Preparation method of compound containing pea protein

Abstract

A preparation method of a compound containing pea protein comprises the following steps: step 1, the pea protein is placed in a ball-milling tank for ball milling, in the ball-milling process, liquid nitrogen is always in an open state, and the temperature is kept between-20 DEG C and-10 DEG C; the ball milling time is 5-50 minutes, so as to obtain the frozen ball-milled modified pea protein; step 2, adding water into dry rice starch to prepare starch milk, then adding water into the frozen ball-milling modified pea protein for dissolving to obtain a frozen ball-milling modified pea protein solution, then adding the frozen ball-milling modified pea protein solution into the starch milk, and stirring at normal temperature to uniformly mix the starch milk and the frozen ball-milling modified pea protein solution to obtain a frozen ball-milling modified pea protein solution; freezing and drying to obtain a pea protein-rice starch compound; or preparing the frozen ball-milled modified pea protein into a frozen ball-milled modified pea protein solution by using a PBS buffer solution, then mixing the frozen ball-milled modified pea protein solution with the blueberry anthocyanin solution, and stirring in a dark place at room temperature to obtain the pea protein-blueberry anthocyanin compound. According to the pea protein-rice starch compound disclosed by the invention, the retrogradation value, the gel hardness, the gelatinization enthalpy value and the short-range orderliness of the rice starch are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing and relates to a method for preparing a composite containing pea protein. Background Art

[0002] Pea (Pisum sativum L.) is one of the world's major legumes, widely grown in more than 90 countries around the world. Pea has a very comprehensive nutritional composition, including protein (20%-25%), carbohydrates (24%-49%), starch (60%-65%), fat (1.5%-2.0%), B vitamins and various minerals such as potassium, phosphorus, magnesium, and calcium. Due to its unique advantages such as high yield, high availability, and low cost, pea is often used as a source of industrial protein.

[0003] Pea protein has a complete range of amino acids and a balanced ratio. Except for the relative lack of sulfur-containing amino acid methionine, other essential amino acids can meet the recommendations of FAO / WHO, making it a high-quality plant protein. Pea protein is rich in lysine, threonine and tryptophan, which is higher than cereal protein. In industrial applications, it can be used with cereal protein to make up for the low lysine content in cereal foods. Many studies have shown that pea protein and its hydrolysates have a variety of biological activities, including antioxidant, anti-hypertensive, anti-inflammatory, cholesterol-lowering and intestinal flora activity regulation, and some activities can be further enhanced by chemical or combined treatment methods. Therefore, regular intake of foods rich in pea protein can help reduce the risk of cardiovascular disease and diabetes, and has an improvement effect on a variety of cancers (breast cancer, kidney cancer and colon cancer, etc.). In addition, compared with soy protein, pea protein is relatively high in nutritional value and has no genetically modified non-food allergens, which is conducive to the development of natural foods that meet clean label standards.

[0004] In summary, pea protein has great development prospects as a substitute for traditional protein ingredients (animal protein and soy protein) due to its low allergenicity, high nutritional value and availability, and the fact that it comes from sustainable crops. It has attracted much attention from researchers and is welcomed by the food industry. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a composite containing pea protein.

[0006] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for preparing a composite containing pea protein, comprising the following steps:

[0007] Step 1: placing the pea protein in a ball mill for ball milling. During the ball milling process, the liquid nitrogen is always turned on and the temperature is maintained between -20°C and -10°C. The ball milling time is 5 min to 50 min to obtain frozen ball milled modified pea protein.

[0008] Step 2: Add water to dry rice starch to prepare starch milk, then add water to dissolve frozen ball-milled modified pea protein to obtain a frozen ball-milled modified pea protein solution, then add the frozen ball-milled modified pea protein solution to the starch milk, stir at room temperature to mix the starch milk and the frozen ball-milled modified pea protein solution evenly, and freeze-dry to obtain a pea protein-rice starch complex; alternatively, prepare the frozen ball-milled modified pea protein into a frozen ball-milled modified pea protein solution using PBS buffer, and then mix it with the blueberry anthocyanin solution, stir in the dark at room temperature to obtain a pea protein-blueberry anthocyanin complex.

[0009] The preferred technical solution is: during ball milling, the rotation speed of the ball mill is set to 400-600 r / min, and the rotation direction is alternated every 4-6 minutes.

[0010] The preferred technical solution is: the concentration of starch milk is 3-8g / 100g, and the mass of rice starch is 5-15% of the mass of frozen ball-milled modified pea protein.

