present invention, the means of homogenization preferably comprises ultrasonic homogenization. The tea residue protein nano solution is obtained after the ultrasonic homogenization.

The time for ultrasonic homogenization is preferably 5-20 min, more preferably 8-15 min, and even more preferably 10min; the power of the ultrasonic homogenization is preferably 200-400W, more preferably 280-350W, and even more preferably 300W; the frequency of the ultrasonic homogenization is preferably 25Hz. In the present invention, the ultrasonic homogenization is preferably a batch ultrasonic homogenization, and more preferably an ultrasonic 3s batch 3s. According to the invention, the tea residue protein nanoparticles are completely dissolved and uniformly dispersed in the PBS solution through ultrasonic homogenization, so that the crosslinking with epsilon-polylysine is better realized.

In the present invention, the solvent of the anthocyanin nano solution is preferably 0.01M PBS buffer. In the present invention, the mass-to-volume ratio of the anthocyanin nanophase solution to the solvent (20 to 100) mg to (80 to 100) mL, more preferably (50 to 80) mg to (85 to 100) mL, still more preferably 60mg:100mL. The pH value of the PBS buffer solution is preferably 2-6. The advantages of the PBS buffer are discussed above and will not be described further herein.

In the invention, the epsilon-polylysine nano solution comprises epsilon-polylysine nano particles and a buffer solution; the preparation method of the epsilon-polylysine nano solution preferably comprises the following steps: and mixing the epsilon-polylysine nano particles with a buffer solution to obtain an epsilon-polylysine nano solution. The mass-volume ratio of the epsilon-polylysine nanoparticles to the buffer solution is preferably (2-10) mg: (80 to 100) mL, more preferably (6 to 9) mg: (92-100) mL, more preferably 8mg:100mL.

The buffer solution of the present invention preferably includes a PBS buffer solution, and the concentration of the PBS buffer solution is preferably 0.01 to 0.06M, more preferably 0.01 to 0.03M, and still more preferably 0.01M. The PBS buffer solution with the concentration is helpful for obtaining a stable epsilon-polylysine nano solution system, and the high concentration of the PBS buffer solution can influence the epsilon-polylysine nano solution system.

After the tea residue protein nano solution, the epsilon-polylysine nano solution and the anthocyanin nano solution are obtained, the mixed solution of the tea residue protein nano particles and the anthocyanin is mixed with the epsilon-polylysine nano solution, and the mixture is homogenized to form the tea residue protein-epsilon-polylysine-anthocyanin nano compound. The tea residue protein-epsilon-polylysine-anthocyanin nano-composite is preferably formed by adding anthocyanin nano-solution into tea residue protein nano-solution, mixing, adding epsilon-polylysine nano-solution and homogenizing; more preferably, the anthocyanin nano solution is slowly added into the tea residue protein nano solution, uniformly stirred and then added with the epsilon-polylysine nano solution. The slow addition is preferably 1mL/min to 5mL/min, more preferably 2mL/min to 4mL/min, and even more preferably 3mL/min. Although the tea residue protein nano solution, the epsilon-polylysine nano solution and the anthocyanin nano solution are all in the nano grade, when the tea residue protein nano solution, the epsilon-polylysine nano solution and the anthocyanin nano solution are polymerized, nano particles are formed under proper conditions, otherwise the tea residue protein and the polylysine can also form particles with larger particle sizes so as to flocculate and precipitate.

The stirring mode is not particularly limited in the invention, and a conventional mode can be adopted. The anthocyanin nano solution is added into the tea residue protein nano solution in order to allow the anthocyanin nano solution to be attached to macromolecular tea residue protein, and then the epsilon-polylysine nano solution is added to be complexed and crosslinked with the tea residue protein, so that the anthocyanin exists in a network structure formed by the crosslinking and complexing of the epsilon-polylysine and the tea residue protein, and a tea residue protein-epsilon-polylysine-anthocyanin nano compound is formed, thereby achieving the effect of protecting the anthocyanin. If the net structure of the complex of epsilon-polylysine and tea-residue protein is prepared first and then anthocyanin is added, the anthocyanin can not be well embedded, so that the effect of protecting the anthocyanin cannot be achieved. The method provided by the invention is used for embedding anthocyanin by utilizing the polyelectrolyte composite action of tea residue protein and epsilon-polylysine so as to improve the stability of anthocyanin. The polyelectrolyte compounding process belongs to in-situ polymerization process, and is one in-situ synthesis process.

