CN113264980A

CN113264980A - High-purity tea seed meal protein and tea saponin and preparation method thereof

Info

Publication number: CN113264980A
Application number: CN202110552490.6A
Authority: CN
Inventors: 龙辉
Original assignee: Hunan Wanxiang Biotechnology Co ltd
Current assignee: Hunan Wanxiang Biotechnology Co ltd
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-08-17
Anticipated expiration: 2041-05-20
Also published as: CN113264980B

Abstract

The invention discloses a high-purity tea seed meal protein and tea saponin and a preparation method thereof, and the preparation method comprises the following steps: the method comprises the following steps of crushing, sieving, soaking and deironing: crushing and sieving the degreased tea seed meal, soaking the degreased tea seed meal in an ethanol water solution and removing iron to obtain a mixture A; the acidification and extraction are as follows: acidifying and heating the mixture A for extraction to obtain ethanol extract of acid tea saponin and final precipitate; performing alkaline wall breaking and extraction: performing alkaline heating and wall-breaking extraction to obtain extractive solution of the mixture; step four, precipitation, alcohol extraction and drying: the extract of the mixture is subjected to isoelectric precipitation, water washing, alcohol extraction and drying to obtain a high-purity tea seed meal protein product; fifthly, calcium precipitation, washing and drying: performing calcium precipitation, washing, acidification and drying on an ethanol extracting solution of the acid tea saponin to obtain a high-purity tea saponin product; the method has the advantages of low cost, high extraction rate of protein and tea saponin, full utilization of raw materials, high purity of protein and tea saponin, light color of product, and mechanization.

Description

High-purity tea seed meal protein and tea saponin and preparation method thereof

Technical Field

The invention belongs to the technical field of comprehensive utilization of oil meal, and particularly relates to a high-purity tea seed meal protein and tea saponin and a preparation method thereof.

Background

The tea seed meal contains 15-25% of protein. The protein in the tea seed meal mainly exists in a protein body in a cell wall, and inherent divalent and trivalent metal ions in the tea seed meal, such as calcium, magnesium, iron, zinc, manganese and copper ions, can be combined with the protein to generate a water-insoluble protein-metal ion compound, so that the extraction rate of the tea seed meal protein extracted by a common alkali liquor extraction method is very low, the extraction rate is generally 50-60%, and the obtained protein usually contains polyphenol components such as tea saponin, tannin and the like, is low in purity, dark in color and astringent in taste, and still cannot be eaten or cannot be fed, so that the research on the extraction method of the tea seed meal protein is necessary to obtain the edible and feeding tea seed meal protein with high extraction rate and high purity.

Tea saponin, which is a typical glycoside substance, is a mixture of various glycoside components. Tea saponin has acidity, and can be called tea saponin acid, and the structure of tea saponin is shown in the following formula.

The connection of different aglycones and different organic acids and the different connection modes lead to a series of tea saponin monomers with similar structures and a plurality of varieties. The Japanese scholars Qingshan Xinxianlang is separated from tea tree seeds for the first time to obtain tea saponin, and aglycone and saccharide are obtained through a hydrolysis experiment so as to determine the chemical formula of the tea saponin. In 1952, the crystal of tea saponin was first separated from tea seeds by Shishimen and Shangyang of university of Tokyo, Japan, and the melting point was determined to be 224-₅₇H₉₀O₂₆。

The pure product of tea saponin is milk white or light yellow powder, has strong hygroscopicity, and the aqueous solution of the tea saponin is acidic to methyl red reaction (tea saponin acid). The acidic tea saponin is insoluble in cold water, absolute ethyl alcohol and absolute methyl alcohol, insoluble in organic solvents such as ethyl ether, acetone, benzene, petroleum ether and the like, slightly soluble in warm water, ethyl acetate and carbon disulfide, and well soluble in hot water, alkaline water solution, aqueous ethyl alcohol, aqueous methyl alcohol, n-butyl alcohol, glacial acetic acid, pyridine and dried vinegar, and can be extracted by the media. Under the condition that the pH value is more than pK (the dissociation constant of carboxyl of glucuronic acid in the tea saponin), the tea saponin acid can be converted into tea saponin acid salt, tea saponin sodium and tea saponin potassium, the solubility of the tea saponin acid salt, the tea saponin acid salt and the tea saponin potassium in water is higher, and the tea saponin in the tea seed meal can be extracted by utilizing the principle; the salt of tea saponin acid with divalent metal ion and trivalent metal ion, such as calcium tea saponin, magnesium tea saponin, ferrous tea saponin and ferric tea saponin, is the precipitate of water insoluble white cloud. Therefore, the chelation and precipitation of the inherent and foreign polluted divalent metal ions and trivalent metal ions in the tea seed meal are beneficial to improving the yield of the tea saponin. Through repeated data analysis and experimental comparison, the applicant finds that most researchers confuse the fundamental differences of tea saponin, tea saponin acid and tea saponin acid salt.

Because the molecular structure simultaneously has hydrophilic saccharide and hydrophobic aglycone, the tea saponin has good surface activity and is a natural surfactant; in addition, the tea saponin has an inhibiting effect on various microorganisms and also has an application prospect of a preservative. The tea saponin also has the action of like biological hormone, and has promoting effect on growth of animals and plants. In addition, the tea saponin also has physiological activities of inhibiting alcohol absorption, protecting gastrointestinal tract, resisting hypertension, resisting permeability and diminishing inflammation, etc. Therefore, the market demands a high-purity tea saponin product.

Because the yield of tea seeds is small, the production of tea oil mainly adopts a squeezing process. At present, tea seed meal (also called tea seed cake and tea seed cake) obtained after tea seed is squeezed to extract tea seed oil and degreased tea seed meal obtained after residual oil is extracted from the tea seed meal by a leaching method are basically in a waste state, so that environmental pollution and resource waste are caused. Through repeated and careful experimental study, the applicant discovers that the tea seed meal contains iron filings for the first time, and the analysis reason is that the iron filings exist, which not only affect the color of the tea saponin product, but also are not beneficial to the full extraction of the tea saponin because the screw roller of the screw press slightly falls off due to the repeated friction and extrusion of high pressure in the process of squeezing and extracting oil by the screw press after shelling. The tea seed meal, the defatted tea seed meal, the tea seed hulls and the tea seed cattails contain 5-20% of tea saponin and other useful components such as protein, polysaccharide, flavone and the like, so that how to synthesize the tea seed meal, the defatted tea seed meal, the tea seed hulls and the tea seed cattails is necessary, urgent and important.

