CN115501346B

CN115501346B - Tea dreg protein-epsilon-polylysine nano material and anthocyanin nano compound and preparation method thereof

Info

Publication number: CN115501346B
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: 2024-04-23
Anticipated expiration: 2042-09-28
Also published as: CN115501346A

Abstract

The invention belongs to the technical field of nanoparticle preparation, and particularly relates to a tea dreg protein-epsilon-polylysine nanomaterial and anthocyanin nanocomposite and a preparation method thereof. The invention provides a tea dreg protein-epsilon-polylysine nano material, which comprises tea dreg protein nano particles and epsilon-polylysine; the tea dreg protein nano-particles and epsilon-polylysine form a net-shaped compound by utilizing a polyelectrolyte compounding method. The example results show that after the anthocyanin is embedded in the tea dreg protein-epsilon-polylysine nano material, the anthocyanin thermal stability is improved by 15%, the light stability is improved by 14%, and the accurate slow control of the anthocyanin gastrointestinal tract is realized.

Description

Tea dreg 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 dreg protein-epsilon-polylysine nanomaterial and anthocyanin nanocomposite and a preparation method thereof.

Background

The tea residue contains 20% -30% of tea protein, wherein the tea protein mainly comprises gluten and prolamine. The tea dreg protein belongs to vegetable protein, has rich amino acid composition, does not contain cholesterol, and is very suitable for eating. Researches show that the tea protein has better functions such as blood pressure reduction, blood sugar reduction, radiation protection and the like, so that the tea protein has higher development and utilization values. At present, the tea leaf protein is mainly applied to the aspects of preparing health-care foods, feeds, antioxidants and microcapsules, and just like 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 three types of tea residues.Lwt-Food Science and Technology,110,324-331), the tea leaf protein nano-particles are used as the stabilizer of Pickering emulsion to maintain the oxidation stability of the grease, so that the application effect is good. At present, tea dreg proteins are not reported as nano-delivery carriers of anthocyanin. Anthocyanin is an active substance which is extremely unstable and has low bioavailability and is particularly sensitive to the environment. Anthocyanin is easily influenced by environmental factors such as temperature, oxygen, enzyme, light and ascorbic acid, so that the structure of the anthocyanin is damaged, and the absorptivity of the anthocyanin in the gastrointestinal tract is very low due to the easily damaged structure, so that how to solve the stability of the anthocyanin and improve the long-acting absorption of the anthocyanin in the gastrointestinal tract is a problem to be solved at present.

Nano-delivery vehicles (nano-delivery) refer to drug (bioactive substances) delivery systems with average particle diameters of 10-1000 nm prepared by mechanical or chemical methods, 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, and the delivery of the nano-carrier is the mode with the widest application range for the delivery mode of anthocyanin.

The nano-delivery vehicles of anthocyanin in the prior art mainly comprise soybean protein, beta-lactoglobulin, chitosan, sodium alginate and the like, and just like the prior art (Chen,Z.,Wang,C.,Gao,X.,Chen,Y.,Santhanam,R.K.(2019).Interaction characterization of preheated soy protein isolate with cyanidin-3-O-glucoside and their effects on the stability of black soybean seed coat anthocyanins extracts.Food Chemistry,271,266-273.) discloses that the anthocyanin shows good heat and oxidation stability by adopting the soybean protein modified by heating as a wall material and simultaneously adopting the anthocyanin as a core material. Meanwhile, the nano materials such as soybean protein, beta-lactoglobulin, chitosan and sodium alginate improve the stability of anthocyanin to a certain extent, but after the anthocyanin is embedded by the soybean protein, the beta-lactoglobulin, the chitosan and the sodium alginate, the improvement range of the thermal stability of anthocyanin is limited, and the materials such as the soybean protein, the beta-lactoglobulin, the chitosan and the sodium alginate are difficult to reduce the cost through waste utilization.

Therefore, the research needs to be enhanced to solve the technical problem of low improvement range of the thermal stability after the anthocyanin is embedded.

Disclosure of Invention

The invention aims to provide a tea dreg protein-epsilon-polylysine nano material, which is used as a carrier to embed anthocyanin, so that the thermal stability and the light stability of anthocyanin are improved.

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

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

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

The pH value of the mixed solution of the tea dreg protein nano solution and the anthocyanin nano solution and the epsilon-polylysine nano solution is 2-6;

The mass concentration of 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 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 dreg protein nanometer solution, the epsilon-polylysine nanometer solution and the anthocyanin nanometer solution is (1-5) 1:1.

Preferably, the tea leaf protein nano-solution comprises tea leaf protein nano-particles and a buffer solution; the mass volume ratio of the tea dreg protein 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 preparation method of the tea leaf protein nano solution comprises the following steps: mixing and homogenizing the tea leaf protein nano-particles with a buffer solution to obtain a tea leaf protein nano-solution.

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

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 grain size of the tea dreg protein-epsilon-polylysine-anthocyanin nano-composite is 100-200 nm.

Preferably, the homogenization comprises ultrasonic homogenization, wherein the ultrasonic homogenization is carried out for 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 dreg protein-epsilon-polylysine nano material, which comprises tea dreg protein nano particles and epsilon-polylysine; the tea dreg protein nano-particles and epsilon-polylysine form a net-shaped compound by utilizing a polyelectrolyte compounding method. According to the invention, tea-leaf proteins can carry negative charges at an isoelectric point pH=3, and epsilon-polylysine can carry positive charges at an isoelectric point pH=9.74; the tea dreg protein with negative charges and the epsilon-polylysine with positive charges are subjected to polyelectrolyte complex reaction to realize the complexing and crosslinking of the epsilon-polylysine and the tea dreg protein to generate nano particles, and the nano particles can realize the effective embedding of anthocyanin under the conditions of a certain concentration and pH value. And the tea dreg raw materials are wide in source, and the invention extracts tea dreg proteins in tea dreg waste materials and develops products, so that the residual value of tea dreg is greatly improved, and the comprehensive utilization and sustainable development of biological base materials are facilitated. The example results show that after the anthocyanin is embedded in the tea dreg protein-epsilon-polylysine nano material, the anthocyanin thermal stability is improved by 15%, the light stability is improved by 14%, and the accurate slow control of the anthocyanin gastrointestinal tract 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 that are required to be used in the embodiments will be briefly described below.