The preparation method comprises the steps of adding an epsilon-polylysine nano solution into a mixed solution of tea leaf protein nanoparticles and anthocyanin, and homogenizing to form the tea leaf protein-epsilon-polylysine-anthocyanin nano compound. In the present invention, the volume ratio of the tea residue protein nano solution, the epsilon-polylysine nano solution, and the anthocyanin nano solution is preferably (1 to 5): 1, more preferably (1.5 to 3): 1, and more preferably 2. In the present invention, the pH value of the tea leaf residue protein nano solution, the anthocyanin nano solution, and the epsilon-polylysine nano solution after mixing is 2 to 6, more preferably 4 to 5.5, and still more preferably 5. According to the invention, tea residue protein can carry negative charges above isoelectric point pH =3, and epsilon-polylysine is positively charged below isoelectric point pH = 9.74; tea-leaf protein with negative charges and epsilon-polylysine with positive charges carry out polyelectrolyte complex reaction to realize the complexing and crosslinking of the epsilon-polylysine and the tea-leaf protein to generate nano particles. The tea residue protein is food-grade, safe and non-toxic, the polylysine has a good antibacterial effect and is non-toxic, the tea residue protein and the polylysine are used as wall materials, the anthocyanin is used as a core material, and the particles with the particle size of nanometer level are formed by a polyelectrolyte composite method, so that the tea residue protein is safe and non-toxic. The tea leaf protein-epsilon-polylysine-anthocyanin nano composite prepared by the invention is in a liquid state by naked eye observation, can be seen to be in a suspended state by electron microscope observation, and can be in a solid state after freeze drying.

In the present invention, the particle size of the tea residue protein-epsilon-polylysine-anthocyanin nanocomposite is preferably 100 to 200nm.

In the invention, the homogenizing mode preferably comprises ultrasonic homogenizing, and after the ultrasonic homogenizing, the tea residue protein-epsilon-polylysine-anthocyanin nano compound is obtained.

The time for ultrasonic homogenization is preferably 5-20 min, and more preferably 10min; the power of the ultrasonic homogenization is preferably 200-400W, more preferably 300W, and the frequency of the ultrasonic homogenization is preferably 25Hz. In the present invention, the ultrasonic homogenization is preferably intermittent ultrasonic homogenization, and more preferably ultrasonic 3s intermittent 3s. The ultrasonic homogenizing instrument is preferably an ultrasonic pulverizer. The homogenization can fully mix the protein, the nano particles can be uniformly dispersed, and the anthocyanin is promoted to enter the structure of the protein epsilon-polylysine.

After the tea-leaf residue protein-epsilon-polylysine-anthocyanin nano compound is obtained, 50mM KCl solution is preferably added into the tea-leaf residue protein-epsilon-polylysine-anthocyanin nano compound. The KCl solution can keep the stability of the tea residue protein-epsilon-polylysine-anthocyanin nano compound, so that the nano particles do not generate aggregation and precipitation. 50mM trace KCl as an additive does not harm human body, so that the KCl solution is not required to be specially removed when the tea residue protein-epsilon-polylysine-anthocyanin nano composite is applied.

The preparation method of the tea residue protein-epsilon-polylysine-anthocyanin nano compound provided by the invention has the advantages that the applied raw materials are safe, the tea residue protein is food-grade, the tea residue protein is safe and nontoxic, the polylysine has a good antibacterial effect and is nontoxic, the operation is convenient and fast, and the industrialization is easy to realize.

Compared with the prior art, the invention also has the following advantages:

firstly, the anthocyanin is embedded by utilizing the polyelectrolyte composite action of the tea residue protein and the epsilon-polylysine to improve the stability of the anthocyanin, and the whole preparation method is simple and quick in process. The tea residue raw material has wide sources, and along with the increase of the consumption of instant tea in China in recent years, a large amount of extracted tea residue waste is difficult to treat, and protein in the tea residue waste is extracted and product development is carried out, so that the residual value of the tea residue is greatly improved, and the comprehensive utilization and sustainable development of biological base materials are facilitated.

Secondly, the tea residue protein-epsilon-polylysine-anthocyanin nano compound embeds anthocyanin, improves the stability of anthocyanin to light and heat, effectively improves the processing stability of anthocyanin, enables the anthocyanin to have the characteristics of accurate and slow control in gastrointestinal tracts, solves the problems of poor stability and low utilization rate of anthocyanin, and greatly expands the application range of anthocyanin in the food processing process. In the embedding material in the prior art, the stability of embedded anthocyanin to heat at 90 ℃ for 2.5 hours is improved by about 10 percent, while the thermal stability of embedded anthocyanin is improved by 11 to 15 percent by the tea residue protein-epsilon-polylysine-anthocyanin nano compound.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1 preparation of tea leaf residue protein-epsilon-polylysine-anthocyanin nanocomposite

1. Tea residue protein extraction

(1) According to the mass volume ratio of the tea residues to boiling water of 1g to 50mL, the tea residues are leached by the boiling water for 15min, cotton cloth with the particle size of 550 mu m is used for filtering tea residue leaching liquor to obtain the tea residues, and the tea residues are leached by the boiling water and filtered to obtain the tea residues, wherein the tea residues are preferably repeated for 3 times. When the boiling water extraction is repeated, the volume of the boiling water added in each time is the same. The residual polyphenols can be removed by filtering tea residue leaching solution after repeating the extraction process for 3 times; drying the tea leaves at 65 ℃, and then crushing the tea leaves through a 180-micron mesh screen to obtain tea leaf powder;

(2) And (2) dispersing the tea residue powder obtained in the step (1) in a NaOH solution with the concentration of 0.1M for alkali extraction, wherein the solid-to-liquid ratio of the tea residue powder to the NaOH solution is 1 (w/v, g/mL), the alkali extraction is continuously heated for 90min at the temperature of 90 ℃, then the centrifugal separation is carried out for 15min at 8000rpm, and the supernatant of the tea residue powder is reserved.