The extraction and characteristics of the protein in the tea seed meal are researched by scholars such as Dinghua and bear rescue. Chinese patent CN201910463338.3 (a method for extracting and refining tea saponin), CN201911119365.5 (a method for extracting tea saponin from camellia oleifera fruit shell), CN201910615565.3 (a method for extracting tea saponin), CN202010475217.3 (a process for extracting tea saponin from camellia oleifera seed cake and its application in amino acid facial soap), CN201710839054.0 (a method for improving the dissolution rate of water-extracted tea saponin), CN201811402817.6 (a method for extracting tea saponin from camellia oleifera seeds), CN201611008220.4 (a method for simultaneously recovering ethanol and tea saponin from the aqueous phase after extracting camellia oleifera seed oil by ethanol water extraction), CN201810246415.5 (a separation method for improving the yield of tea saponin), CN201910046926.7 (a method for extracting tea saponin), CN201910463338.3 (a method for extracting and refining tea saponin), CN201810864405.8 (a method for preparing tea saponin), CN201910615565.3 (a method for extracting tea saponin), CN201910802169.1 (a method for refining high-purity tea saponin), CN201911196405.6 (a method for extracting tea saponin from camellia oleifera seed oil and tea saponin) and CN201711296589.4 (a method for efficiently decontaminating tea saponin Preparation method of saponin), which respectively relates to the processes and technologies of extraction, preparation, purification and refining of tea saponin, however, the patents have the following disadvantages: 1. the extraction rate of protein is low, and the property of the tea saponin is not known sufficiently, so the adopted technical scheme has the congenital defect; 2. the developed tea seed meal protein and tea saponin products have low purity, dark color and heavy astringent taste, so the tea seed meal protein and tea saponin products are not advanced in technology and unreasonable in economy. Therefore, the continuous research on the extraction and refining technology of the tea seed meal protein and the tea saponin, the development of a new technology and a new process with high extraction rate and high purity of the protein and the tea saponin, and the method have important social, economic and practical significance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the high-purity tea seed meal protein and tea saponin, and the protein and the tea saponin have the advantages of high purity, light product color, outstanding product characteristics, high product added value and mechanized preparation.

The invention also aims to provide a preparation method of the high-purity tea seed meal protein and the tea saponin, which is feasible, has the advantages of high protein and tea saponin extraction rate, low cost, full utilization of raw materials, convenient operation, easy selection and matching of equipment, low equipment investment and mechanization.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a high-purity tea seed meal protein and tea saponin and a preparation method thereof comprise the following steps:

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by a stainless steel crusher and sieving the degreased tea seed meal by a standard sieve of 40-100 meshes to obtain degreased tea seed meal; adding an ethanol aqueous solution into the obtained degreased tea seed meal, stirring and soaking at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition and expansion by using an iron remover after stirring and soaking are finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, adjusting the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.1-3.3 by using a hydrochloric acid solution, continuously stirring and refluxing for extraction, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of the acid tea saponin; adding an ethanol aqueous solution into the obtained precipitate b1, and performing secondary and third reflux extraction respectively according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate b 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain the ethanol extract of the acid tea saponin for later use.

(3) Alkaline wall breaking and extraction: adding softened water into the precipitate b3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture for 1.0-2.0 hours at 70-80 ℃ by using a sodium hydroxide solution under the condition of stirring, and performing centrifugal separation after stirring to obtain an extracting solution c1 and tea seed meal residue d1 of the mixture; adding softened water into the obtained tea seed dregs d1, respectively carrying out second extraction and third extraction and centrifugal separation under the condition of stirring to respectively obtain a mixture extracting solution c2, a mixture extracting solution c3 and tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution c3 to obtain a mixture extracting solution for later use.

(4) Isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH of the extracting solution of the mixture obtained in the step three to 4.5 by using a hydrochloric acid solution, and continuously stirring for 0.5-1.0 hour to obtain a crude tea seed meal protein precipitate; respectively carrying out 1 st and 2 nd water washing and centrifugal separation on softened water in the obtained crude tea seed meal protein precipitate under the condition of stirring, and discarding supernatant containing water-soluble components to obtain the crude washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding ethanol in percentage by volume into the washed crude tea seed meal protein precipitate, stirring at room temperature for 0.5-1.5 hours, performing centrifugal separation after stirring, and removing supernatant to obtain the crude tea seed meal protein precipitate subjected to first alcohol washing; adding absolute ethyl alcohol into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring at room temperature for 0.5-1.5 hours, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringented and impurity-removed tea seed meal protein precipitate at the temperature of below 60 ℃ by using a drying method until the moisture percentage content is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain a white high-purity tea seed meal protein product.

(5) Precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.0-6.0 by using calcium hydroxide, adjusting the temperature to 0-20 ℃, continuing stirring for 0.5-1.5 hours, performing centrifugal separation after stirring, and discarding supernatant to obtain a crude product of the calcium tea saponin precipitate; adding an ethanol water solution into the obtained crude product of the calcium tea saponin sediment, adjusting the temperature to be 0-20 ℃, continuously stirring for 0.5-1.5 hours, centrifugally separating after stirring, and removing supernatant to obtain the crude product of the calcium tea saponin sediment subjected to first alcohol washing; adding absolute ethyl alcohol into the obtained tea saponin calcium precipitate crude product subjected to primary alcohol washing, adjusting the temperature to be 0-20 ℃, continuously stirring for 0.5-1.5 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water into the alcohol-washed tea saponin calcium precipitate, adjusting the temperature to be 50-70 ℃, stirring and dispersing, adjusting the pH of the mixed solution to be 3.1-3.3 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to be 0-20 ℃, stirring at constant temperature for 5.0-15.0 hours, performing centrifugal separation after stirring, and discarding supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water into the obtained acid tea saponin precipitate crude product, stirring and dispersing, controlling the pH value of the mixed solution to be 3.1-3.3 and the temperature to be 0-20 ℃, continuing stirring for 0.5-1.5 hours, performing centrifugal separation after stirring, and removing a washing solution containing water-soluble impurities to obtain the acid tea saponin precipitate; drying the acid tea saponin precipitate at the temperature of below 60 ℃ by using a drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain a white high-purity tea saponin product.

Preferably, the degreased tea seed meal in the step is tea seed meal with the oil content of less than or equal to 1.0% obtained after tea seed is squeezed by a squeezing method to extract tea seed oil, and residual oil in the squeezed tea seed meal is removed by a leaching method; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the iron remover is used in food industry or chemical industry.

Preferably, in the step, the mass ratio of the degreased tea seed meal powder to an ethanol aqueous solution is 1:3-7, and the ethanol volume percentage content in the ethanol aqueous solution is 75-85%; the stirring and soaking time is 3-11 hours.

Preferably, the hydrochloric acid in the step two is industrial-grade, chemically pure or analytically pure hydrochloric acid; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the mass ratio of the precipitate b1 to the ethanol water solution is 1:2-6, and the ethanol volume percentage content in the ethanol water solution is 75-85%; the temperature of the reflux extraction is 40-70 ℃, and the time is 0.5-1.5 hours.

