CN117843711A

CN117843711A - High-performance leaf protein and preparation method thereof

Info

Publication number: CN117843711A
Application number: CN202311838540.2A
Authority: CN
Inventors: 缪铭; 冯文娟; 黄文选; 李赟高; 黄志超; 高思聪
Original assignee: Qinhuangdao Senyuan Investment Group Co ltd; Jiangnan University
Current assignee: Qinhuangdao Senyuan Investment Group Co ltd; Jiangnan University
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-04-09

Abstract

The invention discloses high-performance leaf protein and a preparation method thereof, and belongs to the technical field of deep processing of plant leaf protein. The invention adopts a wet method to crush leaves, destroy the connecting protein among veins, mesophylls and leaf epidermis, open the barrier for wrapping the protein, carry out ultrasonic alcohol washing and stirring on grass slag with the protein accounting for 90 percent, carry out enzymolysis by using cellulase and pectase, carry out ultrafiltration and nanofiltration to concentrate enzymolysis liquid for desalination and desugarization, and dry to obtain the leaf protein. The preparation method provided by the invention has the advantages that the yield of the leaf protein reaches more than 76%, the purity reaches more than 86.5%, and meanwhile, the essential amino acid content is high, so that the leaf protein has better solubility, emulsifying property and foamability.

Description

High-performance leaf protein and preparation method thereof

Technical Field

The invention relates to high-performance leaf protein and a preparation method thereof, and belongs to the field of plant leaf protein deep processing technology research.

Background

Plant leaves contain 1.2% -8.2% (average about 3.5% fresh weight) of protein, and the use of leaf protein as human food appears to be a useful alternative in the face of food safety and sustainability issues. The phyllanthus niruri is a new plant variety hybridized with Rumex k-1 Rumex and Rumex, has the advantages of perennial growth, long service life, high yield, strong adaptability and the like in the field of livestock raising, and is a new food raw material at present.

The proteins in the green leaves mainly comprise lipoproteins (membrane bodies), structural proteins (mainly chloroplasts and mitochondria), photoactive pigment binding proteins and enzymes, the soluble proteins mainly comprise enzymes and cytoplasmic proteins, the proteins account for 55% -65% of the total proteins, and the structural proteins account for 35% -45%. The existing extraction methods suitable for large-scale extraction of leaf proteins mainly comprise a heating method (direct or fractional), an acid precipitation method (or heating first and then acid precipitation), an alkali precipitation method, a microbial fermentation method, an alkali dissolution and acid precipitation method and the like.

Wherein, the direct heating method is usually heating at about 80-90 ℃, the extraction rate and purity are lower (respectively 3.8-33.1%, 49.0-53.2%), and green protein is precipitated, so the sample is greenish in color and has grass fishy smell; higher temperatures also often denature proteins, decreasing solubility, with a concomitant decrease in other properties, ultimately limiting the range of applications of the product in the food field. The fractional thermal precipitation precipitates the green proteins first by low temperature and then precipitates the albumin at high temperature, and although the purity of the albumin may be improved (40.5% -88.7%), the yield of the albumin is low (24.5% -40.4%), and part of the albumin is precipitated while precipitating the green proteins. The acid precipitation method and the alkali precipitation method are respectively suitable for alkaline leaves and acidic leaves, the extraction rate is 26.2-58.2%, the purity is 51.5-63.7%, and the product may have unpleasant odor left by the type of acid and alkali adopted, and meanwhile, the color of the product is dark. The microbial fermentation method generally involves natural fermentation and introduction of exogenous bacteria for fermentation, so that the feed liquid reaches a certain acidity and automatic precipitation (pH 3.6-4.5), and the yield and purity can reach 29.86% -32.5%/39.0% -57.19%, respectively. This method has a great problem in that it is required to prevent some harmful substances from being generated by some unfavorable growth of mixed bacteria, which significantly increases the investment of production equipment and detection equipment. The yield and the extraction rate of the alkali-soluble acid precipitation method are 6.8% -57.87%/9.4% -87.38%, a large amount of acid and alkali are consumed in the large-scale production, the combination of polyphenol and protein is promoted in the alkali-soluble process, brown substances are generated, and the color of the final product is not facilitated. While alkali treatment typically reduces the digestibility of the protein (up to 40%).

Patent CN1943391B discloses the use of a thermo-coagulation method, ultrafiltration method to isolate proteins; patent CN102334586B discloses the use of isoelectric precipitation and acetone decolorization, but the problem of residual organic solvent in this technique is serious; patent CN102302083B discloses the extraction of tea proteins by an alkaline method, which also requires decolorization with hydrogen peroxide, followed by precipitation with hydrochloric acid. The patent CN107712525A extracts the method of the protein and beverage made of the protein, disclose juice, and bleach and enzymolysis to juice and residue mixture, get the method of the protein through centrifugal dialysis, this technology has steps are loaded down with trivial details, there are potential safety hazards to use reagent, etc.; patent CN112956591a discloses the extraction of vegetable proteins and the manufacture of crude protein feeds by beating to 80 mesh chips, using food grade NaOH solution and H ₂ O ₂ The protein powder is obtained by decoloring and drying the solution, and the technology has the problems of easy oxidization and high cost.