(3) And (3) adjusting the pH value of the supernatant of the tea powder in the step (2) to 3.0 by using an HCl solution with the concentration of 0.1M, enabling a large amount of crude protein precipitates to appear in the supernatant of the tea powder, centrifuging at 4500r/min for 30min, collecting the crude protein precipitates in the supernatant of the tea powder, and then washing the crude protein precipitates of the tea powder by using pure water until the pH value of the crude protein of the tea powder is neutral. And finally, freeze-drying the crude tea residue protein powder at the temperature of-50 ℃ for 72 hours to obtain tea residue protein nanoparticles, and storing the tea residue protein nanoparticles in a refrigerator at the temperature of 4 ℃ for later use.

2. Preparation of tea residue protein (TP) nano solution

8mg of the prepared tea residue protein nanoparticles are dispersed in 100ml PBS buffer solution with the concentration of 0.01M and ultrasonically homogenized in an ultrasonic crusher. Continuously ultrasonically homogenizing for 10min at the power of 300W and the frequency of 25Hz for 3s and 3s to obtain tea leaf protein nano solution with the concentration of 0.08mg/mL for later use.

3. Preparation of epsilon-polylysine nano solution

An epsilon-polylysine powder (8 mg) was dispersed in 100mL of 0.01M PBS buffer to prepare a polylysine solution (0.08 mg/mL).

4. Preparation of anthocyanin (Anthocyanins, ACNs for short) nano solution

Anthocyanin 60mg was dispersed in 100mL of 0.01M PBS buffer to prepare a 0.6mg/mL anthocyanin nano solution.

5. Preparation of tea residue protein-epsilon-polylysine-anthocyanin nano composite (ACNs-TP-PLL for short) solution

And (3) placing 20mL of the tea dreg protein nano solution obtained in the step (2) into a beaker, slowly adding 10mL of the anthocyanin nano solution obtained in the step (4), uniformly stirring, then adding 10mL of the epsilon-polylysine nano solution obtained in the step (3), and carrying out ultrasonic homogenization under an ultrasonic pulverizer, wherein the ultrasonic homogenization power is 300w, the frequency is 25Hz, and the tea dreg protein-epsilon-polylysine-anthocyanin nano compound is obtained by carrying out continuous ultrasonic homogenization for 10min for 3s and 3s on and off. In order to improve the stability of the tea leaf residue protein-epsilon-polylysine-anthocyanin nano compound, the nano particles are kept not to generate aggregation precipitates, a 50mM KCl solution is added into the tea leaf residue protein-epsilon-polylysine-anthocyanin nano compound for ion balance, and the tea leaf residue protein-epsilon-polylysine-anthocyanin nano compound is obtained after freeze drying. The particle size of the tea residue protein-epsilon-polylysine-anthocyanin nano compound is 100-200 nm. The pH value of the mixed solution of the tea leaf protein nano solution and the anthocyanin nano solution is 5 after the mixed solution and the epsilon-polylysine nano solution are mixed.

Comparative example 1 preparation of tea residue protein-anthocyanin nanocomposite (ACNs-TP) solution

20mL of the tea residue protein nano solution prepared in example 1 was placed in a beaker, 10mL of the anthocyanin nano solution prepared in example 1 was slowly added thereto, and stirred, and 10mL of 0.01M PBS buffer was added thereto.

Comparative example 2 preparation of tea leaf protein-epsilon-polylysine nanocomposite (TP-PLL) solution

And (2) placing 20mL of tea leaf residue protein nano solution into a beaker, slowly adding 10mL of epsilon-polylysine solution, stirring, adding 10mL of 0.01M PBS buffer solution, and performing ultrasonic homogenization under an ultrasonic pulverizer, wherein the ultrasonic homogenization power is 300w, the frequency is 25Hz, and the tea leaf residue protein-epsilon-polylysine-anthocyanin nano compound is obtained by opening and closing for 3s and continuously performing ultrasonic homogenization for 10 min.

Comparative example 3 Anthocyanin (ACNs) Nanoalves preparation

Application example 1

The hydrolysis method of the proteolytic amino acid salt comprises the following steps: 100mg of a dry powder sample was weighed into a hydrolysis tube, and 10mL of 6mol/L HCl (analytical grade) was added, followed by N-charging ₂ The hydrolysis pipe cover is covered tightly; the hydrolysis tube is placed in an oven at 105 ℃ for hydrolysis for 22-24h; the volume of the hydrolyzed sample is fixed to 50mL by ultrapure water (distilled water of Dreches); 1mL of the hydrolysis sample with constant volume is taken to be put in a small beaker and is dried in a vacuum drying oven at 60 ℃ until the sample is dry; after 1mL of 0.02mol/L HCl (guaranteed reagent) was added to the beaker and redissolved, the mixture was passed through a 0.22 μm disposable water film and loaded into a sample bottle for amino acid analysis.