FIG. 1 is an amino acid composition of tea leaf protein prepared in example 1;

FIG. 2-1 is a scanning electron microscope image of the TP-PLL prepared in comparative example 2, wherein A1, A2, A3 are respectively the magnified electron microscope images of the TP-PLL nanocomposite at 8000 times, 15000 times, 40000 times;

FIG. 2-2 is a scanning electron microscope image of ACNs-TP-PLL prepared in example 1, wherein B1, B2, B3 are respectively the electron microscope images of ACNs-TP-PLL nanocomposite at 8000 times, 15000 times, 40000 times;

FIGS. 2-3 are scanning electron microscope images of ACNs-TP (tea leaf protein anthocyanin nanocomposite) prepared in comparative example 1, wherein C1, C2, and C3 are magnified electron microscope images of ACNs-TP nanocomposite at 8000 times, 15000 times, and 40000 times, respectively;

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

FIGS. 2-5 are electron microscope images at 15000 magnification of the TP-PLL prepared in comparative example 2, ACNs-TP-PLL prepared in example 1, ACNs-TP prepared in comparative example 1, ACNs prepared in comparative example 3;

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

FIG. 4 is an ultraviolet 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 shows ACNs retention of ACNs-TP-PLL prepared in example 1 and ACNs prepared in comparative example 3 in simulated gastric fluid and simulated intestinal fluid environments, 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, D is different K ⁺ ion concentration intensity;

FIG. 6-2 is a graph of ACNs-TP-PLL particle size at different pH values and at different K ⁺ ionic strengths, where A is ACNs-TP-PLL particle size at different pH values and B is ACNs-TP-PLL particle size at different K ⁺ ionic strengths;

FIG. 7 shows anthocyanin retention rates for ACNs-TP-PLL prepared in example 1, ACNs prepared in comparative example 3, ACNs-TP, ACNs-PLL and ACNs-SPI prepared in comparative example 1.

Detailed Description

The source of the tea leaf protein nano-particles is not particularly limited, and the tea leaf protein nano-particles with any source can be used. The grain size of the tea leaf protein is preferably nano-scale.

In the invention, the preparation method of the tea leaf protein nano-particles preferably comprises the following steps: performing alkali extraction on tea residue powder to obtain tea residue powder supernatant, and performing acid precipitation on the tea residue powder supernatant to obtain crude tea residue powder protein; and drying the coarse tea dreg powder protein to obtain tea dreg protein nano-particles.

The tea dreg powder is preferably prepared by the following steps: extracting tea leaves with boiling water, filtering to obtain tea leaves, and drying and pulverizing the tea leaves to obtain tea leaves powder. In the invention, the mass ratio of tea leaves to boiling water is preferably (1-5) g: (30-60) mL, more preferably (1-5) g: (40-55) mL; more preferably 1g:50mL. The boiling water extraction time of the present invention is preferably 5 to 20 minutes, more preferably 10 to 18 minutes, and still more preferably 15 minutes. The boiling water extraction and filtration are preferably repeated, the tea residue is obtained by the specific boiling water extraction and filtration, and the tea residue is obtained by the boiling water extraction and filtration, and the method is preferably repeated for 2-4 times, more preferably 3 times, and the method is preferably extracted for 3 times, so that the extraction rate of the tea residue protein can be improved, and the loss in the extraction process of the tea residue protein is reduced. When the boiling water leaching is repeated, the volume of the boiling water added each time is the same.

After the tea residue is boiled and leached, the invention preferably filters the leached feed liquid to obtain the tea residue. The invention is not particularly limited to the manner of filtration described, as is well known to those skilled in the art. In the present invention, the filtering means is preferably a cotton cloth filtering the leaching solution with 550 μm, and the filtering of the present invention can remove the residual polyphenol substances in the tea residue.

After the tea residue is obtained, the tea residue is preferably dried and crushed to obtain tea residue powder. In the present invention, the temperature of the drying is preferably 60 to 80 ℃, more preferably 62 to 70 ℃, and even more preferably 65 ℃. The invention preferably dries until the tea residue can be broken into powder; in the embodiment of the invention, the drying time is preferably 72 hours. The pulverizing method is not particularly limited, and conventional method can be adopted. After the tea dreg powder is obtained, the crushed tea dreg powder is preferably screened by a 180 mu m screen.

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

After the alkali extraction, the invention preferably carries out centrifugation on the alkali extraction liquid to obtain tea residue powder supernatant. The rotational speed of the centrifugation according to the present invention is preferably 4500 to 8500rpm, more preferably 6000 to 8300rpm, and still more preferably 8000rpm; the time for the centrifugation is preferably 10 to 30 minutes, more preferably 13 to 20 minutes, and still more preferably 15 minutes.

After the tea residue powder supernatant is obtained, the invention prefers acid precipitation of the tea residue powder supernatant to obtain crude tea residue powder protein. The acid precipitation method preferably uses HCl solution to adjust the pH value of the supernatant of the tea dreg powder until a large amount of crude protein precipitation of the tea dreg powder is obtained. The concentration of the HCl solution according to 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 supernatant of the tea dreg powder regulated by the method is preferably 2.0-3.4, more preferably 2.5-3.2, and even more preferably 3.0.