Therefore, the interaction between the leaf protein and the monosaccharide, the polysaccharide, the fiber and the lipid is further destroyed by adopting a mild means on the basis of crushing, the soluble protein is released, the protein in a dissolved state is ensured to have better color under the conditions of non-high temperature and non-high acid-base concentration induction, and the development of the method for obtaining the protein with low cost, low pollution, high yield, high purity and higher physicochemical property has great economic benefit and practical value.

Disclosure of Invention

Aiming at the problems, the invention provides the high-quality food She Caoshe protein and the preparation method thereof, which have mild temperature and pH conditions, protect the original structure of the protein, maintain the high-solubility oil drop model characteristics that the polar amino acid residues of the protein are positioned on the surface of the protein and the nonpolar hydrophobic side chains are buried in the molecules, and effectively solve the problems of high energy consumption, high acid and alkali consumption and poor protein properties and appearance quality of the current extraction method. In addition, the invention avoids the problems of low protein precipitation rate and low final purity caused by acid induction and thermal induction by adding a desalting concentration step.

The invention adopts a wet grinder to primarily crush at the crushing stage, the connection of the epidermis, mesophyll and vein is destroyed, the cell wall structure in mesophyll cells is primarily destroyed, and the grain size of grass slag is controlled to be 40-60 meshes. At this time, more than 95% of leaf proteins are still present in the grass residue (filter residue), and the remaining 5% are in the grass juice (filtrate). Therefore, the invention abandons the mode of taking grass juice (40% -75% of total leaf protein) and hay powder as raw materials in the conventional leaf protein production, adopts wet grass residues as raw materials for production, reduces the weight of a treatment system (leaf water content of 90%) compared with the direct use of grass pulp, and simultaneously avoids the interference caused by organic acid, other polyphenols and other substances in the grass juice during the subsequent enzyme treatment. The ultrasonic alcohol washing process effectively damages the cell wall and cell membrane structure, dissolves out micromolecular pigment, and pre-damages the chloroplast structure with the largest stored protein, thereby being beneficial to the dissolution of macromolecular protein during enzymolysis during further treatment. The ethanol can be recycled as the decolorized solution; the grass juice rich in polyphenol can be further processed as a byproduct, and clear liquid obtained in the ultrafiltration process can be mixed with subsequent treatment liquid to achieve the effect of recycling.

The first object of the present invention is a process for the preparation of leaf proteins, comprising the steps of:

s1, pretreatment of raw materials: crushing the grass of the leaf-eating grass to 40-60 meshes, adding 0.3-0.9% w/v sodium sulfite and 0.1-0.4% w/v crosslinked polyvinylpyrrolidone, and centrifuging to obtain grass residues;

s2, ultrasonic alcohol washing: adding ethanol and inorganic acid into the grass residue obtained in the step S1 to carry out ultrasonic stirring, wherein the ultrasonic power is 100-300 w, and the ultrasonic time is 1-2 h; centrifugally separating grass slag and alcohol washing liquid after ultrasonic treatment, drying and crushing the grass slag washed by alcohol to obtain grass slag powder;

s3, enzyme membrane reaction: putting the grass slag powder obtained in the step S2 into a buffer solution system, wherein the liquid-to-material ratio is 1: 30-60, adding cellulase and pectase, stirring uniformly, and carrying out enzymolysis for 10-16 hours; filtering the enzymolysis liquid by using an ultrafiltration membrane, and collecting a permeate;

s4, nanofiltration: filtering and concentrating the permeate liquid obtained in the step S3 by adopting a ceramic nanofiltration membrane to obtain concentrated liquid;

s5, drying: and (3) drying the concentrated solution obtained in the step (S4) to obtain the leaf protein powder.

In one embodiment, the fineness of the grass slag powder after crushing in S is 100 to 150 meshes.

In one embodiment, the concentration of the inorganic acid in S2 is 6M-10M, and the addition amount is 2% -5% v/v.

In one embodiment, the concentration of ethanol in S2 is 80% -90%, and the liquid-to-material ratio of grass slag to ethanol is 1:6 to 10.

In one embodiment, the type of inorganic acid in S2 may be formic acid, acetic acid, phosphoric acid, hydrochloric acid, or the like.

In one embodiment, the ultrasound temperature in S2 is 40-60 ℃.

In one embodiment, the drying conditions in S2 are hot air drying at a temperature of 30 to 60 ℃.

In one embodiment, the ratio of the grass meal to the buffer in S3 is 1:40 to 60.

In one embodiment, the enzymolysis in S3 is performed in a reaction tank, the enzymolysis reaction temperature is 50-70 ℃, the circulation flow is 10-30L/h, and the permeate is collected every 4-6 h.

In one embodiment, the dosage of the cellulase in the S3 is 30U-60U/g substrate, the dosage of the pectase is 10U-30U/g substrate, and the stirring speed in the enzymolysis process is 200-300 r/min.

In one embodiment, the phosphate buffer in S3 is phosphate buffer, tris-hcl buffer.

In one embodiment, the ultrafiltration membrane in S3 has a molecular weight cut-off of 5-20 Kda and a membrane pressure of 0.1-0.6 MPa.