Preferably, the sodium hydroxide in step three is industrial-grade, chemically pure or analytically pure sodium hydroxide; the mass ratio of the precipitate b3 to the softened water is 1: 6-10; the mass ratio of the tea seed meal residue d1 to the softened water is 1: 2.5-5.5, and the conditions of the second extraction and the third extraction are as follows: the pH value of the mixed solution is 11.1-11.5, the extraction temperature is 55-65 ℃, and the extraction stirring time is 0.5-1.0 hour.

Preferably, the hydrochloric acid in step four is industrial-grade, chemically pure or analytically pure hydrochloric acid; the ethanol is industrial grade, food grade, chemically pure or analytically pure ethanol; the drying method is a vacuum drying method or a normal pressure hot air drying method;

preferably, in step four, the mass ratio of the crude tea seed meal protein precipitate to the softened water in step a is 1: 2-4; the pH value of the mixed solution during washing is 4.4-4.5, the washing temperature is 35-45 ℃, and the washing stirring time is 0.5-1.0 hour; and B, the mass ratio of the tea seed meal protein precipitate crude product washed in the step B to the ethanol water solution is 1:2-4, the volume percentage of ethanol in the ethanol water solution is 60-70%, and the mass ratio of the crude tea seed meal protein precipitate obtained by the first ethanol washing to the absolute ethanol is 1: 2-4.

Preferably, the calcium hydroxide in step fifthly is industrial-grade, chemically pure or analytically pure calcium hydroxide; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the hydrochloric acid is industrial grade, chemically pure or analytically pure hydrochloric acid; the drying method is a vacuum drying method or a normal pressure hot air drying method;

preferably, the mass ratio of the crude product of calcium theasapogeninate precipitate to the aqueous ethanol solution in the step A is 1: 2-5; the volume percentage of ethanol in the ethanol water solution is 75-85%, and the mass ratio of the crude product of the calcium theasapogeninate precipitate obtained by the first ethanol washing to the absolute ethanol is 1: 2-4; the mass ratio of the tea saponin calcium precipitate washed by alcohol in the step B to the softened water is 1: 1.5-4.5; the mass ratio of the acid tea saponin precipitate crude product to the softened water is 1: 1.5-3.5.

In addition, the invention also discloses high-purity tea seed meal protein and tea saponin prepared by any one of the preparation methods.

The technical concept of the invention is as follows:

the invention utilizes macromolecular substances such as macromolecular protein and polysaccharide inherent in degreased tea seed meal, starch, pectin and the like which are insoluble in ethanol aqueous solution with higher concentration, iron filings in the degreased tea seed meal after soaking, softening and swelling can be separated by an iron remover, divalent and polyvalent metal ions combined with protein, pectin and tea saponin in the degreased tea seed meal can be converted into hydrochloride so as to release and dissociate the protein, the pectin and the tea saponin, carboxyl of glucuronic acid in tea saponin molecules can be sealed into carboxylic acid and carboxyl in protein molecules can be sealed into carboxylic acid so as to convert the tea saponin into alcohol-soluble acid tea saponin, convert the protein into alcohol-insoluble protein hydrochloride with positive net charge and seal the carboxyl of the pectin, the alcohol aqueous solution can extract alcohol-soluble components including acid tea saponin, polyphenol, tannin, flavone and caffeine, the protein originally positioned in the cell wall can be extracted by breaking the wall and can be precipitated under the isoelectric point, the water-soluble macromolecular components including pectin, polysaccharide, gelatinized starch and the like carried in the tea seed meal protein precipitated at the isoelectric point can be separated by washing, mycotoxin, fatty acid, acid tea saponin, pigment, polyphenol, tannin, flavone, caffeine and other alcohol-soluble components mixed in the tea seed meal protein precipitated at the isoelectric point can be extracted and separated by alcohol liquid, the alcohol-soluble acid tea saponin can be calcified into alcohol-insoluble tea saponin calcium precipitate, the pigment, the tannin, the polyphenol, the flavone and other components in the tea saponin calcium precipitate have alcohol solubility, the tea saponin calcium can be acidified into the property of acid tea saponin which is insoluble in cold water, the degreased tea seed meal is crushed and sieved, is soaked in ethanol aqueous solution, and is subjected to iron removal to obtain an ethanol soaking mixture of degreased tea seed meal after iron removal, acidifying, heating, extracting and centrifugally separating to obtain ethanol extract and final precipitate of the acid tea saponin; and finally, carrying out alkaline heating and wall breaking on the precipitate to obtain a mixture extract of the protein, and carrying out isoelectric point precipitation, washing for impurity removal, alcohol washing for detoxification, decolorization, impurity removal and drying on the extract to obtain a white high-purity tea seed meal protein product. Neutralizing the ethanol extract of the acid tea saponin with calcium hydroxide, calcifying, precipitating, washing with ethanol water solution and ethanol, centrifuging to obtain alcohol-washed calcium tea saponin precipitate, acidifying to obtain cold water insoluble acid tea saponin, cooling, precipitating, washing with water, centrifuging to obtain acid tea saponin precipitate, drying, dewatering, pulverizing, quantitatively packaging, and sealing to obtain white high-purity tea saponin product.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method comprises the steps of crushing defatted tea seed meal by a stainless steel crusher, controlling the iron filings carried into the tea seed meal by the screw press and oil extraction by using an iron remover after macromolecular substances such as protein, polysaccharide, starch, pectin and the like in the defatted tea seed meal are controlled to be in an insoluble state by using a higher-concentration ethanol aqueous solution, soaking the defatted tea seed meal to fully expand and disperse the defatted tea seed meal powder, so that the problem that the macromolecular substances such as protein, polysaccharide, starch, pectin and the like and the tea saponin are difficult to separate is solved, the problem that the iron rust carried into the defatted tea seed meal is crushed by a common crusher, the iron rust and the inherent polyphenol substances in the tea seed meal react to change color, the iron rust and the tea saponin react to generate water-insoluble iron tea saponin so as to reduce the extraction rate of the tea saponin, the iron rust and the protein react to generate water-insoluble protein-iron ion compound so as to reduce the extraction rate of the protein, and the iron filings carried into the tea seed meal by the oil extraction by the screw press can not be removed generally, The iron chips and the polyphenol react to change color, the iron chips react with the tea saponin in the subsequent extraction process to generate water-insoluble iron tea saponin to reduce the extraction rate of the tea saponin, the iron chips react with the protein in the subsequent extraction process to generate water-insoluble protein-iron ion compounds to reduce the extraction rate of the protein and other technical problems and difficulties, and the technical effects of easily separating protein, polysaccharide, starch, pectin and other macromolecular substances from the tea saponin, reducing the subsequent ethanol decoloration load and difficulty, improving the color and purity of subsequent protein products and tea saponin products and improving the yield of subsequent protein and tea saponin products are mainly achieved.