After acid precipitation, the invention preferably carries out centrifugation on the supernatant liquid of the tea slag powder obtained by precipitation to obtain crude protein of the tea slag powder. The rotational speed of the centrifugation according to the present invention is preferably 3000 to 5000rpm, more preferably 4000 to 4800rpm, and still more preferably 4500rpm; the time for the centrifugation is preferably 10 to 30 minutes, more preferably 20 to 30 minutes, and still more preferably 30 minutes. The invention preferably utilizes pure water to wash the tea dreg powder crude protein until the pH value of the crude protein is neutral.

After washing by pure water, the invention preferably dries coarse protein of tea dreg powder to obtain tea dreg protein nano-particles. 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 72 hours.

The instant tea and the milk tea are very popular at the present stage, and a large amount of tea residue waste is generated, and the tea residue protein nano particles are extracted by deep processing of the tea residue by-product to serve as wall materials of anthocyanin embedding materials, so that the added value of the tea can be effectively improved, the amino acid content of the tea residue protein is also richer compared with that of other proteins, various proteins required by human bodies are contained, and the wall materials serving as the anthocyanin embedding materials are safer and more reliable compared with polysaccharide. The invention is the first to extract tea dreg protein and epsilon-polylysine to prepare the nano material.

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

The source of the anthocyanin is not particularly limited, and the anthocyanin can be obtained by adopting a conventional commercial product. The particle size of anthocyanin is preferably nano-scale.

the mass concentration of tea leaf protein in the tea leaf protein nano solution is 0.02-0.125 mg/mL, 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 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 the concentration range, the tea dreg protein nano-particles and epsilon-polylysine can be crosslinked to form a net structure, and flocculation and precipitation can be generated when the pH value and the concentration range are exceeded.

In the invention, the mixed solution of the tea leaf protein nano solution and the anthocyanin nano solution is preferably obtained by adding the anthocyanin nano solution into the tea leaf protein nano solution and mixing.

In the present invention, the tea leaf protein nano-solution preferably comprises tea leaf protein nano-particles and a buffer solution; the mass volume ratio of the tea dreg protein nano-particles to the buffer solution is preferably (2-10) mg: (80-100) mL, more preferably (5-9) mg: (90-100) mL, more preferably 8mg:100mL. The buffer of the present invention preferably includes a phosphate buffer, and the concentration of the phosphate buffer 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, so that the PBS buffer solution can keep the pH of a system relatively stable, meanwhile, the PBS buffer solution can better dissolve and protect solutes relative to distilled water, has a better salt balance effect, and has more stable and reliable pH adjusting result relative to distilled water.

In the invention, the preparation method of the tea leaf protein nano-solution preferably comprises the following steps: mixing and homogenizing the tea leaf protein nano-particles with a buffer solution to obtain a tea leaf protein nano-solution.

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

In the present invention, the means of homogenization preferably includes ultrasonic homogenization. After ultrasonic homogenization, the tea dreg protein nano-solution is obtained.

The ultrasonic homogenization time 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 intermittent ultrasonic homogenization, and further preferably ultrasonic 3s intermittent 3s. According to the invention, tea dreg protein nano particles are completely dissolved and uniformly dispersed in PBS solution by ultrasonic homogenization, so that cross-linking 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 invention, the mass volume ratio of the anthocyanin nano solution to the solvent is (20-100) mg (80-100) mL, more preferably (50-80) mg (85-100) mL, and 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 are not described in detail herein.

In the present 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 epsilon-polylysine nano solution. The mass volume ratio of the epsilon-polylysine nano-particles to the buffer solution is preferably (2-10) mg: (80-100) mL, more preferably (6-9) mg: (92-100) mL, more preferably 8mg:100mL.

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

After obtaining tea leaf protein nano solution, epsilon-polylysine nano solution and anthocyanin nano solution, mixing the mixed solution of tea leaf protein nano particles and anthocyanin with epsilon-polylysine nano solution, and homogenizing to form tea leaf protein-epsilon-polylysine-anthocyanin nano composite. The invention is preferably that the tea dreg protein nano-solution is added with anthocyanin nano-solution for mixing, and then epsilon-polylysine nano-solution is added for homogenizing to form tea dreg protein-epsilon-polylysine-anthocyanin nano-composite; more preferably, the anthocyanin nano solution is slowly added into the tea dreg protein nano solution, and then the epsilon-polylysine nano solution is added after the anthocyanin nano solution is uniformly stirred. The slow addition of the catalyst is preferably 1mL/min to 5mL/min, more preferably 2mL/min to 4mL/min, and even more preferably 3mL/min. While the tea leaf protein nanosolution, epsilon-polylysine nanosolution, and anthocyanin nanosolution are all on the order of nanometers, nanoparticles must be formed under appropriate conditions when they polymerize, otherwise the tea leaf protein and polylysine may also form larger particle size particles to flocculate and precipitate.

The stirring method of the present invention is not particularly limited, and a conventional method may be adopted. According to the invention, the anthocyanin nano solution is added into the tea dreg protein nano solution to enable the anthocyanin nano solution to attach to the macromolecular tea dreg protein, and then the epsilon-polylysine nano solution is added to be in complexation and cross-linking with the tea dreg protein, so that anthocyanin exists in a reticular structure formed by the epsilon-polylysine and the tea dreg protein in a complexation and cross-linking way, and a tea dreg protein-epsilon-polylysine-anthocyanin nano compound is formed, so that the effect of protecting anthocyanin is achieved. If the network structure of the complex of epsilon-polylysine and tea dreg protein is prepared first and then anthocyanin is added, the anthocyanin can not be well embedded, so that the effect of protecting the anthocyanin can not be achieved. According to the invention, anthocyanin is embedded by utilizing the polyelectrolyte composite effect of tea dreg protein and epsilon-polylysine, so that the stability of anthocyanin is improved. The polyelectrolyte compounding method belongs to an in-situ polymerization method and is an in-situ synthesis method.