In one embodiment, the nanofiltration membrane in S4 has a molecular weight cut-off of 300-500 Da and a membrane pressure of 0.6-1.0 MPa.

In one embodiment, the drying in S5 may be vacuum drying or vacuum freeze drying.

In one embodiment, the method of preparing a leaf protein comprises the steps of:

s1, pretreatment of raw materials: pulverizing fresh leaves, adding 0.3-0.6% of sodium sulfite and 0.2-0.4% of crosslinked polyvinylpyrrolidone before pulverizing, centrifuging for 15-30 min to obtain grass residues;

s2, ultrasonic alcohol washing: and (3) mixing the grass slag liquid obtained in the step (S1) in a ratio of 1: 6-8 adding 80-90% ethanol and 2-5% v/v 6M-10M hydrochloric acid to carry out ultrasonic treatment at 45-60 ℃ for 1-2 h with the ultrasonic power of 100-300W; centrifuging for 15-30 min at 5000-7000 r/min after ultrasonic treatment to separate grass slag and alcohol washing liquid, drying the grass slag with hot air at 30-50 ℃, and crushing the grass slag and sieving the crushed grass slag with a 100-150 mesh sieve to obtain grass slag powder;

s3, enzyme membrane reaction: putting the grass slag powder obtained in the step S2 into a phosphate buffer solution or a Tris-hydrochloric acid buffer solution system, wherein the liquid-to-material ratio is 1: 40-60, adding 30-50U/g substrate cellulase and 10-20U/g substrate pectase, stirring uniformly, and performing enzymolysis for 8-20 h; the enzyme reaction temperature is 50-70 ℃, the enzymolysis liquid is filtered by a ceramic ultrafiltration membrane with the molecular weight of 5-15 Kda, the ultrafiltration pressure is 0.1-0.5 MPa, the enzymolysis liquid circulates at the flow rate of 10-20L/h, and the permeate liquid is collected every 4-5 h;

s4, nanofiltration: filtering and concentrating the permeate liquid obtained in the step S3 by adopting a ceramic nanofiltration membrane with the pressure of 400-500 Da, wherein the membrane pressure is 1.0-1.5 MPa, so as to obtain concentrated liquid;

s5, drying: and (3) carrying out vacuum drying or freeze drying on the concentrated solution obtained in the step (S4) to obtain the leaf protein powder.

The second object of the invention is to provide a leaf protein, which is prepared by any one of the above methods.

A third object of the present invention is to provide the use of the leaf protein described above for the preparation of a food product.

In one embodiment, the leaf protein described above may act as an emulsifier or a foaming agent.

In one embodiment, the leaf proteins described above can be used to prepare bread, beverages, and the like.

Advantageous effects

Compared with grass juice and grass powder used in the conventional production of leaf proteins, the invention adopts a wet method to crush leaves, destroy connecting proteins among veins, mesophylls and leaf epidermis, open a barrier for wrapping the proteins, carry out ultrasonic alcohol washing and stirring on grass residues with the protein accounting for 90%, carry out enzymolysis by using cellulase and pectase, carry out ultrafiltration and nanofiltration, concentrate enzymolysis liquid for desalination and desugaring, and dry to obtain the leaf proteins.

The leaf protein preparation method provided by the invention avoids the influence of organic acid, phenols and the like in juice on the subsequent extraction process of protein; the permeability of cell membranes is changed in the ultrasonic alcohol washing process, so that small pigment molecules are promoted to be rapidly dissolved, more than 90% of pigments in weeding residues can be removed, the loss caused by large-scale dissolution of macromolecular proteins is avoided in the operation process, and meanwhile, compared with other decolorization modes (strong oxidants), ethanol is adopted for decolorization, the method is milder; ethanol can be recovered as a decolorized solution; the grass juice rich in polyphenol can be further processed as a byproduct, and clear liquid obtained in the ultrafiltration process can be mixed with subsequent treatment liquid to achieve the effect of improving the yield. The invention ensures the whiteness and purity of the leaf protein product to the maximum extent in an economic and effective mode, the yield reaches more than 75 percent, and the purity reaches more than 84.5 percent.

Specifically, the leaf protein prepared by the invention has the following advantages:

(1) The yield of the leaf protein reaches 76% at maximum, and the purity is 86.5%;

(2) The leaf protein contains 18 amino acids, and the ratio of essential amino acids reaches 40.28%;

(3) The appearance quality is similar to that of commercial vegetable protein soy protein and pea protein;

(4) The aqueous solution has higher solubility under different pH values, the solubility under the acidic condition (pH 4.5) is 12 percent, the solubility under the neutral condition (pH 6.5) is 97 percent, and the solubility under the alkaline condition (pH 8.5) is 100 percent.