[0011] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0012] (1) Effects of cryo-ball milling modification on the physicochemical and functional properties of pea protein

[0013] The cryo-milling treatment changed the spatial structure of pea protein, significantly reduced the particle size, and changed the surface hydrophobicity and fluorescence intensity, which shows that cryo-milling is conducive to the unfolding of the internal structure of the protein, thereby improving the functional properties of pea protein. When the ball milling time is 20 minutes, the solubility of pea protein increases from 3.08±0.12% to 12.37±0.21%, the emulsification increases by 47.59%, and the emulsification stability increases by 11.17%, so the ball milling treatment for 20 minutes may be the most suitable modification condition.

[0014] (2) Effects of frozen ball-milled modified pea protein on the gelatinization and digestibility of rice starch

[0015] Compared with rice starch, the pea protein-rice starch composite with pea protein added had lower recovery value, gel hardness, gelatinization enthalpy and order degree; compared with the unmodified pea protein-rice starch composite system, the effect of the ball-milled pea protein-rice starch composite system was more significant (P<0.05), and the gelatinization enthalpy of the pea protein-rice starch composite modified by ball milling for 20 minutes decreased by 13.72%, and the mass fractions of slowly digestible starch and resistant starch increased by 3.74% and 2.05%, respectively. Before ball milling for 20 minutes, the effect of pea protein on rice starch changed greatly. When the ball milling time exceeded 20 minutes, the effect was no longer as obvious as before, which may be related to the change of pea protein particle size. Fourier transform infrared spectroscopy results showed that hydrogen bonding was the main driving force for the interaction between pea protein and rice starch. Scanning electron microscopy also showed that the conformation of the pea protein-rice starch composite treated by ball milling changed, which may be the main reason affecting the gelatinization characteristics and digestibility of rice starch.

[0016] (3) Effect of frozen ball-milled modified pea protein on the color stability of blueberry anthocyanins

[0017] Heat treatment and light treatment experiments proved that ball-milled pea protein had a certain protective effect on the color stability of blueberry anthocyanins, among which pea protein ball-milled for 20 minutes had the best effect. Fluorescence spectroscopy, infrared spectroscopy and ultraviolet spectroscopy verified the interaction between the two: pea protein and blueberry anthocyanins had a high affinity, belonging to static quenching, and the binding site was about 1; blueberry anthocyanins changed the secondary structure of pea protein, and the complex showed a more stable structure, while affecting the microenvironment of amino acid residues. The research results provide inspiration for the interaction between pea protein and anthocyanins, and provide direction for further practical application in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Effect of cryoball milling on particle size distribution of pea protein.

[0019] Figure 2 To investigate the effect of cryo-ball milling on the nitrogen solubility index of pea protein.

[0020] Figure 3 To investigate the effect of cryoball milling on the Zeta potential of pea protein.

[0021] Figure 4 To investigate the effects of cryoball milling on the emulsification activity and emulsification stability of pea protein.

[0022] Figure 5 To investigate the effect of cryoball milling on the surface hydrophobicity of pea protein.

[0023] Figure 6To investigate the effect of cryoball milling on the free thiol content of pea protein.

[0024] Figure 7 This study investigates the effect of cryo-ball milling on the fluorescence absorption spectrum of pea protein.

[0025] Figure 8 This study shows the effect of cryoball milling on SDS-PAGE of pea protein.

[0026] Fig. 9 To investigate the effect of cryo-ball milling on the microstructure of pea protein.

[0027] Fig.10 Infrared spectra of pea protein-starch after different ball milling treatments.

[0028] Fig.11 Deconvoluted spectra of pea protein-starch after different ball milling treatments.

[0029] Fig.12 Effect of ball-milled modified pea protein on the microstructure of rice starch.

[0030] Fig.13 This is the fluorescence spectrum of pea protein-blueberry anthocyanin complex at 25°C.

[0031] Fig.14 This is the Stern-Volmer curve of the interaction between pea protein and blueberry anthocyanins under different temperature conditions.

[0032] Fig.15 This is the fitting diagram of the binding constant of pea protein and blueberry anthocyanin under different temperature conditions.

[0033] Fig.16 This is the synchronous fluorescence spectrum of the interaction between pea protein and blueberry anthocyanin (Δλ=15nm).

[0034] Fig.17 This is the synchronous fluorescence spectrum of the interaction between pea protein and blueberry anthocyanin (Δλ=60nm).