The invention adds epsilon-polylysine nano-solution into the mixed solution of tea dreg protein nano-particles and anthocyanin, and homogenizes to form tea dreg protein-epsilon-polylysine-anthocyanin nano-composite. In the present invention, the volume ratio of the tea leaf protein nanosolution, the epsilon-polylysine nanosolution and the anthocyanin nanosolution is preferably (1-5): 1:1, more preferably (1.5-3): 1:1, and even more preferably 2:1:1. In the present invention, the pH value of the tea leaf protein nanosolution, anthocyanin nanosolution and epsilon-polylysine nanosolution after mixing is 2 to 6, more preferably 4 to 5.5, still more preferably 5. According to the invention, tea-leaf proteins can carry negative charges at an isoelectric point pH=3, and epsilon-polylysine can carry positive charges at an isoelectric point pH=9.74; and carrying out polyelectrolyte complex reaction on the tea dreg proteins with negative charges and the epsilon-polylysine with positive charges to realize complexing crosslinking of the epsilon-polylysine and the tea dreg proteins to generate nano particles. The tea dreg protein is food-grade, safe and nontoxic, and polylysine has good antibacterial effect and is nontoxic, the tea dreg protein and polylysine are used as wall materials, and anthocyanin is used as a core material to form particles with the particle size of nanometer grade by using a polyelectrolyte compounding method, so that the tea dreg protein is safe and nontoxic. The tea dreg protein-epsilon-polylysine-anthocyanin nano-composite prepared by the method is in a liquid state by naked eyes, 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 invention, the grain size of the tea leaf protein-epsilon-polylysine-anthocyanin nano-composite is preferably 100-200 nm.

In the invention, the homogenization mode preferably comprises ultrasonic homogenization, and after the ultrasonic homogenization, the tea dreg protein-epsilon-polylysine-anthocyanin nano-composite is obtained.

The ultrasonic homogenization time is preferably 5-20 min, 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 further preferably ultrasonic 3s intermittent 3s. The ultrasonic homogenizing apparatus of the present invention is preferably an ultrasonic pulverizer. The homogenization can fully mix the materials, the nano particles can be uniformly dispersed, and anthocyanin is promoted to enter the structure of protein epsilon-polylysine.

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

The tea dreg protein-epsilon-polylysine-anthocyanin nano-composite preparation method provided by the invention has the advantages that the applied raw materials are safe, the tea dreg protein is food-grade, the tea dreg protein is safe and nontoxic, the polylysine has 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 has the following advantages:

According to the invention, anthocyanin is embedded by utilizing the polyelectrolyte composite effect of tea dreg protein and epsilon-polylysine, so that the stability of anthocyanin is improved, and the whole preparation method is simple and quick in process. The tea residue raw material sources are wide, 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 developed, 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 dreg protein-epsilon-polylysine-anthocyanin nano-composite embeds anthocyanin, improves the stability of anthocyanin to light and heat, effectively improves the processing stability of anthocyanin, ensures that anthocyanin has the characteristic of precise slow control in the gastrointestinal tract, solves the problems of poor stability and low availability of anthocyanin, and greatly expands the application range of anthocyanin in the food processing process. The thermal stability of the anthocyanin after embedding in the embedding material in the prior art for 2.5 hours at the temperature of 90 ℃ is improved by about 10 percent, and the thermal stability of the anthocyanin after embedding in the tea dreg protein-epsilon-polylysine-anthocyanin nano-composite disclosed by the invention is improved by 11-15 percent.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

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

1. Tea dreg protein extraction

(1) The method is characterized in that the tea residue is leached with boiling water for 15min according to the mass volume ratio of 1g to 50mL of the tea residue to obtain tea residue, cotton cloth with 550 mu m is used for filtering tea residue leaching liquid to obtain the tea residue, and the tea residue is leached with boiling water and filtered to obtain the tea residue. When repeatedly leaching with boiling water, the volume of boiling water added is the same each time. The residual polyphenol substances can be removed by filtering the tea residue leaching solution after repeating the extraction process for 3 times; drying the tea residue at 65deg.C, and pulverizing and sieving with 180 μm sieve to obtain tea residue powder;

(2) Dispersing the tea dreg 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 dreg powder to the NaOH solution is 1:50 (w/v, g/mL), continuously heating the tea dreg powder for 90 minutes at the temperature of 90 ℃, and centrifugally separating the tea dreg powder for 15 minutes at 8000rpm, so as to keep tea dreg powder supernatant.

(3) And (3) regulating the pH value of the tea slag powder supernatant in the step (2) to 3.0 by using an HCl solution with the concentration of 0.1M, wherein a large amount of crude protein precipitation occurs in the tea slag powder supernatant, centrifuging for 30min at 4500r/min, collecting the crude protein precipitation of the tea slag powder supernatant, and then washing the crude protein precipitation of the tea slag powder by using pure water until the pH value of the crude protein of the tea slag powder is neutral. And finally, freeze-drying the coarse tea dreg powder protein, wherein the freeze-drying temperature is-50 ℃, the freeze-drying time is 72 hours to obtain tea dreg protein nano-particles, and storing the tea dreg protein nano-particles in a refrigerator at 4 ℃ for standby.

2. Tea dreg protein (TP) nano solution preparation

8Mg of the prepared tea leaf protein nano-particles are dispersed in 100mLPBS buffer solution with the concentration of 0.01M and are subjected to ultrasonic homogenization in an ultrasonic crusher. The ultrasonic homogenizing power is 300W, the frequency is 25Hz, the continuous ultrasonic homogenizing is carried out for 10min for 3s after 3s are turned on, and the tea dreg protein nano solution with the tea dreg protein concentration of 0.08mg/mL is obtained for standby.