(5) Has excellent emulsifying property and emulsifying stability under different pH values, and has emulsifying capacity of 14m under acidic condition (pH 4.5) ² /g, 29m under neutral conditions (pH 6.5) ² /g, 40m under alkaline conditions (pH 8.5) ² /g；

(6) Excellent foamability and foamability stability at various pH values, foamability of 110% under acidic conditions (pH 4.5), foamability of 140% under neutral conditions (pH 6.5), and foamability of 147% under alkaline conditions (pH 8.5);

(7) The average particle size is small, the average particle size under acidic condition (pH 4.5) is 69.7nm, the average particle size under neutral condition (pH 6.5) is 84.1nm, and the average particle size under alkaline condition (pH 8.5) is 93.7nm;

(8) Has good digestion characteristics, and the digestion rate reaches 88.4 percent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a comparison of the appearance of the inventive leaf protein with several commercial proteins (RPI for leaf protein, SPI for soy protein isolate, PPI for pea protein isolate, RPI for whey protein isolate, EPI for egg white protein, naCas for sodium caseinate; supra);

FIG. 2 shows the molecular weight of leaf proteins, indicated by the left column as leaf protein RPI, and the right column as Marker;

FIG. 3 is a graph of the solubility of a leaf protein of the present invention and several commercial proteins;

FIG. 4 is a graph of emulsifying capacity and emulsion stability of the leaf proteins of the present invention and several commercial proteins;

FIG. 5 is a graph of foaming capacity and foaming stability of the leaf proteins of the present invention and several commercial proteins;

FIG. 6 is a graph of average particle size of leaf proteins of the present invention and several commercial proteins.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

Protein detection method in the examples:

1. the protein content is detected by adopting a national standard GB/T6432-2018 Kjeldahl nitrogen determination method so as to represent the purity of the protein.

2. Calculation of protein yield

Extraction yield of crude protein= (content of crude protein in leaf protein/content of protein in fresh leaf) ×100%.

3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed under reducing conditions, and protein samples of 2mg/mL were prepared according to 1:4, adding protein loading buffer solution in proportion, uniformly mixing, boiling the mixture for 5 minutes, and centrifuging at 10000r/min for 5 minutes. Thereafter, 10. Mu.L of the supernatant and 10. Mu.L of trichromatic pre-stained standard protein were loaded in different lanes on a gel prepared by a Shanghai subunit enzyme Biotechnology Co., ltd (Shanghai China) PAGE gel rapid preparation kit (4% -15%). Electrophoresis was performed at 200V for about 1 hour, then treated with a staining solution (0.1% coomassie brilliant blue R250, 25% isopropyl alcohol and 10% glacial acetic acid) for about 1 hour, and decolorized in a decolorization solution (10% acetic acid solution, 5% ethanol) until protein bands were clear.

4. Determination of protein amino acid composition

And determining the amino acid content of various proteins by adopting an OPA pre-column derivatization reverse-phase high performance liquid chromatography-ultraviolet detection method.

OPA pre-column derivatization reversed-phase high performance liquid chromatography analysis: mobile phase a (ph=7.2): 27.6mmol/L sodium acetate-triethylamine-tetrahydrofuran (volume ratio 500:0.11:2.5); mobile phase B (ph=7.2): 80.9mmol/L sodium acetate-methanol-acetonitrile (volume ratio 1:2:2); agilent Hy.persil ODS column (5 μm,4.0 mm. Times.250 mm) was eluted with a gradient, the elution procedure being: 0min,8% b;17min,50% B;20.1min,100% b;24.0min,0%

B. The flow rate of the mobile phase was 1.0mL/min and the column temperature was 40 ℃. The ultraviolet detector (VWD) detects 338nm in wavelength and proline at 262 nm. The amino acid content is quantified by an external standard method.

5. Determination of protein colour

The color properties of the protein samples were determined using a colorimeter (CR-400,Konica Minolta Sensing,Inc., osaka, japan). L (brightness), a (red) and b (yellow) are used to describe color characteristics.

6. Determination of protein emulsifying ability and emulsion stability

A1% strength protein sample solution was adjusted to the corresponding pH (4.5, 6.5, 8.5) with 0.1mol/L NaOH/HCl. Placing at different temperatures (10deg.C, 25deg.C, 40 deg.C), adding 6mL of protein solution, adding 2mL of soybean oil, and stirring at 10000r/min for 2min. After 10s and 30min of rest, respectively, the lower 50uL of emulsion was removed, 5mL of 0.1% SDS solution was added, and after thoroughly mixing, the absorbance was measured at 500nm, using SDS solution as a blank. The calculation formulas of the emulsifying capacity and the emulsifying stability are as follows:

emulsion stability=a ₃₀ /A ₀

Wherein A is ₀ Absorbance at rest for 30 s; a is that ₃₀ Absorbance at rest for 30 min; c is the protein concentration;is the volume fraction of oil.

7. Determination of protein foamability and foamability stability

The 1% strength sample solution was adjusted to a range of pH values (4.5, 6.5, 8.5) with 0.1mol/L NaOH/HCl, placed at various temperatures (10 ℃, 25 ℃,40 ℃) and stirred at 10000r/min for 1min. After 0 and 30min, respectively, the total volume was determined. Foaming ability and foam stability calculation formula:

foaming Capacity= (A) _0min －B)/B×100％

Foam stability= (a _30min －B)/(A _0min －B)×100％

Wherein A is _0min 、A _30min Total volume at rest 0, 30min after stirring: b is the liquid volume before stirring.

8. Measurement of protein particle size

The particle size distribution of the protein was determined using a Nano Brook Omni instrument. The 3mg/mL protein solution was adjusted to different pH values (4.5, 6.5, 8.5) with 1M HCl and NaOH. The test was performed at a temperature of 25℃and the data was collected three times at a rate of 60 seconds per cycle.