The amino acid content of the tea residue protein prepared in example 1 was measured by the above-described proteolytic amino acid hydrolysis method, and the results are shown in table 1 and fig. 1. As can be seen from FIG. 1, the amino acid types of the tea residue proteins are abundant, and the amino acid types and contents of the tea residue proteins are shown in Table 1.

TABLE 1 amino acid species and content of tea residue protein

Application example 2

The surface microstructures of the ACNs-TP-PLL solution prepared in example 1, the ACNs solution prepared in comparative example 3, the ACNs-TP solution prepared in comparative example 1, and the TP-PLL solution prepared in comparative example 2 were observed using a Scanning Electron Microscope (SEM). The concrete method is as follows:

the ACNs-TP-PLL prepared in example 1 was freeze-dried at-54 ℃ for 48 hours to obtain freeze-dried ACNs-TP-PLL nano solid particles, which were then attached to an SEM stub using a double-sided cellophane tape, then gold-palladium was applied to the ACNs-TP-PLL, and after photographing, the average ACNs-TP-PLL size was determined using digital image analysis, electron microscope operating parameter voltage was 3kv, and operating distance was 8.1mm.

The ACNs solution prepared in comparative example 3, ACNs-TP solution prepared in comparative example 1, and TP-PLL solution prepared in comparative example 2 were observed by using the same technical scheme as that of ACNs-TP-PLL described above, and the results are shown in fig. 2.

In FIG. 2-1, it can be seen from A1, A2 and A3 that TP-PLL is a dense network structure, and anthocyanin can be complexed in the network structure of TP-PLL, so as to achieve the purpose of stabilizing anthocyanin, and the size of TP-PLL particles reaches nanometer level as shown by a ruler in SEM picture of TP-PLL.

In fig. 2-2, B1, B2, and B3 show that the dense network structure of ACNs-TP-PLL compared to A1, A2, and A3 has a sheet structure of anthocyanin, and that anthocyanin is well wrapped in the network structure of the shell material.

In fig. 2-3, it can be known from C1, C2, and C3 that a single tea residue protein is in a relatively uniform granular structure, and in the ACNs-TP nano-composite, the tea residue protein interacts with each other to cover and wrap the flaky anthocyanin nanoparticles.

In fig. 2 to 4, it can be seen from D1, D2, and D3 that the anthocyanin nanoparticles have a sheet-like uneven structure.

FIGS. 2-5 show the microscopic nano-structure of TP-PLL, ACNs-TP and ACNs, and the addition of PLL (. Epsilon. -polylysine) causes a huge change in TP to become a complex structure with a network structure.

As can be seen from FIGS. 2-1 to 2-5, the anthocyanin and tea residue protein raw materials are nanoscale; although the tea residue protein-epsilon-polylysine forms a composite net structure in the preparation process, flocculation precipitation does not exist, and the nano-scale tea residue protein-epsilon-polylysine still can be seen by an electron microscope; finally, the prepared anthocyanin-tea residue protein-epsilon-polylysine nanoparticles are detected by a laser particle size analyzer to be nano-sized under different pH and ion conditions, and are presumed to belong to small molecules, enter a cavity structure of the tea residue protein-epsilon-polylysine to form a more compact composite network structure, but still keep the soluble characteristic under proper conditions and do not precipitate.

Application example 3ACNs-TP-PLL Fourier Infrared Spectroscopy

The freeze-dried solid of TP nano solution prepared in example 1, the freeze-dried solid of ACNs-TP-PLL solution prepared in example 1, the freeze-dried solid of ACNs nano solution prepared in comparative example 3, the freeze-dried solid of ACNs-TP solution prepared in comparative example 1 were subjected to Fourier transform infrared spectroscopySolid, 1mg of the solid after freeze-drying of the TP-PLL solution prepared in comparative example 2 and 100mg of KBr were mixed and pressed at 20MPa to prepare pellets, which were then subjected to 4000cm ^-1 ～500cm ^-1 Range scan with 4cm resolution ^-1 The number of scans was 64, see FIG. 3. The freeze-drying temperature is-54 deg.C, and the time is 48h.

As can be seen from FIG. 3, TP is 1637cm ^-1 And 1458cm ^-1 Amide I band and amide II band, respectively, and 2924cm ^-1 The characteristic C-H stretching vibration of protein, TP-PLL is 946cm ^-1 The peak of (A) is the out-of-plane bending vibration of C-H, and the wavelength of formed ACNs-TP-PLL is 1637cm ^-1 、2924cm ^-1 、1457cm ^-1 Both have absorption peaks and are similar to the characteristic peaks of TP-PLL and TP, which may be due to the attractive binding between TP and PLL through hydrogen bonds and opposite charges, but ACNs are 1636.90cm ^-1 The peak of C = O, C-H stretching vibration on benzene ring, the shift of characteristic peak after the complex embedding with TP-PLL, and some characteristic peaks of ACNs such as 1448cm ^-1 、1199cm ^-1 All disappeared, indicating that ACNs had been embedded.