3. Epsilon-polylysine nano solution preparation

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

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

0.6Mg/mL anthocyanin nanosolution was prepared by dispersing 60mg anthocyanin in 100mL of 0.01M PBS buffer.

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

Placing 20mL of tea dreg protein nano solution in the step 2 into a beaker, slowly adding 10mL of anthocyanin nano solution in the step 4, uniformly stirring, adding 10mL of epsilon-polylysine nano solution 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 3s is started and 3s is stopped, so that continuous ultrasonic homogenization is carried out for 10min, and a tea dreg protein-epsilon-polylysine-anthocyanin nano compound is obtained. In order to improve the stability of the tea leaf protein-epsilon-polylysine-anthocyanin nano-composite, the nano-particles are kept from generating aggregation and precipitation, 50mM KCl solution is added into the tea leaf protein-epsilon-polylysine-anthocyanin nano-composite for ion balance, and the tea leaf protein-epsilon-polylysine-anthocyanin nano-composite is obtained after freeze drying. The grain size of the tea dreg protein-epsilon-polylysine-anthocyanin nano-composite is 100-200 nm. The pH value of the mixed solution of the tea dreg protein nano solution and the anthocyanin nano solution and the epsilon-polylysine nano solution after being mixed is 5.

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

20ML of the tea leaf 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 grounds protein-epsilon-polylysine nanocomposite (TP-PLL) solution

Placing 20mL of tea leaf protein nano solution into a beaker, slowly adding 10mL of epsilon-polylysine solution, stirring, adding 10mL of 0.01M PBS buffer solution, and carrying out ultrasonic homogenization under an ultrasonic pulverizer, wherein the ultrasonic homogenization power is 300w, the frequency is 25Hz, and 3s is turned on for 3s, and continuous ultrasonic homogenization is carried out for 10min to obtain the tea leaf protein-epsilon-polylysine-anthocyanin nano compound.

Comparative example 3 preparation of Anthocyanin (ACNs) nanosolvent

Application example 1

Proteolytic amino acid salt acid hydrolysis method steps: weighing 100mg of dry powder sample, placing the sample into a hydrolysis tube, adding 10mL of 6mol/L HCl (analytically pure), filling N ₂, and then covering the tube cover of the hydrolysis tube tightly; the hydrolysis tube is put into a baking oven at 105 ℃ for hydrolysis for 22-24 hours; the hydrolyzed sample is fixed to volume to 50mL by ultrapure water (distilled water of the chen type); taking 1mL of the hydrolyzed sample after the volume fixing in a small beaker, and placing the small beaker in a vacuum drying oven at 60 ℃ for drying until the hydrolyzed sample is dry; 1mL of 0.02mol/L HCl (high-grade pure) is added into a beaker, and after being redissolved, a disposable water film of 0.22 mu m is added, and the mixture is filled into a sample bottle for amino acid analysis and determination.

The amino acid content of the tea leaf protein prepared in example 1 was measured by the above proteolytic amino acid hydrochloric 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 leaf proteins are rich, and the amino acid types and the amino acid contents of the tea leaf proteins are shown in Table 1.

TABLE 1 amino acid types and contents of tea leaf proteins

Application example 2

The surface microstructure of ACNs-TP-PLL solution prepared in example 1, ACNs solution prepared in comparative example 3, ACNs-TP solution prepared in comparative example 1, and TP-PLL solution prepared in comparative example 2 was observed using a Scanning Electron Microscope (SEM). The specific 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, the freeze-dried ACNs-TP-PLL was connected to SEM stub by using double-sided cellophane tape, then ACNs-TP-PLL was coated with gold-palladium, and after photographing, the average size of ACNs-TP-PLL was determined by digital image analysis, operating parameter voltage of electron microscope was 3kv, and working distance was 8.1mm.

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 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 is shown from A1, A2 and A3 that TP-PLL has a dense network structure, and the network structure of TP-PLL can complex anthocyanin therein, thereby achieving the aim of stabilizing anthocyanin, and the size of TP-PLL particles reaches nanometer level according to the scale in SEM picture of TP-PLL.

In FIG. 2-2, it is apparent from B1, B2, and B3 that ACNs-TP-PLL has a sheet-like structure of anthocyanin in a dense network structure compared with A1, A2, and A3, and that anthocyanin is well encapsulated in a network structure of shell material.

In FIGS. 2-3, it is evident from C1, C2 and C3 that the single tea leaf protein has a relatively uniform granule-packed structure, and the interaction of tea leaf proteins in ACNs-TP nano-composite covers and encapsulates the platy anthocyanin nano-particles.

In fig. 2 to 4, D1, D2 and D3 show that the anthocyanin nanoparticles have a platelet-shaped heterogeneous structure.

FIGS. 2-5 show that the micro-nanostructures of TP-PLL, ACNs-TP and ACNs, and the addition of PLL (ε -polylysine) caused a dramatic change in TP into a complex structure with a network.

As can be seen from fig. 2-1 to 2-5, both anthocyanin and tea leaf protein materials are nano-scale; in the preparation process, although the tea dreg protein-epsilon-polylysine forms a composite reticular structure, flocculation precipitation does not exist, and the result of an electron microscope can be seen to be still at the nano level; finally, the particle size of the prepared anthocyanin-tea dreg protein-epsilon-polylysine nano-particle is detected by a laser particle size meter under different pH and ion conditions, the nano-particle size is proved to be nano-scale, and the anthocyanin is presumed to belong to small molecules, enter the cavity structure of the tea dreg protein-epsilon-polylysine, form a more compact compound network structure, but still maintain the dissoluble characteristic under proper conditions and do not precipitate.