[0035] Fig.18 This is the infrared spectrum of pea protein and pea protein-blueberry anthocyanin complex.

[0036] Fig.19 This is the UV-visible spectrum of the pea protein-blueberry anthocyanin complex.

[0037] Fig. 20 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0038] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0039] See also Figure 1-20 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0040] The relevant reagents used in the embodiments of the present invention are shown in the following table:

[0041]

[0042]

[0043] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0044] The technical solution of the present invention is further described in detail below with reference to examples.

[0045] Example 1: A method for preparing a composite containing pea protein

[0046] 1. Determination of basic ingredients

[0047] The basic components of pea protein were analyzed: the determination of protein was based on the national standard GB 5009.5-2016 (Kjeldahl nitrogen determination method); the determination of moisture was based on the national standard GB 5009.3-2016 (direct drying method); the determination of fat was based on GB 5009.6-2016 (Soxhlet extraction method); and the determination of ash was based on GB 5009.4-2016 (high temperature calcination method).

[0048] 2. Preparation of frozen ball-milled modified pea protein

[0049] Place an appropriate amount of pea protein in a ball mill and set the speed to 500r / min. To ensure uniform grinding, alternate the rotation direction every 5 minutes. During the ball milling process, liquid nitrogen is always turned on and the temperature is maintained between -20℃ and -10℃. Samples are taken at 10min, 20min, 30min and 40min of ball milling, and marked as PPI 10, PPI 20, PPI30 and PPI 40, with pea protein (PPI 0) that has not been ball milled as a control.

[0050] 3. Determination of the effect of frozen ball milling modification on the physicochemical and functional properties of pea protein

[0051] (1) Determination of color change

[0052] The color of the pea protein powder was measured using a colorimeter. After calibration with the built-in white plate of the instrument, 10 g of pea protein powder was placed in a clean, transparent self-sealing bag. The brightness (L*), red-green (a*), and yellow-blue (b*) of the sample were measured, and the whiteness (W) was calculated using the following formula.

[0053] W = L* - 3b*.

[0054] (2) Determination of particle size distribution

[0055] Take an appropriate amount of pea protein and dissolve it in 0.01mol / L phosphate buffer with a pH value of 7.0. Use a magnetic stirrer at room temperature to stir it at a speed of 500r / min for 1h to fully mix it. Then pour the pea protein solution into the sample cell of the Malvern laser particle size analyzer, mix it at a speed of 1500rpm, and start measuring when the shading reaches between 10% and 15%. Select the general mode for the test mode, repeat the test three times, and use D(0.1), D(0.5), D(0.9), D(3,2) and D(4,3) to analyze the experimental results. Among them, D(0.1), D(0.5) and D(0.9) represent the volume of particles less than or equal to the particle size, accounting for 10%, 50% and 90% of the total volume of the sample, respectively. D(3,2) represents the volume surface area equivalent average diameter (i.e., the weighted average of particle size to surface area), and D(4,3) represents the volume mass equivalent average diameter (i.e., the weighted average of particle size to volume).

[0056] (3) Determination of solubility

[0057] The solubility of protein is expressed by the nitrogen solubility index (NSI). Take an appropriate amount of pea protein, dissolve it in 0.01 mol / L phosphate buffer with a pH value of 7.0, and stir it magnetically at 500 r / min for 1 hour at room temperature to mix it thoroughly. Centrifuge it at 10000 r / min for 10 minutes, and determine the protein content and total protein content of the supernatant by the Kjeldahl method. Calculate the protein solubility according to the following formula.

[0058]

[0059] (4) Determination of Zeta potential

[0060] Take an appropriate amount of pea protein and dissolve it in 0.01mol / L phosphate buffer with a pH value of 7.0. Use a magnetic stirrer at room temperature to stir it at a speed of 500r / min for 1h to fully mix it. Centrifuge the prepared pea protein solution at 10000r / min for 10min at room temperature, and then dilute the supernatant with 0.01mol / L phosphate buffer with a pH value of 7.0 to make the final concentration of pea protein 0.1mg / mL, and filter it through a 0.45μm membrane to determine the Zeta potential. Determination parameters: dispersed refractive index 1.492, dispersed phase refractive index 1.33, test temperature 25℃.