Through 3419cm ^-1 ～3423cm ^-1 The change shows that anthocyanin can interact with tea residue protein firstly, and is connected with the tea residue protein through hydrogen bonds to adsorb anthocyanin on the tea residue protein, so that the tea residue protein and anthocyanin interact firstly, and polylysine is added for crosslinking, so that the stability of ACNs-TP-PLL can be enhanced. Application example 4ACNs-TP-PLL UV spectrogram

After the TP solution prepared in example 1, the ACNs-TP-PLL solution prepared in example 1, the ACNs nano-solution prepared in comparative example 3 and the ACNs-TP solution prepared in comparative example 1 were all protected from light for 0.5h at room temperature, full wavelength scanning was performed, and the results are shown in FIG. 4.

The ultraviolet-visible spectrophotometer is a method for relatively intuitively researching the coating relation between a subject and an object, and the method can say that the inclusion condition of the subject and the object comprises the following steps: (1) Differences before and after the formation of the inclusion compound are illustrated by differences of heights, shapes and positions of absorption peaks; (2) The analysis is performed from the absorption position and intensity of the maximum absorption wavelength. As can be seen from FIG. 4, ACNs have absorption peaks near 280nm and 550nm, and the intensities of the absorption peaks of ACNs coated by TP-PLL are greatly reduced at 280nm and 550nm, which is probably because anthocyanin enters the complex network structure of tea residue protein-epsilon-polylysine, so that anthocyanin nanoparticles are coated, and the intensity of characteristic peaks is reduced.

Example 2

20mL of the ACNs-TP-PLL solution prepared in example 1 was added to a conical flask, the pH of the solution was adjusted to 1.5 with 2M HCl, preheated for 10min in a shaker (37 ℃, 95 rpm/min), and 4mg of pepsin was added to start simulated gastric fluid digestion; after 120min, the pH of the digest was adjusted to 7.2 with 4.0M NaOH, 100mg of porcine bile salt extract was added, mixed well in a shaker for 10min, 8mg of pancreatin was added to begin simulating digestion in the small intestine for 150min. Sampling 2mL every 30min in the process of stomach digestion, sampling 2mL every 30min in the process of intestinal digestion, detecting the content of anthocyanin in digestive juice by adopting a high performance liquid chromatography, wherein the percentage of the content of anthocyanin measured in the digestive juice and the content of total anthocyanin added into the digestive juice initially in different time periods is the retention rate of anthocyanin, and the stability of anthocyanin in the digestive juice is represented.

Comparative example 4

20mL of the nano-sized solution of ACNs prepared in comparative example 3 was added to the Erlenmeyer flask, and the rest of the simulated gastric digestion and simulated intestinal digestion conditions were the same as in example 2.

The anthocyanin retention in the digestive juices of the ACNs-TP-PLL of example 2 and the ACNs of comparative example 4 was calculated over different time periods as follows:

anthocyanin retention (%) in digestive juice = WT/W0X 100%

WT: content of ACNs (mu g/mL) in simulated gastric fluid or simulated intestinal fluid at time T

W0: content of ACNs in digestive juice at the beginning of sample (μ g/mL)

The results are shown in Table 2, table 3 and FIG. 5.

TABLE 2 Retention of anthocyanin in simulated gastric fluid by different subjects

TABLE 3 Retention of anthocyanins in simulated intestinal fluid by different subjects

As can be seen from a and B in fig. 5, the tea residue protein-epsilon-polylysine-anthocyanin nanocomposite ACNs-TP-PLL coated with tea residue protein and epsilon-polylysine shows a trend of first rising and then falling in the anthocyanin concentration of the stomach, and the anthocyanin concentration of the ACNs-TP-PLL in the stomach is higher than that of anthocyanin ACNs because the anthocyanin in the stomach is well embedded in the tea residue protein-epsilon-polylysine composite wall material, so that the anthocyanin shows a partial release trend in the peristalsis process of the stomach, and the released anthocyanin is digested and decomposed under the action of gastric juice, so that the trend of first rising and then falling is shown. In the intestinal tract, tea residue protein-epsilon-polylysine is decomposed into small molecular substances under the action of intestinal pancreatin, so that the polarity of the microenvironment where the anthocyanin is located is enhanced under the interaction of the small molecular substances and protease, and the stability of the anthocyanin in intestinal juice is improved.

Example 3-1 stability test of ACNs-TP-PLL

A. Effect of continuous heating on the stability of ACNs-TP-PLL solutions

The ACNs-TP-PLL solution with pH =5 prepared in example 1 was heated continuously at 90 ℃ for 2.5h, sampled every 0.5h, and the content of anthocyanin in the solution was measured by the pH differential method. The pH differential detection procedure is described in detail below.

B. Effect of illumination on the stability of ACNs-TP-PLL solutions

The ACNs-TP-PLL with pH =5 prepared in example 1 was placed at 25 ℃ under white light irradiation for 10d, and the ACNs content in the sample was measured by pH differential method. The procedure for pH differential detection is as follows.