Application example 3ACNs-TP-PLL Fourier infrared spectrogram

The solid obtained by freeze-drying the TP nano-solution prepared in example 1, the solid obtained by freeze-drying the ACNs-TP-PLL solution prepared in example 1, the solid obtained by freeze-drying the ACNs nano-solution prepared in comparative example 3, the solid obtained by freeze-drying the ACNs-TP solution prepared in comparative example 1, and the solid obtained by freeze-drying the TP-PLL solution prepared in comparative example 2 were mixed with 100mgKBr at 20MPa to prepare granules, and then scanned in a range of 4000cm ^-1～500cm^-1 with a resolution of 4cm ^-1 with a scanning frequency of 64, see FIG. 3. The freeze-drying temperature was-54℃for 48h.

As can be seen from fig. 3, TP is an amide I band and an amide II band at 1637cm ^-1 and 1458cm ^-1, respectively, whereas 2924cm ^-1 is a C-H stretching vibration characteristic of protein, TP-PLL has an absorption peak at wavelength 1637cm ^-1、2924cm^-1、1457cm^-1 after formation of ACNs-TP-PLL at 946cm ^-1, and is similar to TP-PLL and TP characteristic peak, probably due to the bonding between TP and PLL by hydrogen bonding and the opposite attraction of charges, but ACNs is a stretching vibration peak of c=o, C-H on benzene ring at 1636.90cm ^-1, and characteristic peak shift after composite embedding of TP-PLL, and some characteristic peaks of ACNs such as 1448cm ^-1、1199cm^-1 disappear, indicating ACNs has been embedded.

Through 3419cm ^-1～3423cm^-1, the anthocyanin can interact with the tea-leaf protein firstly, and is connected with the tea-leaf protein through hydrogen bonds, so that the anthocyanin is adsorbed on the tea-leaf protein, therefore, the tea-leaf protein and the anthocyanin interact firstly, and then polylysine is added for crosslinking, so that the stability of ACNs-TP-PLL can be enhanced. Application example 4ACNs-TP-PLL ultraviolet spectrogram

The TP solution prepared in example 1, ACNs-TP-PLL solution prepared in example 1, ACNs nm solution prepared in comparative example 3, and ACNs-TP solution prepared in comparative example 1 were all scanned at full wavelength after being protected from light at room temperature for 0.5h, and the results are shown in FIG. 4.

The ultraviolet-visible spectrophotometer is a method for intuitively researching the coating relation between a host and an object, and the inclusion condition of the host and the object which can be illustrated by the method comprises the following steps: (1) Differences before and after clathrate formation are described by differences in the height, shape and position of absorption peaks; (2) Analysis was performed from the absorption position and intensity of the maximum absorption wavelength. It can be seen from FIG. 4 that ACNs has an absorption peak around 280nm and 550nm, and ACNs-TP-PLL after TP-PLL coating, wherein the intensity of the absorption peak of ACNs is greatly reduced at 280nm and 550nm, probably because anthocyanin enters into the complex network structure of tea leaf protein-epsilon polylysine, so that anthocyanin nanoparticles are wrapped to weaken the intensity of characteristic peaks.

Example 2

Into a conical flask was added 20mL of ACNs-TP-PLL solution prepared in example 1, the pH of the solution was adjusted to 1.5 with 2M HCl, and the mixture was preheated in a shaker (37 ℃ C., 95 rpm/min) for 10min, and 4mg of pepsin was added to initiate simulated gastric juice 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 in a shaker for 10min, and 8mg of pancreatin was added to initiate simulated small intestine digestion for 150min. 2mL of the mixture is sampled every 30min in the stomach digestion process, 2mL of the mixture is sampled every 30min in the intestine digestion process, the anthocyanin content in the digestion liquid is detected by adopting a high performance liquid chromatography, and the percentage of the anthocyanin content detected in the digestion liquid in different time periods and the total anthocyanin content initially added into the digestion liquid is the retention rate of the anthocyanin, which indicates the stability of the anthocyanin in the digestion liquid.

Comparative example 4

Into a conical flask was added 20mL of the ACNs nm solution prepared in comparative example 3, and the rest of the simulated stomach digestion and simulated intestine digestion conditions were the same as in example 2.

Anthocyanin retention in the digestive juice was calculated for various time periods for ACNs-TP-PLL of example 2 and ACNs of comparative example 4 as follows:

Anthocyanin retention in digestate (%) =wt/w0×100%

WT: at time T, the content (μg/mL) of ACNs in gastric juice or intestinal juice is simulated

W0: the sample initially contained ACNs% of the digestive juice (μg/mL)

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

TABLE 2 retention of anthocyanins in simulated gastric fluid for different subjects

TABLE 3 retention of anthocyanins in simulated intestinal fluid for different subjects

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

Example 3-1ACNs stability test of TP-PLL

A. effect of sustained heating on ACNs-TP-PLL solution stability

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

B. effect of light on ACNs-TP-PLL solution stability

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

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

The ACNs-TP-PLL solution prepared in example 1 was prepared by taking 6 samples of 80mL each, adjusting the pH of the 6 sample solutions with 0.1M hydrochloric acid or 0.1M sodium hydroxide to make the pH of the ACNs-TP-PLL solution 2, 3, 4, 5 and 6, continuously heating the 6 sample solutions at 90℃for 2.5 hours, and measuring the ACNs content of the samples by pH differential method. The pH differential assay procedure is described below.

D. Effect of K ⁺ ion Strength on thermal stability of ACNs-TP-PLL solution

The ACNs-TP-PLL solution of ph=5 prepared in example 1 was taken as 6 samples, 80mL each, kcl solution was added to the 6 samples, the K ⁺ ion intensity concentration in the 6 samples was 0mM, 50mM, 100mM, 150mM, 200mM, respectively, and then the 6 samples were heat-treated at 90 ℃ for 1 hour, and the content of ACNs in the samples was measured by pH differential method. ACNs-TP-PLL ph=5. The pH differential assay procedure is described below.