9. Determination of in vitro digestion Properties of proteins

In vitro gastrointestinal digestion protocols were performed following standard procedures "information on gastrointestinal food digestion regulates static in vitro simulation".

Specifically, 1g of the sample was mixed with a simulated saliva solution (pH 7, 75U/mL alpha-amylase, 1.5mM NaCl ₂ ) 1, the method comprises the following steps: mixing at 37deg.C for 2min at a ratio of 1 w/w. Gastric juice (pH 3.0, 2000U/m L pepsin, 0.5mM CaCl) ₂ ) 1, the method comprises the following steps: the reaction was completed after 2 hours at a 1v/v ratio. The artificial intestinal juice enzymatic hydrolysis process was simulated, and intestinal phase solution (pH 7, containing 100U/mL pancreatin, 10mM oxgall, containing 0.6mM CaCL) was added to chyme at 1:1v/v to adjust pH 7.0, and the reaction was completed after 2 hours at 37 ℃. Inactivating enzyme at 90deg.C and heating for 15min. The sample was then centrifuged at 7000r/min for 30min at 4℃and the supernatant was collected to determine the protein content.

Digestibility = (protein content in digested supernatant/protein content in raw material liquid) ×100%

The protein content was determined using Kjeldahl nitrogen, consistent with 1.

10. The raw materials used in the examples:

cellulase: from Trichoderma reesei (Trichoderma reesi) with an enzyme activity of 200000U/g, available from Shandong Dabio-engineering Co., ltd;

pectase: from Aspergillus niger (Aspergillus niger), enzyme activity 60000U/g, available from Shandong Dacron bioengineering Co., ltd;

alpha-amylase: enzyme activity 1000000U/g, sigma Aldrich, trade Limited (Shanghai);

pepsin: enzyme activity 2500000U/g, sigma Aldrich, trade Co., ltd;

trypsin: the enzyme activity is 2000000U/g, sigma Aldrich (Shanghai) trade company;

trichromatic pre-dye protein Marker 10 kDa-250 kDa: product number: WJ103L, shanghai enzyme biomedical technologies Co., ltd;

whey protein isolate: the protein content is Roquette freres (france);

ovalbumin: the protein content is more than or equal to 80, and is a national medicine group chemical reagent company;

soy protein isolate: the protein content is 85%, and Shandong Yuxin Biotech Co., ltd;

pea protein isolate: protein content 85%, roquette freres (france);

sodium caseinate: the protein content was not less than 92.5, which was found by tokyo chemical industry Co.

Example 1: leaf protein preparation

Step S1, raw material pretreatment: taking phyllanthus niruri as a leaf protein extraction raw material, finely crushing phyllanthus niruri by using a wet-process crusher until the particle size reaches 40-60 meshes, adding 0.6% w/v of sodium sulfite and 0.2% w/v of crosslinked polyvinylpyrrolidone (PVPP), performing 6000r/min, and centrifuging for 30min to separate grass residues and grass juice;

step S2, ultrasonic alcohol washing: and (3) mixing the grass slag obtained in the step (S1) in a liquid-to-material ratio of 1: adding ethanol with the volume fraction of 90% for ultrasonic-assisted decoloring, wherein the ultrasonic condition is 50 ℃, the ultrasonic treatment is carried out for 2 hours, the power is 300W, meanwhile, 6M hydrochloric acid with the power of 2% v/v is added, centrifuging for 15-30 min at 5000-7000 r/min after ultrasonic treatment, separating grass slag and alcohol washing liquid, drying with hot air at 30-50 ℃, and crushing the dried grass slag to 100 meshes to obtain grass slag powder;

step S3, enzyme membrane reaction: putting the grass slag powder obtained in the step S2 into a phosphate buffer solution system with the pH value of 7.0, wherein the feed-liquid ratio is 1:50, adding cellulase into a reaction tank according to the addition amount of 50U/g substrate and pectase according to the addition amount of 20U/g substrate, and uniformly stirring, wherein the enzymolysis time is 10 hours; the molecular weight cut-off of the ceramic ultrafiltration membrane is 10Kda, the ultrafiltration pressure is 0.2MPa, enzymolysis liquid circulates at a flow rate of 20L/h, permeate liquid is collected every 6h, the cut-off liquid continuously reacts, and the enzymolysis temperature is controlled at 60 ℃ in the reaction process;

step S4, nanofiltration desugaring and desalting: performing desugarization treatment on the clear liquid obtained in the step S3 by adopting a ceramic nanofiltration membrane, wherein the size of the membrane is 500Da, and the pressure of the membrane is 1.0MPa;

step S5, drying: and (3) carrying out vacuum drying on the concentrated solution obtained in the step (S4) to obtain high-quality leaf protein powder.

The high quality leaf protein in this example was 75.0% pure and 85.2% by kjeldahl method.

Example 2: leaf protein preparation

On the basis of example 1, the addition amount of sodium sulfite in step S1 was changed to 0.3% w/v; s2, the ultrasonic power is 200W; and S3, the liquid-material ratio is 1:60, the enzymolysis time is 12h, and the leaf protein is prepared.