(2) The method comprises the steps of converting divalent and polyvalent metal ions combined with protein, pectin and tea saponin in lipid tea seed meal into hydrochloride dissolved in alcohol water by using subacid acidification treatment (pH is 3.1-3.3) so as to release and dissociate protein, pectin and tea saponin, sealing carboxyl of glucuronic acid in tea saponin molecules into carboxylic acid and sealing carboxyl in protein molecules into carboxylic acid so as to convert the tea saponin into alcohol-soluble acid tea saponin, converting protein into alcohol-insoluble protein hydrochloride with positive net charge and sealing carboxyl of pectin, extracting the acid tea saponin and precipitating alcohol-insoluble macromolecular substances such as protein, polysaccharide, pectin and starch by heating subacid (pH is 3.1-3.3), solving the problem that the divalent and polyvalent metal ions inherent in degreased tea seed meal are combined with the protein and the tea saponin to form insoluble substances, the method solves the technical problems that the inherent structure of the tea saponin is damaged and the extraction rate is low due to acid hydrolysis of glycosidic bonds in the tea saponin by heating and extracting under an acidic condition (such as the common pH value of 1-3), and the technical problems and difficulties that the separation of protein and alcohol-soluble tea saponin and alcohol-soluble components such as alcohol-soluble pigment, tannin, polyphenol, flavone and the like is difficult and the like, and mainly achieves the technical effects of fully extracting the tea saponin and the protein, fully separating the tea saponin and the protein, and improving the extraction rate and the purity of the tea saponin and the protein.

(3) The method of the invention extracts the protein originally positioned in the cell wall by using the alkaline heating and wall breaking with higher pH, extracts the water-soluble macromolecular components including pectin, polysaccharide, gelatinized starch and the like carried by the tea seed meal protein precipitated by water washing and isoelectric point, extracts and separates mycotoxin, fatty acid, acid tea saponin, pigment, polyphenol, tannin, flavone, caffeine and other alcohol-soluble components carried by the tea seed meal protein precipitated by isoelectric point by ethanol water solution and absolute ethanol, solves the technical problems of low extraction rate of the tea seed meal protein, difficult detoxification, decoloration and difficult deastringency of the tea seed meal protein, solves the technical problems and difficulties of low purity, dark color, toxicity, heavy astringency and the like of the tea seed meal protein, and mainly achieves the purposes of improving the extraction rate of the tea seed meal protein, fully detoxifying, decoloring, deastringency and impurity removal, improving the purity and sensory quality of the tea seed meal protein.

(4) The method disclosed by the invention has the advantages that the alcohol-soluble acid tea saponin is neutralized and calcified into alcohol-insoluble tea saponin calcium precipitate to be separated from impurities such as pigment, polyphenol, tannin, flavone and the like, mycotoxin, pigment, tannin, polyphenol, flavone and other alcohol-soluble components in the tea saponin calcium precipitate are removed by washing with an ethanol aqueous solution and ethanol, the alcohol-washed tea saponin calcium is acidified into hot-water-soluble acid tea saponin by heating with soft subacid (pH of 3.1-3.3), the acid tea saponin is precipitated by cooling (0-20 ℃), and the problem that the extraction rate of the tea saponin is low due to the fact that insoluble tea saponin is formed by combining divalent and polyvalent metal ions inherent in defatted tea seed meal and the tea saponin is low and the problem that the inherent structure of the tea saponin is damaged due to the acid hydrolysis of a glycosidic bond in the tea saponin by heating and extracting under acidic conditions (such as common pH of 1-3) is solved by washing with water with low-temperature and slightly acidic (pH of 3) and the like The method has the advantages of low extraction rate, solving the problems of difficult separation of alcohol-soluble tea saponin and alcohol-soluble mycotoxin, pigment, tannin, polyphenol, flavone and other substances, solving the technical problems and difficulties of large tea saponin loss and the like caused by washing and separating water-soluble impurities by water at overhigh temperature, and mainly achieving the technical effects of fully extracting the tea saponin, improving the utilization rate and yield of the tea saponin, fully separating the alcohol-soluble impurities, fully removing toxic and colored substances, improving the sensory quality of products, reducing the solubility of acid tea saponin, fully precipitating the acid tea saponin, fully removing the water-soluble impurities, keeping the inherent molecular structure of the acid tea saponin and improving the yield and purity of the acid tea saponin.

(5) Compared with the prior art, the invention has the advantages that the protein and tea saponin products with low purity, deep color, high cost, toxicity, heavy astringency and low extraction rate can be obtained by only using tea seed meal or degreased tea seed meal as raw materials through respective technical treatment in the prior art; the invention realizes the technical breakthrough of using the degreased tea seed meal as the raw material to manufacture the protein and the tea saponin products with high purity, light color, low cost and high extraction rate, the purity of the obtained protein product can reach more than 98.5 percent, the extraction rate can reach more than 90 percent, and the protein is non-toxic, colorless and non-astringent; the purity of the obtained tea saponin product can reach more than 98 percent, and the extraction rate can reach more than 95 percent.

Drawings

FIG. 1 is a process flow chart of the preparation method of high-purity tea seed meal protein and tea saponin according to the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 80-mesh standard sieve to obtain degreased tea seed meal powder; adding 30kg of ethanol aqueous solution with the ethanol volume percentage content of 80% into 6kg of the obtained degreased tea seed meal, stirring for 7 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, regulating the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.2 by using a hydrochloric acid solution, regulating the temperature to 55 ℃, continuing stirring and reflux extraction for 1 hour, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of the acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 4 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 8 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, using sodium hydroxide solution to enable the pH of the mixture to be 12 under the stirring condition, stirring for 1.5 hours at 75 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 of the mixture and tea seed meal residue d 1; adding softened water with the mass being 4 times of that of the obtained tea seed dregs d1, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions that the pH is 11.3, the temperature is 60 ℃ and the stirring time is 0.75 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.45 by using a hydrochloric acid solution, and continuously stirring for 0.75 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 3 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.45, the temperature is 40 ℃ and the stirring time is 0.75 hours, and discarding supernatant containing water-soluble components to obtain the crude product of the water-washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding ethanol water solution with the ethanol volume percentage content of 65 percent and the mass of 3 times of the crude washed tea seed meal protein precipitate into the crude washed tea seed meal protein precipitate, stirring the mixture at room temperature for 1 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude tea seed meal protein precipitate washed by alcohol for the first time; adding absolute ethanol with the mass multiple of 3 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring for 1 hour at room temperature, performing centrifugal separation after stirring, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 55 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 0.69 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.5 by using calcium hydroxide, adjusting the temperature to 10 ℃, continuing stirring for 1 hour, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium theasapogenin precipitate; adding ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 3.5 times of the crude product of the tea saponin calcium precipitate into the crude product of the tea saponin calcium precipitate, adjusting the temperature to 10 ℃, continuing stirring for 1 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the tea saponin calcium precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 3 times into the obtained crude product of the tea saponin calcium precipitate subjected to primary alcohol washing, adjusting the temperature to 10 ℃, continuously stirring for 1 hour, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain the tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 3 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to be 60 ℃, stirring and dispersing, adjusting the pH of the mixed solution to be 3.2 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to be 10 ℃, stirring for 10 hours at constant temperature, performing centrifugal separation after stirring is finished, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 2.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.2 and the temperature to be 10 ℃, continuing stirring for 1 hour, performing centrifugal separation after stirring, and discarding a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 55 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 0.743 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 0.96/6-16.0%; the obtained high-purity tea saponin product is white powder, and the yield is 0.743/6-12.4%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.7% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 17.4% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the protein extraction rate is 91.0% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.5% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 12.79% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 95.5% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 2