Stability test of comparative example 5ACNs

A. stability Effect of sustained heating on ACNs

The ACNs nm solution prepared in comparative example 3, ph=5, was sampled every 0.5h at 90 ℃ for 2.5h, and the anthocyanin content of the solution was detected by pH differential method. The pH differential assay procedure is described below.

B. Stability Effect of illumination on ACNs

The ACNs nm solution prepared in comparative example 3, ph=5, was placed under white light irradiation at 25 ℃ for 10d, and the content of ACNs was measured by pH differential method. The pH differential assay procedure is described below.

C. Influence of pH on the thermal stability of ACNs

The ACNs nm solution prepared in comparative example 3 was continuously heated at pH 2 to 6 at 90℃for 2.5 hours, and the ACNs content of the sample was measured by pH differential method. The pH differential assay procedure is described below.

D. effect of K ⁺ ion strength on ACNs thermal stability

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

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

2mL of 8 samples to be measured in the A treatment, the B treatment, the C treatment, the D treatment in example 3 and the A treatment, the B treatment, the C treatment and the D treatment in comparative example 3 were diluted 5 times with a buffer solution of pH 1.0 and a buffer solution of pH 4.5, respectively, and after being sufficiently mixed, the samples were equilibrated at room temperature for 10 minutes, and the absorbance values of the samples at 520nm and 700nm were measured using pure water as a blank group when the samples to be measured in A, B, C, D in example 3 and A, B, C, D in comparative example 3, respectively.

Buffer configuration at pH 1.0: accurately weighing 1.86gKCl, mixing with 980mL distilled water, correcting pH to 1.0 with concentrated HCI, transferring to a volumetric flask of 1L, and fixing volume with distilled water.

Buffer configuration at pH 4.5: accurately weighing 32.82g of sodium acetate, mixing with 960mL of distilled water, correcting the pH to 4.5 by using concentrated HCI, transferring to a volumetric flask of 1L, and fixing the volume by using distilled water.

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

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

Stability 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 treatment; r0 is the total ACNs content (mg/mL) in the sample under the initial condition;

stability improvement (%) = difference of ACNs-TP-PLL retention mean and ACNs retention mean;

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

TABLE 4-1 stability ACNs Retention Rate and stability improvement data for ACNs-TP-PLL of example 3 and comparative example 5 at different durations of heating

TABLE 4-2 data for improved retention and stability of ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at different light times

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Table 4-3 Retention Rate and stability improvement data for ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at different pH values

Tables 4-4 retention and stability enhancement data for ACNs-TP-PLL of example 3 and ACNs of comparative example 5 at different K ⁺ ionic strengths

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

As can be seen from Table 4-2 and B in FIG. 6-1, ACNs of comparative example 5 showed a greatly improved stability after continuous light irradiation while ACNs-TP-PLL of example 3 showed a greatly improved stability, indicating that tea leaf protein- ε -polylysine was able to provide protection for anthocyanin photostabilization.

As can be seen from Table 4-3 and C in FIG. 6-1, the lower the ACNs pH of comparative example 5, the better the thermal stability is, because ACNs exists mainly in the form of red salt ions at pH less than 2, when the pH is raised, the hydration of C-2 and proton transfer of the acidic hydroxyl groups on anthocyanin by nucleophilic attack of water molecules occur, and there is kinetic and thermodynamic competition between these two reactions, so that the stability of ACNs is lowered when the pH is increased, but ACNs-TP-PLL of example 3 shows excellent thermal stability at pH equal to 5, because the shell material of its nanoparticles can well encapsulate anthocyanin in this network by polyelectrolyte complexation at pH equal to 5, while too low or too high pH can result in anthocyanin not being well encapsulated in the network, too low tea-seed protein precipitation flocculation, and too high pH can result in failure to form good complex.

It is known from table 4-4 and D in fig. 6-1 that the presence of excessive K ⁺ ions damages the thermal stability of ACNs-TP-PLL nano-ions of example 3, because the presence of excessive K ⁺ ions can cause electrostatic shielding effect on the system, and the tea leaf proteins carry negative charges at ph=5 to interact with excessive K ⁺, so that electrostatic shielding generated by tea leaf proteins cannot form a stable complex network structure with epsilon-polylysine, resulting in the decrease of thermal stability of anthocyanin, but K ⁺ ion concentration at 50mM can cause the system to be more stable, because K ⁺ concentration at 50mM is just in the process of a stable structure, and anthocyanin is well embedded in the complex network structure of tea leaf proteins-epsilon-polylysine, while excessive tea leaf proteins do not form excessive complex precipitation due to electrostatic shielding effect generated by small amount of K ⁺, but make the system more stable.

Example 3-2

Particle size analyzer (Delsa Max Pro; beckman Coulter, UK) was used to measure the particle size of ACNs-TP-PLL nanocomposite in solution at different pH conditions of pH=2, pH=3, pH=4, pH=5, pH=6 in C treatment of example 3-1 and the particle size of ACNs-TP-PLL composite in solution at different K ⁺ ion intensities of K ⁺ ion intensity of 0mM, 50mM, 100mM, 150mM, 200mM in D treatment of example 3-1 using dynamic light scattering as follows: the ACNs-TP-PLL solution at pH 5 in treatment C of example 3-1 was diluted 10 times, 1mL was loaded, and 10 measurements were made for each sample, the apparatus was programmed to count and calculate the particle size of 0.02-2000 μm in diameter, and the same assay was performed for the remainder.