[0061] (5) Determination of emulsification activity and emulsification stability

[0062] Take an appropriate amount of pea protein, dissolve it in 0.01mol / L phosphate buffer with a pH value of 7.0, and prepare a pea protein solution with a mass fraction of 0.1% (w / v). Mix the prepared pea protein solution with soybean oil in a ratio of 6:1, use a high-speed shearing machine to shear for 2min at 15000r / min, and pause for 10s every 30s. Then draw 50μL of the emulsion from 5mm from the bottom of the beaker, and add 5mL of 0.1% SDS (w / v) solution. After the two are vortexed and mixed, the absorbance value A0 is measured at 500nm, and a 0.1% SDS solution is used as a blank control. After the emulsion is allowed to stand for 30min, resample it from 5mm from the bottom of the beaker, and measure the absorbance value A30 according to the A0 determination step. Emulsification activity and emulsification stability are calculated according to the following formula.

[0063]

[0064] Where: A0 is the absorbance value measured at 0 min; D is the dilution multiple; C is the protein concentration before emulsion formation (g / mL); is the volume fraction of oil in the emulsion (v / v); A 30 It is the absorbance value measured at 30 min.

[0065] (6) Determination of surface hydrophobicity

[0066] Take an appropriate amount of pea protein and dissolve it in 0.1mol / L PBS solution (pH=7), and dilute the pea protein solution to 0.02, 0.04, 0.06, 0.08, and 0.1mg / mL. Take 4mL of the above dilution and add 20μL of 8mmol / L 8-anilinonaphthalenesulfonate (ANS) solution, and measure the fluorescence intensity of the sample after 10min of light-proof reaction. The measurement conditions are: excitation wavelength 390nm, emission wavelength 470nm, slit width 5nm, voltage 700mV. Plot the fluorescence intensity against protein concentration, and take the slope of the initial stage of the curve as the surface hydrophobicity.

[0067] (7) Determination of free thiol content

[0068] The pea protein sample was dissolved in 0.1 mol / L phosphate buffer (containing 1.2 g / L EDTA, 6.9 g / L glycine, 10.4 g / L Tris, 25 g / L SDS, pH = 8.0). The mixture was shaken and mixed for 30 min, centrifuged at 6000 r / min for 10 min, 5 mL of the supernatant was added with 0.1 mL of Ellman's reagent solution, mixed evenly, and placed at room temperature in the dark for 15 min, and the absorbance of the supernatant and the blank control was measured at 412 nm.

[0069]

[0070] Wherein: free thiol content (μmol / g); 73.53 is the molar absorption coefficient of Ellmann reagent; A412 is the absorbance of the sample at 412nm; D is the dilution factor of the sample; C is the protein concentration of the sample solution (mg / mL).

[0071] (8) Determination of intrinsic fluorescence spectrum

[0072] A 1 mg / mL pea protein solution was prepared with 0.01 mol / L phosphate buffer with a pH value of 7.0. After magnetic stirring for 1 hour, the solution was centrifuged at 10000 r / min for 10 minutes and the supernatant was taken for fluorescence spectrum scanning. Measurement parameters: excitation wavelength of 290 nm, scanning range of 300-500 nm, scanning speed of 1200 nm / min. Phosphate buffer was used as blank.

[0073] (9) Determination of secondary structure

[0074] Weigh 1 mg of pea protein and 100 mg of dry KBr powder in an agate mortar, mix and grind thoroughly, and take an appropriate amount to make tablets. Measure the infrared absorption spectrum in the range of 4000-400 cm-1, with an absorption resolution of 2 cm-1 and 32 scans. Analyze the spectrum using OMNIC software, and analyze the amide I region (1700-1600 cm-1) in the spectrum to determine the relative content of the four secondary structures of the protein.

[0075] (10) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0076] Refer to Xu Zhihan's method and make slight modifications. Take 10mg pea protein sample and dissolve it in 1mL 1M Tris-HCl (pH=6.8), centrifuge at 10000r / min for 10min, take 16μL centrifugal supernatant and add 4μL SDS-PAGE protein loading buffer (5X), heat in a 100℃ water bath for 5min for lysis, cool to room temperature, take 12μL sample mixture and 10μL protein marker and add them to the loading well respectively, and run at a constant voltage of 120V for 45min. After the electrophoresis, use Coomassie Brilliant Blue R-250 to stain for 20min, and then use decolorizing solution to decolorize until the electrophoresis band is clear.

[0077] (11) Scanning electron microscopy (SEM)

[0078] Use a cotton swab to dip a small amount of pea protein powder and evenly apply it on the conductive double-sided tape, spray gold for 30 seconds, and then place the sample disk under a scanning electron microscope and select a magnification of 500 to observe the surface morphology of the pea protein.