C. Effect of pH on the thermal stability of ACNs-TP-PLL solutions

The ACNs-TP-PLL solution prepared in example 1 was sampled into 6 samples of 80mL each, the pH of the 6 sample solutions was adjusted using 0.1M hydrochloric acid or 0.1M sodium hydroxide to 2, 3, 4, 5, and 6 each, and the 6 sample solutions were continuously heated at 90 ℃ for 2.5 hours, and the content of ACNs in the samples was measured by the pH differential method. The procedure for pH differential detection is as follows.

D、K ⁺ Influence of the Ionic Strength on the thermal stability of ACNs-TP-PLL solutions

6 samples of pH =5 ACNs-TP-PLL solution prepared in example 1 were sampled at 80mL each, and Kcl solution was added to each of the 6 samples to make K in the 6 samples ⁺ The concentrations of ionic strength were 0mM, 50mM, 100mM, 150mM, and 200mM, respectively, and then 6 samples were heat-treated at 90 ℃ for 1 hour to determine the ACNs content in the samples by the pH differential method. ACNs-TP-PLL pH =5. The procedure for pH differential detection is as follows.

Comparative example 5 stability test of ACNs

A. Stability Effect of continuous heating on ACNs

The ACNs nano solution prepared in comparative example 3 was sampled every 0.5h under continuous heating at 90 ℃ for 2.5h, and the content of anthocyanin in the solution was measured by pH differential method. The procedure for pH differential detection is as follows.

B. Stability impact of illumination on ACNs

The ACNs nano solution prepared in comparative example 3, pH =5, was placed at 25 ℃ for 10d of white light irradiation, and the ACNs content was measured by pH differential method. The procedure for pH differential detection is as follows.

C. Thermal stability Effect of pH on ACNs

Continuously heating the ACNs nano solution prepared in the comparative example 3 when the pH values are respectively 2-6, wherein the heating temperature is 90 ℃, the heating time is 2.5h, and measuring the content of the ACNs in the sample by using a pH differential method. The procedure for pH differential detection is as follows.

D、K ⁺ Effect of Ionic Strength on the thermal stability of ACNs

The nano solution of ACNs prepared in comparative example 3 was heated at 90 ℃ for 1 hour at pH =5 under different ionic strengths of 0mM, 50mM, 100mM, 150mM, and 200mM, and the content of ACNs in the sample was measured by pH differential method.

The method for detecting the content of anthocyanin in the solution of the example 3 and the solution of the comparative example 5 by a pH differential method comprises the following steps:

2mL of 8 samples to be tested of A treatment, B treatment, C treatment and D treatment in example 3 and A treatment, B treatment, C treatment and D treatment in comparative example 3 were diluted 5-fold with a buffer solution of pH1.0 and a buffer solution of pH4.5, respectively, mixed well and then allowed to equilibrate at room temperature for 10min, and absorbance values at 520nm and 700nm of the samples were measured using pure water as blank groups for the measurement of A, B, C and D samples in example 3 and A, B, C and D samples in comparative example 3.

pH1.0 buffer preparation: 1.86g of KCl was weighed accurately, mixed with 980mL of distilled water, adjusted to pH1.0 with concentrated HCI, transferred to a 1L volumetric flask and made to volume with distilled water.

pH4.5 buffer solution configuration: 32.82g of sodium acetate are weighed out accurately, mixed with 960mL of distilled water, the pH is adjusted to 4.5 with concentrated HCI, transferred to a 1L volumetric flask and brought to volume with distilled water.

The calculation formula of the total content (C) of the ACNs (anthocyanins) in the sample to be detected is shown as follows:

in the formula, apH1.0 is the difference of absorbance values of a sample at the wavelengths of 520nm and 700nm respectively after the sample is diluted by a buffer solution with the pH value of 1.0; apH4.5 is the difference of absorbance values of a sample at the wavelengths of 520nm and 700nm after being diluted by a buffer solution with the pH value of 4.5; MW is the relative molecular mass of cyanidin-3-O-glucoside (449.2 g/mol); DF is the dilution factor of the sample; epsilon is the molar extinction coefficient (26900L/mol cm) of cyanidin-3-O-glucoside ^-1 ) (ii) a l is the optical path length (1 cm).

Stable anthocyanin retention (%) = RT/R0 x 100%; RT in the formula is the total content (mg/mL) of ACNs in the sample after heating or light irradiation treatment; r0 is the total content (mg/mL) of ACNs in the sample under the initial condition;

stability improvement (%) = difference between ACNs-TP-PLL retention average and ACNs retention average;

the results are shown in tables 4-1 to 4-4 and FIG. 6-1.

TABLE 4-1 stability ACNs Retention and stability enhancement data for the ACNs-TP-PLL of example 3 and comparative example 5 at different duration heating times

TABLE 4-2 Retention and stability enhancement data for ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at different illumination times

Tables 4-3 retention and stability enhancement data for ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at different pH' s

Tables 4-4 different K ⁺ Data on retention and stability improvement of ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at Ionic Strength

As can be seen from Table 4-1 and A in FIG. 6-1, the ACNs-TP-PLL has greatly enhanced thermal stability, and anthocyanin in the ACNs-TP-PLL shows relatively high retention rate even under continuous heating for 2.5h, which also provides a technical reference for reducing anthocyanin loss in food processing of anthocyanin foods.