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 100-mesh standard sieve to obtain degreased tea seed meal powder; adding 385kg of ethanol aqueous solution with the ethanol volume percentage content of 85% into 55kg of the obtained degreased tea seed meal, stirring for 11 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition and expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, adjusting the pH value of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.1 by using a hydrochloric acid solution, adjusting the temperature to 70 ℃, continuing stirring and reflux extraction for 1.5 hours, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 85 percent and the mass multiple of 6 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 10 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, using sodium hydroxide solution to enable the pH of the mixture to be 12.5 under the stirring condition, stirring for 2 hours at 80 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 of the mixture and tea seed meal residue d 1; adding softened water with the mass 5.5 times of that of the obtained tea seed dregs d1, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.5, temperature 66 ℃ and stirring time of 1 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.5 by using a hydrochloric acid solution, and continuously stirring for 1 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 4 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.5, the temperature is 45 ℃ and the stirring time is 1 hour, and discarding supernatant containing water-soluble components to obtain the crude product of the washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 70 percent and the mass of 4 times of that of the obtained washed tea seed meal protein precipitate crude product into the water washed tea seed meal protein precipitate crude product, stirring the mixture at room temperature for 1.5 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the tea seed meal protein precipitate crude product subjected to first alcohol washing; adding absolute ethyl alcohol with the mass multiple of 4 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring at room temperature for 1.5 hours, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 50 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 9.35 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 6 by using calcium hydroxide, adjusting the temperature to 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium theasapogenin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 85 percent and the mass multiple of 5 times of the crude product of the tea saponin calcium precipitate into the crude product of the tea saponin calcium precipitate, adjusting the temperature to 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the tea saponin calcium precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 4 times into the obtained crude product of the tea saponin calcium precipitate subjected to primary alcohol washing, adjusting the temperature to 0 ℃, continuously stirring for 1.5 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain the tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 4.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 70 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.1 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 0 ℃, stirring at constant temperature for 15 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 3.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.1 and the temperature to be 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after stirring is finished, and removing a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 50 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 7.16 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 9.35/55-17.0%; the obtained high-purity tea saponin product is white powder, and the yield is 7.16/55-13.0%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 99.0% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 18.3% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the extraction rate of the protein is 92.0% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 99.0% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 13.4% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 96.0% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 3

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 40-mesh standard sieve to obtain degreased tea seed meal powder; adding 90kg of ethanol aqueous solution with the ethanol volume percentage content of 75% into 30kg of the obtained degreased tea seed meal, stirring for 3 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after scrap iron removal for later use;

(2) acidifying and extracting: under the condition of stirring, regulating the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.3 by using a hydrochloric acid solution, regulating the temperature to 40 ℃, continuing stirring and reflux extraction for 0.5 hour, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 75 percent and the mass multiple of 2 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 6 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture with sodium hydroxide solution under the condition of stirring to ensure that the pH of the mixture is 11.5, stirring for 1 hour at 70 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 of the mixture and tea seed meal residue d 1; adding softened water with the mass 2.5 times of that of the obtained tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.1, temperature 55 ℃ and stirring time of 0.5 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.4 by using a hydrochloric acid solution, and continuously stirring for 0.5 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass 2 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.4, the temperature is 35 ℃ and the stirring time is 0.5 hour, and discarding supernatant containing water-soluble components to obtain the crude product of the water-washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 60 percent and the mass of 2 times of that of the obtained washed tea seed meal protein precipitate crude product into the obtained washed tea seed meal protein precipitate crude product, stirring the mixture at room temperature for 0.5 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the tea seed meal protein precipitate crude product subjected to first alcohol washing; adding absolute ethanol with the mass multiple of 2 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring for 0.5 hour at room temperature, performing centrifugal separation after stirring, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 50 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 4.49 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5 by using calcium hydroxide, adjusting the temperature to 20 ℃, continuing stirring for 0.5 hour, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium theasapogenin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 75 percent and the mass multiple of 2 times of the crude product of the tea saponin calcium precipitate into the crude product of the tea saponin calcium precipitate, adjusting the temperature to 20 ℃, continuing stirring for 0.5 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the tea saponin calcium precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 2 times of the crude product of the tea saponin calcium precipitate obtained by the first alcohol washing, adjusting the temperature to 20 ℃, continuously stirring for 0.5 hour, performing centrifugal separation after stirring, removing supernatant, drying the precipitate and removing ethanol to obtain the tea saponin calcium precipitate obtained by the alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 1.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 50 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.3 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 20 ℃, stirring at constant temperature for 15 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 1.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.3 and the temperature to be 20 ℃, continuing stirring for 0.5 hour, performing centrifugal separation after stirring, and removing a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 50 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 3.61 kilograms of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 4.49/30-15.0%; the obtained high-purity tea saponin product is white powder, and the yield is 3.61/30-12.0%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.5% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 16.4% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the extraction rate of the protein is 90.0% by calculating the formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.0% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 12.4% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 95.0% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 4

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 60-mesh standard sieve to obtain degreased tea seed meal powder; adding 780kg of ethanol water solution with the ethanol volume percentage content of 80% into 120kg of the obtained degreased tea seed meal, stirring for 10 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, regulating the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.2 by using a hydrochloric acid solution, regulating the temperature to 65 ℃, continuing stirring and reflux extraction for 1 hour, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of the acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 5.5 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 9.5 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture with sodium hydroxide solution under the condition of stirring to ensure that the pH of the mixture is 12.3, stirring for 1 hour at 75 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 and tea seed meal residue d1 of the mixture; adding softened water with the mass 5 times of that of the tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.4, temperature 60 ℃ and stirring time of 0.75 hours to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.49 by using a hydrochloric acid solution, and continuously stirring for 0.75 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 3.5 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.49, the temperature is 40 ℃ and the stirring time is 0.75 hours, and discarding supernatant containing water-soluble components to obtain the crude product of the water-washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 65 percent and the mass of 3.5 times of that of the obtained washed crude tea seed meal protein precipitate into the water-washed crude tea seed meal protein precipitate, stirring the mixture at room temperature for 1.25 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude tea seed meal protein precipitate subjected to first alcohol washing; adding absolute ethyl alcohol with the mass multiple of 3.5 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring at room temperature for 1.25 hours, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 60 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 20kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.9 by using calcium hydroxide, adjusting the temperature to 5 ℃, continuing stirring for 1.25 hours, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium tea saponin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 4.5 times of the crude product of the calcium theasapogeninate precipitate into the crude product of the calcium theasapogeninate precipitate, adjusting the temperature to 5 ℃, continuing stirring for 1.25 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the calcium theasapogeninate precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 3.5 times into the obtained crude product of the tea saponin calcium precipitate subjected to primary alcohol washing, adjusting the temperature to 5 ℃, continuously stirring for 1.25 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain the tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 4 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 65 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.2 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 5 ℃, stirring at constant temperature for 14 hours, performing centrifugal separation after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 3 times into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.2 and the temperature to be 5 ℃, continuing stirring for 1.25 hours, centrifugally separating after stirring, and discarding a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 55 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 15.35 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 20/120-16.7%; the obtained high-purity tea saponin product is white powder, and the yield is 15.35/120-12.8%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.9% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 18.0% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the extraction rate of the protein is 91.7% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.9% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 13.2% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 95.8% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 5