[0079] 4. Determination of the effect of frozen ball-milled modified pea protein on the gelatinization and digestibility of rice starch

[0080] (1) Preparation of pea protein-rice starch complex

[0081] Accurately weigh 5.0g (dry basis) of rice starch to prepare 5g / 100g of starch milk, then weigh 10% of the starch dry weight of pea protein ball-milled for 0min, 10min, 20min, 30min, and 40min, add the pre-dissolved protein solution to the starch milk, stir the starch and protein blend for 1h using a magnetic stirrer at room temperature, and freeze-dry to obtain a pea protein-rice starch complex, which are labeled as PPI0-RS, PPI10-RS, PPI20-RS, PPI30-RS, and PPI40-RS, respectively, and pure rice starch (RS) without pea protein added is used as a control.

[0082] (2) Paste attributes

[0083] Use a rapid viscosity analyzer to analyze the mixed powder. Accurately weigh 3.5g of the mixed powder and transfer it into a dry and clean sample tube. Then add 25mL of deionized water to the sample tube and directly place the sample tube into the rapid viscosity analyzer for measurement. Record the gelatinization temperature, peak viscosity, minimum viscosity, final viscosity, attenuation value, and regeneration value of the sample.

[0084] (3) Gel properties

[0085] The pea protein-rice starch composite after RVA test was cooled and sealed, and refrigerated at 4°C for 24 hours for texture determination. Test conditions: TPA mode, P 0.5 probe, pre-test rate 1.0 mm / s, test rate 1.0 mm / s, post-test rate 1.0 mm / s; compression degree 50%; trigger force 5g.

[0086] (4) Water absorption capacity, solubility and swelling capacity

[0087] Prepare 40 mL of 2.5% sample suspension (by volume mass) in a 100 mL centrifuge tube. Place a magnetic stirrer in the tube and keep the suspension in a water bath at 90°C for 30 minutes. Centrifuge the suspension at 4000 r / min for 15 min, then decant the supernatant and weigh the swollen particles. Extract 10 mL of sample from the supernatant, place in a petri dish, and dry in an air oven at 120°C for 4 h to achieve constant mass. Water absorption capacity, solubility, and swelling capacity are calculated using the following equations:

[0088]

[0089] (5) Thermodynamic properties

[0090] Thermodynamic properties were tested using a differential scanning calorimeter. A suspension with a sample mass fraction of 30% was prepared with distilled water and equilibrated at 25°C for 24 hours. Accurately weigh 800 mg of sample solution and distilled water and place them in a sample tank and a reference tank. Heat from 25°C to 90°C at a rate of 1°C / min. Record the starting temperature (T0), peak temperature (Tp), end temperature (Tc) and enthalpy change (△H).

[0091] (6) Fourier transform infrared spectroscopy

[0092] The sample powder and potassium bromide powder were weighed at a ratio of 1:100 and placed in an agate mortar for grinding and mixing. The sample slices were prepared by tabletting. The sample to be tested was placed in the instrument and scanned in the range of 4000-400 cm-1. The spectrum was processed using Omnic8.0 software, and the characteristic peaks in the 800-1200 cm-1 segment were deconvoluted to calculate the values ​​of 1047 cm-1 / 1022 cm-1 and 1022 cm-1 / 995 cm-1.

[0093] (7) Scanning electron microscopy

[0094] Use a cotton swab to dip a small amount of sample powder and evenly spread it on the conductive double-sided tape, then adhere it to the carrier, spray gold for 30 seconds, and then place the sample plate under a scanning electron microscope and select a magnification of 2000 to observe the surface morphology of pea protein.

[0095] (8) Determination of digestibility

[0096] The determination of starch digestibility was based on the method of Wang Yu et al., with appropriate modifications. Accurately weigh 400 mg of sample in a 100 mL conical flask, add 15 mL of sodium acetate buffer (pH = 5.2) and 6 glass beads, mix thoroughly, add 5 mL of mixed enzyme solution (α-amylase 100 U / mg, glucosidase 300 U / mL), and perform enzymolysis in a 37°C constant temperature water bath. Take out 0.5 mL of enzymolysis solution at 0, 20, and 120 min of the enzymolysis reaction, add 4.5 mL of anhydrous ethanol to mix and terminate the reaction, centrifuge at 5000 r / min for 10 min, and use a glucose kit to detect the glucose content in the supernatant. The content of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) was calculated using the following formula.