The high quality leaf protein in this example was detected to have a yield of 75.7% and a purity of 84.5%.

Example 3: leaf protein preparation

Based on the example 1, changing the adding amount of the crosslinked polyvinylpyrrolidone in the S1 to 0.4% w/v, and crushing the crosslinked polyvinylpyrrolidone in the S2 to 150 meshes; and S3, the liquid-material ratio is 1:40, enzymolysis time is 16h, and the leaf protein is prepared.

The high quality leaf protein in this example was detected to have a yield of 76% and a purity of 86.5%.

Example 4: leaf protein performance assay

The leaf proteins prepared in examples 1 to 3 have smaller composition and structure differences, are essentially the same, and the proteins prepared in example 1 are taken as examples to detect different properties, and are specifically as follows:

(1) Amino acid composition

The amino acid composition of the leaf proteins obtained in example 1 was examined and compared with commercial Whey Protein Isolate (WPI), egg white protein (EPI), soy Protein Isolate (SPI), pea Protein Isolate (PPI) and sodium caseinate (NaCas).

TABLE 1 amino acid composition of leaf proteins

From Table 1, it is understood that the amino acid composition of leaf protein contains 18 amino acids, the ratio of essential amino acids is 40.28%, which is higher than the specified value (40%) of the world health organization/national food and agricultural organization (WHO/FAO), and the contents of isoleucine, leucine and phenylalanine are particularly rich. The effects of isoleucine include the repair of muscle, control of blood glucose, and the supply of energy to body tissues in concert with leucine and valine. It also increases the production of growth hormone and helps burn visceral fat. Phenylalanine is a precursor substance for catecholamine biosynthesis, and has various effects of inhibiting blood pressure elevation and improving mesenteric arteriole remodeling thereof.

Compared with commercial proteins, the results are shown in Table 2, and the leaf proteins prepared by the invention have higher amino acid scores, and have higher chemical scores than most proteins and only lower than egg white proteins (EPI), thus having very high nutritional value.

TABLE 2 essential amino acid composition of leaf proteins (mg/g pro) compared to commercial proteins

Amino acid score of RPI = amino acid content in RPI/(FAO/WHO) amino acid content;

chemical score = total amino acids/(FAO/WHO) total amino acids.

(2) Leaf protein appearance and color

Comparing the chromaticity of leaf proteins with commercial proteins, figure 1 shows the appearance of leaf proteins. In table 3, each set of experiments was repeated 3 times, expressed as mean ± standard deviation, with a significance analysis (P < 0.05) with SPSS22.0, the different letters of the same column representing differences with significance, P < 0.05. It can be seen in conjunction with the data of table 3 (where RPI represents leaf protein, SPI represents soy protein isolate, PPI represents pea protein isolate, WPI represents whey protein isolate, EPI represents egg white protein, naCas represents sodium caseinate), that the brightness (L) of leaf protein is not significantly different from commercial soy protein isolate and pea protein isolate.

TABLE 3 colorimetric comparison of leaf proteins with commercial proteins

(3) Molecular weight of leaf protein

The molecular weight of the leaf proteins was measured by SDS-PAGE gel electrophoresis.

As a result, it was shown in FIG. 2 that the leaf proteins were mainly composed of protein subunits of two molecular weights of 50kDa and 15kDa, as shown in SDS-PAGE gel electrophoresis.

(4) Leaf protein solubility

The solubility of the leaf proteins of example 1 with commercial proteins was measured and the results were as follows:

FIG. 3 shows the solubility of leaf proteins at 25℃under different pH conditions, as can be seen from the figure, the solubility of leaf proteins under acidic conditions is 12% greater than soy protein isolate, pea protein isolate and sodium caseinate; the solubility under neutral condition reaches 97%, which is larger than other proteins except whey protein; the solubility under alkaline conditions reached 100%, which indicates that the leaf protein reached a fully dissolved state under alkaline conditions (pH 8.5). From the comparison of the solubility of the leaf protein with that of other proteins under different pH conditions, the leaf protein obtained by the method has higher solubility and has obvious advantages compared with commercial vegetable proteins.

(5) Emulsifying ability of leaf protein

The emulsifying capacity of the leaf proteins of example 1 with commercial proteins was examined and the results were as follows:

figure 4 shows the emulsifying capacity of leaf proteins at different pH conditions. As can be seen from the figure, the emulsifying capacity of the leaf protein under acidic conditions is 14m ² And/g, far greater than isolated soy protein, slightly less than sodium caseinate (18 m ² /g); 29m under neutral conditions ² /g, protein isolate from whey (33 m ² /g) and sodium caseinate (32 m ² /g) approaching; 40m under alkaline conditions ² And/g, only below egg white protein and sodium caseinate. The emulsion stability of the leaf protein under acidic condition is 41% which is higher than that of whey protein isolate and sodium caseinate; 41% under neutral conditions and 48% under alkaline conditions, only below sodium caseinate. And observing the emulsion state, the leaf protein emulsion does not show layering phenomenon after being placed for 30min under the condition of 3 pH values, and other proteins show layering phenomena with different degrees. As can be seen from the comparison of the emulsifying property and the emulsifying stability of the leaf protein and other proteins under different pH conditions, the leaf protein obtained by the invention has higher emulsifying property and emulsifying stability, and has remarkable advantages compared with commercial vegetable proteins.