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 40-mesh standard sieve to obtain degreased tea seed meal powder; adding 344kg of ethanol water solution with 80% ethanol volume percentage content into 86kg of the obtained degreased tea seed meal, stirring for 4 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, regulating the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.2 by using a hydrochloric acid solution, regulating the temperature to 45 ℃, continuing stirring and reflux extraction for 1 hour, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of the acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 3 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 7 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, using sodium hydroxide solution to enable the pH of the mixture to be 12 under the stirring condition, stirring for 1.5 hours at 75 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 of the mixture and tea seed meal residue d 1; adding softened water with the mass 3 times of that of the tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.3, temperature 60 ℃ and stirring time of 1 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.42 by using a hydrochloric acid solution, and continuously stirring for 1 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 4 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.42, the temperature is 40 ℃ and the stirring time is 1 hour, and discarding supernatant containing water-soluble components to obtain the crude product of the washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 65 percent and the mass of 2.5 times of that of the obtained washed tea seed meal protein precipitate crude product, stirring for 1 hour at room temperature, performing centrifugal separation after stirring is finished, and removing supernatant to obtain the tea seed meal protein precipitate crude product washed with alcohol for the first time; adding absolute ethyl alcohol with the mass multiple of 2.5 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring for 1 hour at room temperature, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 55 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 13.15 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.6 by using calcium hydroxide, adjusting the temperature to 15 ℃, continuing stirring for 1 hour, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium theasapogenin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 2.5 times of the crude product of the tea saponin calcium precipitate into the crude product of the tea saponin calcium precipitate, adjusting the temperature to 15 ℃, continuing stirring for 1 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the tea saponin calcium precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 2 times into the obtained crude product of the tea saponin calcium precipitate subjected to primary alcohol washing, adjusting the temperature to 15 ℃, continuously stirring for 1 hour, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain the tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 3.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 55 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.2 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 15 ℃, stirring at constant temperature for 6 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 2 times into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH of the mixed solution to be 3.2 and the temperature to be 15 ℃, continuing stirring for 1 hour, performing centrifugal separation after stirring is finished, and removing a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 55 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 10.41 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 13.15/86-15.3%; the obtained high-purity tea saponin product is white powder, and the yield is 10.41/86-12.1%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.6% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 16.7% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the extraction rate of the protein is 90.5% by calculating the formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.2% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 12.5% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the people's republic of China exit inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 95.3% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 6

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 100-mesh standard sieve to obtain degreased tea seed meal powder; adding 1800kg of ethanol aqueous solution with the ethanol volume percentage content of 85% into 300kg of the obtained degreased tea seed meal, stirring for 9 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after scrap iron removal for later use;

(2) acidifying and extracting: under the condition of stirring, adjusting the pH value of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.1 by using a hydrochloric acid solution, adjusting the temperature to 60 ℃, continuing stirring and reflux extraction for 1.5 hours, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 85 percent and the mass multiple of 5 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 9 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture for 1.75 hours at 80 ℃ by using sodium hydroxide solution under the condition of stirring, and performing centrifugal separation after stirring to obtain an extracting solution c1 and tea seed dregs d1 of the mixture; adding softened water with the mass being 4.5 times of that of the obtained tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH being 11.5, temperature being 65 ℃ and stirring time being 1 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH of the extracting solution of the mixture obtained in the step three to 4.48 by using a hydrochloric acid solution, and continuously stirring for 1 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 4 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.48, the temperature is 45 ℃ and the stirring time is 1 hour, and discarding supernatant containing water-soluble components to obtain the crude product of the washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 70 percent and the mass of 4 times of that of the obtained washed tea seed meal protein precipitate crude product into the water washed tea seed meal protein precipitate crude product, stirring the mixture at room temperature for 1.5 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the tea seed meal protein precipitate crude product subjected to first alcohol washing; adding absolute ethyl alcohol with the mass multiple of 2.5 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring at room temperature for 1.5 hours, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 60 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 49.46 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.8 by using calcium hydroxide, adjusting the temperature to 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium tea saponin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 85 percent and the mass multiple of 4 times of the crude product of the tea saponin calcium precipitate into the crude product of the tea saponin calcium precipitate, adjusting the temperature to 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the tea saponin calcium precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 2 times of the crude product of the tea saponin calcium precipitate obtained by the first alcohol washing, adjusting the temperature to 0 ℃, continuously stirring for 1.5 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate and removing ethanol to obtain the tea saponin calcium precipitate obtained by the alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 4.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 70 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.1 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 0 ℃, stirring at constant temperature for 13 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 3.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.1 and the temperature to be 0 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after stirring is finished, and removing a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 55 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 38.11 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 49.46/300-16.5%; the obtained high-purity tea saponin product is white powder, and the yield is 38.11/300-12.7%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.9% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 17.8% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the protein extraction rate is 91.6% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.7% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 13.1% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006, and calculating the extraction rate of the tea saponin to be 95.8% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

Example 7

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 40-mesh standard sieve to obtain degreased tea seed meal powder; adding 975kg of ethanol aqueous solution with the ethanol volume percentage content of 75% into 195kg of the obtained degreased tea seed meal, stirring for 5 hours at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after scrap iron removal for later use;