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

TABLE 4-5 particle sizes of ACNs-TP-PLL in solutions at different pH conditions

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

Tables 4-6 particle size of ACNs-TP-PLL in solutions of different K ⁺ ionic strengths

From a in fig. 6-2 and table 4-5, ACNs-TP-PLL has a smaller particle size (p < 0.05) at pH 5-6 because TP can crosslink with PLL at pH 5-6 to become more dense, so that hydrophobic groups in tea-leaf protein drain more water to form a larger amount and dense cavity structure load ACNs to reduce particle size, whereas nanoparticle particles are larger at pH 2-4 because TP and PLL and ACNs are in a free state, tea-leaf protein has started to flocculate at ph=2, tea-leaf protein has a near isoelectric point (pi=3.0) due to charge close to zero, and electrostatic action is reduced between particles to promote aggregation of nanoparticles to increase particle size. Alkaline conditions are unfavorable for the adsorption of anthocyanin by tea dreg proteins.

From B in FIG. 6-2 and Table 4-6, it is clear that, at an ionic strength of 0, K ⁺, since the system is mainly composed of polylysine with strong positive charges and tea-leaf protein with negative charges, they can continuously form a network structure through electrostatic interaction to load ACNs more, so that the average particle size is also relatively large (p < 0.05) 159+ -9 nm, but after a small amount of K ⁺ ions is added, the particle size of ACNs-TP-PLL is also reduced (p < 0.05), because the addition of a small amount of K ⁺ ions can prevent the mutual electrostatic interaction between tea-leaf protein and polylysine, so that the system balance between tea-leaf protein and polylysine is gradually achieved, which is likely to be more stable to the system, and therefore, the controllable loading of nanoparticles can be performed through a small amount of K ⁺ ions.

Example 4

ACNs-TP-PLL prepared in example 1, ACNs prepared in comparative example 3, ACNs-TP, ACNs-PLL and ACNs-SPI prepared in comparative example 1 were pH 5.0 and then heated at 90℃for 1 hour, and anthocyanin retention was calculated using the above formula, and the results are shown in Table 5 and FIG. 7. From Table 5 and FIG. 7, ACNs-TP-PLL prepared in example 1 had the highest retention, which can be shown that ACNs and TP nanomaterials have better thermal stability effect as wall material embedding anthocyanin than prior art embedding material soy protein.

The preparation method of ACNs-PLL is as follows: 8mg of epsilon-polylysine powder was dispersed in 100mL of 0.01M PBS buffer to prepare 0.08mg/mL epsilon-polylysine solution for later use. 20mL of the epsilon-polylysine nano-solution is placed in a beaker, 10mL of the anthocyanin nano-solution prepared in example 1 is slowly added and stirred, and 10mL of 0.01M PBS buffer solution is added to obtain ACNs-PLL.

The preparation method of ACNs-SPi is as follows: soy protein nanoparticles 8mg were dispersed in 100mLPBS buffer at a concentration of 0.01M and sonicated in a sonicator. Ultrasonic homogenizing is carried out for 10min at power of 300W and frequency of 25Hz for 3s and 3s for 3s, and soybean protein nano solution with soybean protein concentration of 0.08mg/mL is obtained for standby.

20ML of the soybean protein nano solution is placed in a beaker, 10mL of the anthocyanin nano solution prepared in the example 1 is slowly added and stirred, and 10mL of 0.01M PBS buffer solution is added to obtain ACNs-SPI.

TABLE 5 anthocyanin retention under heating 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 anthocyanin retention rate of ACNs-TP-PLL under different heating conditions is highest, which shows that after the anthocyanin is embedded in the tea dreg protein-epsilon-polylysine nano material prepared by the invention, the thermal stability of the anthocyanin is improved.

Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

1. The tea dreg protein-epsilon-polylysine nano material is characterized by comprising tea dreg protein nano particles and epsilon-polylysine; the tea leaf protein nano-particles and epsilon-polylysine form a net-shaped compound; the preparation method of the tea dreg protein nano-particles comprises the following steps: extracting tea leaves with boiling water and filtering to obtain tea leaves, and drying and crushing the tea leaves to obtain tea leaves powder; performing alkali extraction and acid precipitation on the tea residue powder to obtain crude protein of the tea residue powder; and drying the coarse tea dreg powder protein to obtain tea dreg protein nano-particles.

2. A tea leaf protein-epsilon-polylysine-anthocyanin nanocomposite, wherein tea leaf protein nanoparticles and epsilon-polylysine as claimed in claim 1 are used as wall materials and anthocyanin is used as a core material.

3. The method for preparing the tea leaf protein-epsilon-polylysine-anthocyanin nano-composite according to claim 2, which is characterized by comprising the following steps: mixing the mixed solution of the tea dreg protein nano solution and the anthocyanin nano solution with the epsilon-polylysine nano solution, and homogenizing to form a tea dreg protein-epsilon-polylysine-anthocyanin nano compound;

4. The preparation method according to claim 3, wherein 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 dreg protein nano solution, the epsilon-polylysine nano solution and the anthocyanin nano solution is (1-5) 1:1.

5. The method of claim 4, wherein the tea leaf protein nano-solution comprises tea leaf protein nano-particles and a buffer; the mass volume ratio of the tea dreg protein 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.

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

7. The method of claim 4, wherein 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;

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

9. The preparation method of claim 4, wherein the grain size of the tea leaf protein-epsilon-polylysine-anthocyanin nanocomposite is 100-200 nm.

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

11. The method of claim 10, wherein homogenizing comprises ultrasonic homogenizing for a period of 10 minutes at a power of 300W.

12. The preparation method according to claim 3, wherein the mass concentration of tea leaf protein in the tea leaf protein nano-solution is 0.08 mg/mL; the mass concentration of anthocyanin in the anthocyanin nano solution is 0.6 mg/mL; the mass concentration of epsilon-polylysine in the epsilon-polylysine nano solution is 0.08 mg/mL.