(6) Foaming ability of leaf protein

The foaming capacity of the leaf proteins of example 1 and commercial proteins was measured and the results were as follows:

figure 5 shows the foaming capacity of leaf proteins at different pH conditions. From the figure, the foaming capacity of leaf proteins was 110% under acidic conditions, 140% under neutral conditions and 147% under alkaline conditions. The foaming stability of leaf proteins under acidic conditions was 91%, 87% under neutral conditions and 91% under alkaline conditions. As can be seen from the comparison of the emulsifying property and the emulsifying stability of the leaf protein and other proteins under different pH conditions, the leaf protein obtained by the invention has higher foaming property and foaming stability, and has remarkable advantages compared with commercial vegetable proteins.

(7) Particle size of leaf protein

figure 6 shows the average particle size of leaf proteins at different pH conditions. As is clear from the graph, the average particle size of the leaf protein under acidic conditions was 69.7nm, the average particle size under neutral conditions was 84.1nm, and the average particle size under alkaline conditions was 93.7nm. The average particle size of the leaf protein is relatively small compared to other commercial proteins, facilitating its dissolution and dispersion.

(8) Digestion assay

table 4 shows the digestibility of leaf proteins with commercial proteins, and the results show that the extracted leaf proteins of the invention have higher digestibility than soy protein isolate, pea protein isolate and sodium caseinate, and can be used as a good source of supplemental proteins.

Table 4 comparison of digestibility of leaf proteins with commercial proteins

Comparative example 1: changing the crushed grain size

On the basis of example 1, the wet-milled particle size in step S1 was changed to 10 mesh and 80 mesh to prepare leaf proteins.

The detection shows that the yield of the leaf protein obtained by crushing with 10 meshes is 30.2 percent and the purity is 56.5 percent; the yield of the leaf protein obtained by 80-mesh crushing is 45.8%, and the purity is 77.6%.

Comparative example 2: sodium sulfite and crosslinked polyvinylpyrrolidone are not added

Based on example 1, the step of adding sodium sulfite and cross-linked polyvinylpyrrolidone in step S1 was omitted to prepare leaf protein.

Through detection, the yield of the leaf protein in the comparative example is 50.6%, the purity is 62.4%, and the color of the leaf protein is darker and the chromaticity is poorer.

Comparative example 3: does not carry out ultrasonic alcohol washing

Based on the embodiment 1, the ultrasonic alcohol washing step in the step S2 is omitted, and the grass residues obtained in the step 1 are directly dried and crushed to be less than 100 meshes to prepare the leaf proteins.

The detection shows that the yield of the leaf protein in the comparative example is 40.1% and the purity is 48.5%.

Comparative example 5: changing the ultrasonic feed-liquid ratio

Based on the embodiment 1, the ratio of the ultrasonic alcohol washing feed liquid in the step S2 is changed to 1:4, preparing the leaf protein.

The detection shows that the yield of the leaf protein in the comparative example is 55.6%, and the purity is 63.5%.

Comparative example 6: changing ultrasonic power, time

(1) Varying ultrasonic power

On the basis of the embodiment 1, the ultrasonic alcohol washing power in the step S2 is changed to 50w and 400w respectively, so as to prepare the leaf protein.

Through detection, when the ultrasonic alcohol washing power is 50w, the yield of the prepared leaf protein is 50.5%, and the purity is 56.7%; when the ultrasonic alcohol washing power is 400w, the yield of the prepared leaf protein is 64.5%, and the purity is 55.4%.

(2) Varying ultrasound time

Based on the embodiment 1, the ultrasonic alcohol washing time in the step S2 is changed to 0.5h and 3h respectively, so as to prepare the leaf protein.

Through detection, the ultrasonic alcohol washing time is 0.5h, the yield of the prepared leaf protein is 48.7%, and the purity is 57.4%; ultrasonic alcohol washing time is 3h, the yield of the prepared leaf protein is 65.2%, and the purity is 54.2%

Comparative example 7: changing the enzymolysis liquid-material ratio

Based on the embodiment 1, the ratio of the enzymatic hydrolysis feed liquid in the step S3 is changed to 1:30 and 1:70, preparing the leaf protein.

Through detection, the feed liquid ratio is changed to 1: the yield of the leaf protein prepared by 30 is 52.7 percent, and the purity is 70.5 percent; the feed liquid ratio is changed into 1: the yield of the leaf protein prepared by 70 is 51.5 percent and the purity is 64.2 percent.

Comparative example 8: changing the size of the ultrafiltration membrane

Based on example 1, the ultrafiltration membrane in step S4 was changed to 50kDa in size to prepare leaf protein.

Through detection, the yield of the leaf protein in the comparative example is 50.4%, the purity is 76.5%,

comparative example 9: changing the size of nanofiltration membrane

Based on example 1, the nanofiltration membrane in step S4 was changed to 1kDa in size to prepare leaf protein.

The detection shows that the yield of the leaf protein in the comparative example is 57.2%, and the purity is 70.3%.