As can be seen from Table 4-2 and B in FIG. 6-1, the stability of the ACNs of comparative example 5 is continuously decreased after continuous illumination of light while the stability of the ACNs-TP-PLL of example 3 is greatly enhanced, indicating that the tea residue protein-epsilon-polylysine can provide protection for the photostability of anthocyanin.

As can be seen from tables 4-3 and C in FIG. 6-1, the lower the pH of ACNs of comparative example 5, the better the thermal stability is, because ACNs exist mainly in the form of red molten salt ions at a pH of less than 2, and when the pH value is increased, the hydration of the molten salt at C-2 and the proton transfer of the acidic hydroxyl group on anthocyanin by the nucleophilic attack of water molecules occur, and there are kinetic and thermodynamic competition between the two reactions, so that the stability of ACNs is reduced due to the increase of pH, but the ACNs-TP-PLL of example 3 shows excellent thermal stability at a pH of 5, because the shell material of the nanoparticles can well encapsulate anthocyanin in the network through polyelectrolyte complexation at a pH of 5, and the low or high pH can cause the anthocyanin not to be well encapsulated in the network, the low tea residue protein to flocculate, and the high pH can lead to the failure to form better formulations.

From Table 4-4 and D in FIG. 6-1, the excess K can be found ⁺ The presence of ions would destroy the thermal stability of the ACNs-TP-PLL nano-ions of example 3 due to excessive K ⁺ The existence of ions can cause the system to generate electrostatic shielding effect, and the tea residue protein carries negative charge and can react with excessive K when the pH is =5 ⁺ Interact with the protein to ensure that the tea residue protein generates electrostatic shielding and cannot form a stable complex network structure with epsilon-polylysine, so that the heat stability of anthocyanin is reduced, but K ⁺ An ionic concentration of 50mM makes the system more stable, since K ⁺ When the concentration is 50mM, the system is just in the process of stabilizing the structure, at the moment, anthocyanin is well embedded in the complex network structure of tea residue protein-epsilon-polylysine, and excessive tea residue protein is mixed with a small amount of K ⁺ The electrostatic shielding effect is generated, so that the phenomenon of excessive complex precipitation is avoided, and the system is more stable.

Examples 3 to 2

Particle size of ACNs-TP-PLL nanocomposites in solution at different pH conditions of pH =2, pH =3, pH =4, pH =5, pH =6 in the C treatment of example 3-1 and K in the D treatment of example 3-1 were measured using dynamic light scattering using a particle size analyzer (Delsa Max Pro; beckman Coulter, UK) ⁺ The ionic strength was 0mM, 50mM, 100mM, 150mM, 200mM different K ⁺ The specific determination method of the particle size of the ACNs-TP-PLL compound in the ionic strength solution is as follows: the ACNs-TP-PLL solution at pH 5 in treatment C of example 3-1 was diluted 10 times, loaded 1mL, measured 10 times per sample, the apparatus was programmed to count and calculate the size of particles having a diameter of 0.02 to 2000 μm, and the same assay method was processed for the rest.

The results are shown in tables 4-5, tables 4-6 and FIG. 6-2.

TABLE 4-5 particle size of ACNs-TP-PLL in solution at different pH

pH value	2	3	4	5	6
						ACNs-TP-PLL particle size (nm)	790±63	418±18	147±4	138±7	232±17

Tables 4-6 different K ⁺ Particle size of ACNs-TP-PLL in ionic strength solution

From a in fig. 6-2 and table 4-5, it can be seen that ACNs-TP-PLL had a smaller particle size (p < 0.05) at pH 5-6 because TP was able to crosslink with PLL at pH 5-6 more densely, allowing the hydrophobic groups in the tea residue protein to drain more water to form a larger and dense cavity structure to carry ACNs and thus reduce the particle size, while the particles of nanoparticles were larger at pH 2-4 because TP was free from PLL and ACNs, the tea residue protein had already started to flocculate at pH =2, the tea residue protein was close to zero in the vicinity of the isoelectric point of the tea residue protein (PI = 3.0), and the electrostatic interaction between the particles was reduced, promoting aggregation of the nanoparticles and thus increasing the particle size. The alkaline condition is not favorable for the adsorption of anthocyanin by tea residue protein.

As can be seen from B in FIG. 6-2 and tables 4-6, K ⁺ When the ionic strength is 0, the particle size is relatively large (p is relatively large) because polylysine with strong positive charges and tea residue protein with negative charges in the system can continuously form a net structure through electrostatic interaction to load more ACNs<0.05 Average particle size 159. + -.9 nm, but with addition of a small amount of K ⁺ The particle size of the ACNs-TP-PLL decreases with the ions (p)<0.05 Because of a small amount of K) ⁺ The ion addition can prevent the mutual electrostatic interaction between the tea-residue protein and the polylysine, so that the tea-residue protein and the polylysine gradually reach the system balance, which can be more stable to the system, and therefore, a small amount of K can be used ⁺ Ions to effect controlled loading of nanoparticles.