(2) acidifying and extracting: under the condition of stirring, regulating the pH value of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.3 by using a hydrochloric acid solution, regulating the temperature to 50 ℃, continuing stirring and reflux extraction for 0.5 hour, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 75 percent and the mass multiple of 3.5 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 8 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture with sodium hydroxide solution under the condition of stirring to ensure that the pH of the mixture is 11.5, stirring for 1 hour at 70 ℃, and performing centrifugal separation after stirring to obtain an extracting solution c1 of the mixture and tea seed meal residue d 1; adding softened water with the mass 5.5 times of that of the obtained tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.4, temperature 55 ℃ and stirring time of 0.5 hour to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to be 4.44 by using a hydrochloric acid solution, and continuously stirring for 0.5 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 4 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.44, the temperature is 35 ℃ and the stirring time is 1 hour, and discarding supernatant containing water-soluble components to obtain the crude product of the water-washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 60 percent and the mass of 2.5 times of that of the obtained washed crude tea seed meal protein precipitate into the water-washed crude tea seed meal protein precipitate, stirring the mixture at room temperature for 0.5 hour, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude tea seed meal protein precipitate subjected to first alcohol washing; adding absolute ethanol with the mass multiple of 2 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring for 0.5 hour at room temperature, performing centrifugal separation after stirring, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 60 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 30.82 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.3 by using calcium hydroxide, adjusting the temperature to 20 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium tea saponin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 75 percent and the mass multiple of 2.5 times of the crude product of the calcium theasapogeninate precipitate into the crude product of the calcium theasapogeninate precipitate, adjusting the temperature to 20 ℃, continuing stirring for 1.5 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the calcium theasapogeninate precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 2 times of the crude product of the tea saponin calcium precipitate obtained by the first alcohol washing, adjusting the temperature to 20 ℃, continuously stirring for 1.5 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate and removing ethanol to obtain the tea saponin calcium precipitate obtained by the alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 4.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to be 60 ℃, stirring and dispersing, adjusting the pH of the mixed solution to be 3.3 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to be 20 ℃, stirring at constant temperature for 7 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 2.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.3 and the temperature to be 20 ℃, continuing stirring for 1 hour, performing centrifugal separation after stirring, and discarding a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 50 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 23.80 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 30.82/195-15.8%; the obtained high-purity tea saponin product is white powder, and the yield is 23.80/195-12.2%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.9% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 17.2% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the protein extraction rate is 91.0% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.3% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 12.6% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006 of the people's republic of China, and calculating the extraction rate of the tea saponin to be 95.4% by a formula extraction rate (product quality x purity)/(raw material quality x raw material tea saponin content).

Example 8

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a 80-mesh standard sieve to obtain degreased tea seed meal powder; adding 2750kg of ethanol aqueous solution with the ethanol volume percentage content of 80% into 500kg of the obtained degreased tea seed meal, stirring for 8 hours at room temperature, removing scrap iron in the degreased tea seed meal after sufficient liquid absorption and expansion by using an iron remover after stirring is finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(2) acidifying and extracting: under the condition of stirring, adjusting the pH of the ethanol soaking mixture of the degreased tea seed meal powder without iron filings obtained in the step to 3.2 by using a hydrochloric acid solution, adjusting the temperature to 65 ℃, continuing stirring and reflux extraction for 1.25 hours, and performing centrifugal separation after extraction to obtain ethanol extracting solution a1 and precipitate b1 of acid tea saponin; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 4.5 times of the obtained precipitate b1, and respectively carrying out secondary reflux extraction and tertiary reflux extraction according to the same method of the primary reflux extraction to respectively obtain ethanol extract a2 of the acid tea saponin, ethanol extract a3 of the acid tea saponin and precipitate 3; mixing the ethanol extract a1 of the acid tea saponin, the ethanol extract a2 of the acid tea saponin and the ethanol extract a3 of the acid tea saponin to obtain an ethanol extract of the acid tea saponin for later use;

(3) alkaline wall breaking and extraction: adding softened water with the mass multiple of 8.5 times into the precipitate 3 obtained in the second step, stirring and dispersing uniformly, stirring the mixture for 1 hour at 75 ℃ by using sodium hydroxide solution under the condition of stirring, and performing centrifugal separation after stirring to obtain an extracting solution c1 and tea seed meal residue d1 of the mixture; adding softened water with the mass 5.5 times of that of the obtained tea seed dregs d1 into the mixture, respectively carrying out second extraction and third extraction and centrifugal separation under the conditions of pH 11.4, temperature 60 ℃ and stirring time of 0.75 hours to respectively obtain a mixture extracting solution c2, a mixture extracting solution 3 and a separated tea seed dregs d3, and mixing the mixture extracting solution c1, the mixture extracting solution c2 and the mixture extracting solution 3 to obtain a mixture extracting solution for later use;

(4) isoelectric point precipitation, secondary alcohol extraction and drying

A. Isoelectric precipitation and water washing: under the condition of stirring, regulating the pH value of the extracting solution of the mixture obtained in the step three to 4.47 by using a hydrochloric acid solution, and continuously stirring for 0.75 hour to obtain a crude tea seed meal protein precipitate; adding softened water with the mass being 4 times of that of the crude product of the tea seed meal protein precipitate into the crude product of the tea seed meal protein precipitate, respectively carrying out first and second water washing and centrifugal separation under the conditions that the pH is 4.47, the temperature is 40 ℃ and the stirring time is 0.75 hours, and discarding supernatant containing water-soluble components to obtain the crude product of the water-washed tea seed meal protein precipitate;

B. secondary alcohol extraction and drying: adding an ethanol water solution with the ethanol volume percentage content of 65 percent and the mass of 4 times of that of the obtained washed tea seed meal protein precipitate crude product into the water washed tea seed meal protein precipitate crude product, stirring the mixture at room temperature for 1.25 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the tea seed meal protein precipitate crude product subjected to first alcohol washing; adding absolute ethanol with the mass multiple of 4 times into the obtained crude tea seed meal protein precipitate subjected to primary alcohol washing, stirring at room temperature for 1.25 hours, performing centrifugal separation after stirring is finished, and removing supernatant to obtain tea seed meal protein precipitate subjected to detoxification, decolorization, deastringency and impurity removal; drying the detoxified, decolored, deastringent and impurity-removed tea seed meal protein precipitate at 60 ℃ by using a vacuum drying method until the percentage content of water is less than or equal to 5%, crushing, quantitatively packaging and sealing to obtain 83.9 kg of white high-purity tea seed meal protein product;

(5) precipitating with calcium, washing and drying

A. Calcium precipitation and alcohol washing: under the condition of stirring, adjusting the pH value of the ethanol extract of the acid tea saponin obtained in the second step to 5.7 by using calcium hydroxide, adjusting the temperature to 5 ℃, continuing stirring for 1.25 hours, performing centrifugal separation after stirring, and removing supernatant to obtain a crude product of calcium tea saponin precipitate; adding an ethanol water solution with the ethanol volume percentage content of 80 percent and the mass multiple of 4.5 times of the crude product of the calcium theasapogeninate precipitate into the crude product of the calcium theasapogeninate precipitate, adjusting the temperature to 5 ℃, continuing stirring for 1.25 hours, performing centrifugal separation after the stirring is finished, and removing supernatant to obtain the crude product of the calcium theasapogeninate precipitate subjected to primary alcohol washing; adding absolute ethanol with the mass multiple of 3.5 times into the obtained crude product of the tea saponin calcium precipitate subjected to primary alcohol washing, adjusting the temperature to 5 ℃, continuously stirring for 1.25 hours, performing centrifugal separation after stirring, removing supernatant, drying the precipitate, and removing ethanol to obtain the tea saponin calcium precipitate subjected to alcohol washing;