Comparative example 10: enzymatic extraction of leaf proteins

The method for extracting leaf protein by using an enzyme method can refer to a reference of a process study for extracting alfalfa leaf protein by using a cellulase method, and comprises the following steps:

step S1, raw material pretreatment: pulping the leaves of She Caoxian with a weight ratio of 2% of salt solution to 1:3 for 2-3 min;

step S2, enzymolysis: at pH 4.86, temperature 55.65 ℃, the enzyme addition amount of cellulase and pectase is 7:3, adding enzyme with the weight of 7.22% of fresh leaves, performing enzymolysis for 10 hours in a water bath oscillator, inactivating enzyme, and filtering to obtain filtrate, namely leaf protein extract;

step S3, acid precipitation: adjusting pH of the filtrate to 4.0 with hydrochloric acid, flocculating for 8min in a constant temperature water bath oscillator, and centrifuging for 10min at 5000r/min to obtain leaf protein;

and S4, drying the leaf protein in a vacuum freeze dryer to obtain the leaf protein powder.

The detection shows that the yield of the leaf protein in the comparative example is 40.2%, and the purity is 58.5%.

Comparative example 11: extraction of leaf proteins by salt extraction and acid precipitation

The leaf protein is extracted by salt extraction and acid precipitation, and the specific steps are as follows:

step S1, raw material pretreatment: mixing She Caoxian leaf with 0.5M phosphate buffer solution with pH of 7.5 at a liquid-to-material ratio of 1:10, heating to boil, cooling to 25deg.C, homogenizing at 14000r/min for 2min, centrifuging at 4000r/min for 10min, and separating to obtain supernatant;

step S2, isoelectric ammonium sulfate precipitation: adjusting the pH of the solution to 5.5, adding 85% w/v ammonium sulfate into the supernatant, stirring for 60min at 4000r/min, and precipitating to obtain leaf protein;

and S3, drying the leaf protein in a vacuum freeze dryer to obtain the leaf protein powder.

The detection shows that the yield of the leaf protein in the comparative example is 38.5%, and the purity is 64.2%.

Comparative example 12: extraction of gratophyllum by alkali dissolution and acid precipitation

The leaf protein is extracted by an alkali-dissolution and acid-precipitation method, and the specific steps are as follows:

step S1, raw material pretreatment: drying She Caoxian leaves at 60deg.C, pulverizing, grinding, and sieving with 100 mesh sieve;

step S2, alkali dissolution extraction: adding 2M sodium hydroxide at a feed-liquid ratio of 1:40 (w/v) to adjust the pH to 12.0, extracting for 2.0h at 50 ℃; centrifuging at 5000r/min for 20min after extraction to obtain supernatant;

step S3, acid precipitation: adding 2M hydrochloric acid into the obtained supernatant, adjusting pH to 3.0, precipitating at 4deg.C for 30min, centrifuging for 20min at 5000r/min to obtain precipitate;

step S4, drying: and (3) drying the precipitate obtained in the step (S3) to obtain the leaf protein powder.

The detection shows that the yield of the leaf protein in the comparative example is 40.5%, and the purity is 67.5%.

The method for measuring purity and yield in the above comparative example is the same as in example 1, and it can be seen from comparative examples 10 to 12 that the extraction method of the present invention is higher in extraction yield and purity than the conventional leaf protein extraction method.

The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing leaf protein, comprising the steps of:

s2, ultrasonic alcohol washing: adding ethanol and inorganic acid into the grass residue obtained in the step S1 to carry out ultrasonic stirring, wherein the ultrasonic power is 100-300 w, and the ultrasonic time is 1-2 h; centrifugally separating grass slag and alcohol washing liquid after ultrasonic treatment, drying the grass slag and crushing to obtain grass slag powder;

s3, enzyme membrane reaction: putting the grass slag powder obtained in the step S2 into a buffer solution system, wherein the liquid-to-material ratio is 1: 40-60, adding cellulase and pectase, stirring uniformly, and carrying out enzymolysis for 10-16 h; filtering the enzymolysis liquid by using an ultrafiltration membrane, and collecting a permeate;

2. The preparation method according to claim 1, wherein the fineness of the grass slag powder after crushing in S2 is 100 to 150 mesh.

3. The preparation method according to claim 1, wherein the liquid-to-material ratio of grass slag to ethanol in S2 is 1:6 to 10.

4. The process according to claim 1, wherein the concentration of the inorganic acid in S2 is 6M to 10M.

5. The preparation method according to claim 1, wherein the dosage of cellulase in S3 is 20U-60U/g substrate, the dosage of pectase is 10U-30U/g substrate, and the stirring speed in the enzymolysis process is 200-300 r/min.

6. The preparation method according to claim 1, wherein the ultrafiltration membrane in S3 has a molecular weight cut-off of 5-20 Kda and a membrane pressure of 0.1-0.6 MPa.

7. The preparation method according to claim 1, wherein the nanofiltration membrane in S4 has a molecular weight cut-off of 300-500 Da and a membrane pressure of 0.6-1.0 MPa.

8. The leaf protein of any one of the methods of claims 1-7.

9. An emulsifier or foaming agent comprising the leaf protein of claim 8.

10. Use of the leaf protein of claim 8 in the preparation of a food product.