Example 4

The ACNs-TP-PLL prepared in example 1, the ACNs prepared in comparative example 3, the ACNs-TP, ACNs-PLL and ACNs-SPi prepared in comparative example 1 were heated at 90 ℃ for 1 hour with a pH of 5.0, and anthocyanin retention was calculated using the above formula, and the results are shown in Table 5 and FIG. 7. As can be seen from Table 5 and FIG. 7, the highest retention rate of ACNs-TP-PLL prepared in example 1 can be obtained, which indicates that the ACNs and TP nano-materials have better thermal stability effect as wall materials for embedding anthocyanin than the soy protein as embedding material in the prior art.

The preparation method of the ACNs-PLL comprises the following steps: epsilon-polylysine powder (8 mg) was dispersed in 100mL of 0.01M PBS buffer to prepare an Epsilon-polylysine solution (0.08 mg/mL) for use. 20mL of the epsilon-polylysine nano solution was placed in a beaker, 10mL of the anthocyanin nano solution prepared in example 1 was slowly added thereto and stirred, and 10mL of 0.01M PBS buffer was added thereto to obtain ACNs-PLL.

The preparation method of the ACNs-SPi comprises the following steps: 8mg of soy protein nanoparticles were dispersed in 100ml PBS buffer at a concentration of 0.01M and homogenized ultrasonically in an ultrasonicator. Continuously ultrasonically homogenizing for 10min at the power of 300W and the frequency of 25Hz for 3s and 3s to obtain the soybean protein nano solution with the concentration of the soybean protein of 0.08mg/mL for later use.

20mL of the soy protein nano solution was placed in a beaker, 10mL of the anthocyanin nano solution prepared in example 1 was slowly added thereto and stirred, and 10mL of 0.01M PBS buffer was added thereto to obtain ACNs-SPi.

TABLE 5 anthocyanin Retention under heating conditions for different samples

ACNs-TP	ACNs-PLL	ACNs-TP-PLL	ACNs	ACNs-SPi
					60±0.63	65±0.92	72±0.58	57±0.41	57±2.4

As can be seen from Table 5, the highest anthocyanin retention rate of ACNs-TP-PLL under different heating conditions indicates that the thermal stability of anthocyanin is improved after anthocyanin is embedded in the tea residue protein-epsilon-polylysine nano material prepared by the invention.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A tea-residue protein-epsilon-polylysine nano material is characterized by comprising tea-residue protein nano particles and epsilon-polylysine; the tea residue protein nanoparticles and epsilon-polylysine form a network complex.

2. The tea residue protein-epsilon-polylysine-anthocyanin nano compound is characterized in that tea residue protein nano particles and epsilon-polylysine in claim 1 are used as wall materials, and anthocyanin is used as a core material.

3. The method for preparing the tea residue protein-epsilon-polylysine-anthocyanin nanocomposite as claimed in claim 2, which comprises: mixing the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution with the epsilon-polylysine nano solution, and homogenizing to form a tea residue protein-epsilon-polylysine-anthocyanin nano compound;

the mass concentration of the tea-leaf protein in the tea-leaf protein nano solution is 0.02-0.125 mg/mL; the mass concentration of anthocyanin in the anthocyanin nano solution is 0.2-1.25 mg/mL; the mass concentration of the epsilon-polylysine in the epsilon-polylysine nano solution is 0.02-0.125 mg/mL.

4. The preparation method according to claim 3, wherein the mixed solution of the tea residue protein nano solution and the anthocyanin nano solution is obtained by adding the anthocyanin nano solution to the tea residue protein nano solution and mixing;

5. The preparation method according to claim 4, wherein the tea residue protein nano solution comprises tea residue protein nano particles and a buffer solution; the mass volume ratio of the tea-leaf protein nanoparticles to the buffer solution is (2-10) mg: (80-100) mL; the buffer solution comprises PBS buffer solution, and the concentration of the PBS buffer solution is 0.01-0.06M.

6. The preparation method according to claim 4 or 5, wherein the preparation method of the tea residue protein nano solution comprises the following steps: and mixing and homogenizing the tea leaf protein nanoparticles and the buffer solution to obtain the tea leaf protein nano solution.

7. The method for preparing according to claim 4, wherein the epsilon-polylysine nano-solution comprises epsilon-polylysine nano-particles and a buffer;

8. The preparation method according to claim 4, wherein the solvent of the anthocyanin nano solution is 0.01M PBS buffer, and the mass-to-volume ratio of the anthocyanin to the solvent (20-100) mg: (80-100) mL.

9. The preparation method according to claim 4, wherein the particle size of the tea residue protein-epsilon-polylysine-anthocyanin nano-composite is 100 to 200nm.

10. The method according to claim 3, wherein the homogenizing comprises ultrasonic homogenizing, wherein the ultrasonic homogenizing is performed for 5-20 min at a power of 200-400W and at a frequency of 25Hz.