B. acidifying, washing and drying: adding softened water with the mass multiple of 4.5 times into the obtained alcohol-washed tea saponin calcium precipitate, adjusting the temperature to 65 ℃, stirring and dispersing, adjusting the pH of the mixed solution to 3.1 by using a hydrochloric acid solution under the condition of stirring, adjusting the temperature of the mixture to 5 ℃, stirring at constant temperature for 12 hours, centrifugally separating after stirring, and removing the supernatant containing water-soluble impurities to obtain an acid tea saponin precipitate crude product; adding softened water with the mass multiple of 3.5 times of the crude product of the acid form tea saponin precipitate into the crude product of the acid form tea saponin precipitate, stirring and dispersing, controlling the pH value of the mixed solution to be 3.1 and the temperature to be 5 ℃, continuing stirring for 1.25 hours, performing centrifugal separation after stirring, and removing a washing solution containing water-soluble impurities to obtain the acid form tea saponin precipitate; drying the acid tea saponin precipitate by a vacuum drying method at 55 ℃ until the percentage content of water is less than or equal to 5 percent, crushing, quantitatively packaging and sealing to obtain 62.51 kg of white high-purity tea saponin products.

The obtained high-purity tea seed meal protein product is white powder, and the yield is 83.9/500-16.8%; the obtained high-purity tea saponin product is white powder, and the yield is 62.51/500-12.5%.

The protein purity (conversion coefficient 6.25) of the obtained high-purity tea seed meal protein product is 98.6% by adopting a first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, the protein content (conversion coefficient 6.25) in the used tea seed meal is 18.1% by adopting the first Kjeldahl method in the determination of the protein in the food of national standard GB/T5009.5-2003, and the protein extraction rate is 91.4% by calculating the formula extraction rate (product quality x purity)/(raw material quality x raw material protein content).

The method comprises the steps of determining the purity of the tea saponin product to be 98.9% by adopting an acid hydrolysis-acetone Soxhlet extraction-constant weight method in the chemical industry standard HG/T4492-2013 of the people's republic of China natural nonionic surfactant tea saponin, determining the content of the tea saponin in the used tea seed meal to be 13.0% by adopting an 80% ethanol extraction method in the national standard GB/T35131-2017 of the people's republic of China tea seed cake and meal and an acid-base hydrolysis method in the determination of the saponin content in the tea saponin in the export of the inspection and quarantine industry standard SN/T1852-2006 of the people's republic of China, and calculating the extraction rate of the tea saponin to be 95.2% by a formula extraction rate (product quality multiplied by purity)/(raw material quality multiplied by raw material tea saponin content).

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A high-purity tea seed meal protein and tea saponin and a preparation method thereof are characterized by comprising the following steps:

(1) crushing, sieving, soaking and removing iron: crushing the degreased tea seed meal by using a stainless steel crusher and sieving the degreased tea seed meal by using a standard sieve of 40-100 meshes to obtain degreased tea seed meal; adding an ethanol aqueous solution into the obtained degreased tea seed meal, stirring and soaking at room temperature, removing scrap iron in the degreased tea seed meal after full imbibition and expansion by using an iron remover after stirring and soaking are finished, and obtaining an ethanol soaking mixture of the degreased tea seed meal after the scrap iron is removed for later use;

(4) Isoelectric point precipitation, secondary alcohol extraction and drying

(5) Precipitating with calcium, washing and drying

2. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the degreasing tea seed meal is squeezed tea seed meal obtained after tea seed oil is squeezed and extracted through a squeezing method, and the tea seed meal with the oil content less than or equal to 1.0% is obtained after residual oil in the squeezed tea seed meal is removed through a leaching method; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the iron remover is used in food industry or chemical industry.

3. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the mass ratio of the degreased tea seed meal to an ethanol aqueous solution is 1:3-7, and the volume percentage content of ethanol in the ethanol aqueous solution is 75-85%; the stirring and soaking time is 3-11 hours.

4. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the hydrochloric acid is industrial grade, chemically pure or analytically pure hydrochloric acid; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the mass ratio of the precipitate b1 to the ethanol water solution is 1:2-6, and the ethanol volume percentage content in the ethanol water solution is 75-85%; the temperature of the reflux extraction is 40-70 ℃, and the time is 0.5-1.5 hours.

5. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the sodium hydroxide in the step three is industrial-grade, chemically pure or analytically pure sodium hydroxide; the mass ratio of the precipitate b3 to the softened water is 1: 6-10; the mass ratio of the tea seed meal residue d1 to the softened water is 1: 2.5-5.5, and the conditions of the second extraction and the third extraction are as follows: the pH value of the mixed solution is 11.1-11.5, the extraction temperature is 55-65 ℃, and the extraction stirring time is 0.5-1.0 hour.

6. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: step four, the hydrochloric acid is industrial grade, chemically pure or analytically pure hydrochloric acid; the ethanol is industrial grade, food grade, chemically pure or analytically pure ethanol; the drying method is a vacuum drying method or a normal pressure hot air drying method.

7. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: in the step A, the mass ratio of the tea seed meal protein precipitate crude product to the softened water is 1: 2-4; the pH value of the mixed solution during washing is 4.4-4.5, the washing temperature is 35-45 ℃, and the washing stirring time is 0.5-1.0 hour; and B, the mass ratio of the tea seed meal protein precipitate crude product washed in the step B to the ethanol water solution is 1:2-4, the volume percentage of ethanol in the ethanol water solution is 60-70%, and the mass ratio of the crude tea seed meal protein precipitate obtained by the first ethanol washing to the absolute ethanol is 1: 2-4.

8. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the calcium hydroxide in the step fifthly is industrial-grade, chemically pure or analytically pure calcium hydroxide; the ethanol is industrial grade or food grade, chemically pure or analytically pure ethanol; the hydrochloric acid is industrial grade, chemically pure or analytically pure hydrochloric acid; the drying method is a vacuum drying method or a normal pressure hot air drying method.

9. The method for preparing high purity tea seed meal protein and tea saponin according to claim 1, wherein the method comprises the following steps: the mass ratio of the crude product of calcium theasapogeninate precipitate to the aqueous ethanol solution in the step A is 1: 2-5; the volume percentage of ethanol in the ethanol water solution is 75-85%, and the mass ratio of the crude product of the calcium theasapogeninate precipitate obtained by the first ethanol washing to the absolute ethanol is 1: 2-4; the mass ratio of the tea saponin calcium precipitate washed by alcohol in the step B to the softened water is 1: 1.5-4.5; the mass ratio of the acid tea saponin precipitate crude product to the softened water is 1: 1.5-3.5.

10. A high purity tea seed meal protein and tea saponin prepared by the preparation method according to any one of claims 1-9.