[0097] RDS=[(M 20 -M0×0.9)] / M TS ×100%.

[0098] SDS=[(M 120 -M 20 ×0.9)] / M TS ×100%.

[0099] RS = (1 - RDS - SDS) x 100%.

[0100] Where: M0 is the amount of glucose before hydrolysis (mg); M 20 is the amount of glucose released within 20 min (mg); M 120 is the amount of glucose released within 120 min (mg); MTS is the total starch mass (mg).

[0101] 5. Determination of the effect of frozen ball-milled modified pea protein on the color stability of blueberry anthocyanins

[0102] (1) Preparation of pea protein-blueberry anthocyanin complex

[0103] Pea protein without frozen ball milling modification, pea protein with frozen ball milling modification for 20 min, and pea protein with frozen ball milling modification for 40 min were selected, and pea protein solution and blueberry anthocyanin solution (BAS) were prepared using 0.02 mol PBS buffer (pH 6.3). The two were mixed in different proportions and stirred for 1 h at room temperature in the dark to obtain pea protein-blueberry anthocyanin complexes of different concentrations, which were named PPI0-BAS, PPI20-BAS, and PPI40-BAS respectively.

[0104] (2) Determination of the color stability of blueberry anthocyanins by different ball-milled modified pea proteins

[0105] The stability of blueberry anthocyanins was evaluated by heat treatment and light treatment. The heat stability experiment was to place the sample in a 50mL centrifuge tube, heat it in a constant temperature water bath at 80℃ for 2h away from light, and then quickly cool it to room temperature in an ice water bath. The brightness (L*), red-green (a*), and yellow-blue (b*) of the sample were measured using a fully automatic colorimeter. The chromaticity value (C*), hue angle (Hueo) and color difference value (ΔE) were calculated by the following formula. The color loss (%) was evaluated by comparing the color difference value (ΔE) before and after the sample treatment. The concentration of blueberry anthocyanins was 0.5mg / mL, and the concentration of pea protein was 0, 0.5, 1, and 1.5mg / mL, respectively.

[0106] The light stability test is to place the samples under sunlight, take samples on days 0, 1, 3 and 5, and then evaluate the storage stability by comparing the color loss of the samples before and after treatment. The concentration of blueberry anthocyanin is 0.5 mg / mL. The concentration of pea protein is fixed at 1 mg / mL.

[0107]

[0108] Among them, L0*, a0*, and b0* are the chromaticity values ​​of the pigment solution that has not been degraded.

[0109] (3) Determination of fluorescence spectrum

[0110] The intrinsic fluorescence spectra of the pea protein-blueberry anthocyanin complex were measured at 25°C, 35°C, and 45°C. The final concentration of pea protein was 10 μM, the final concentration of blueberry anthocyanin was 0, 10, 20, 30, 40, and 50 μM, the slit width was 10.0 nm, the excitation wavelength was 280 nm, and the emission wavelength was 300-450 nm.

[0111] The fluorescence quenching mechanism of blueberry anthocyanins on pea protein, as well as the binding constant (KA) and the number of binding sites (n) were analyzed by the Stern-Volmer equation and the double logarithmic Stern-Volmer equation. The formula is as follows:

[0112]

[0113] Where F0 and F are the fluorescence intensities of the protein in the absence and presence of the quencher, respectively, and K SV is the Stern-Volmer dynamic quenching constant, [Q] is the concentration of free small molecule quencher, K q is the rate constant of the quenching process of biomacromolecules, τ0 represents the lifetime of the fluorophore in the absence of a quencher, which is approximately 10-8s, and Ks and n represent the binding constant and number of binding sites between the quencher and the fluorophore, respectively.

[0114] (4) Determination of synchronous fluorescence spectra

[0115] The synchronous fluorescence spectra were scanned at Δλ=15 nm (excitation wavelength range: 270-320 nm) and Δλ=60 nm (excitation wavelength range: 240-320 nm), respectively, and the buffer solution and the quencher solution of the same concentration were used as blanks for subtraction.

[0116] (5) Fourier transform infrared spectroscopy

[0117] The prepared sample solution was freeze-dried to obtain sample powder, mixed with KBr and crushed into thin sheets. -1 Scanning Fourier transform infrared spectroscopy.

[0118] (6) Ultraviolet absorption spectrum

[0119] The UV absorption spectra of pea protein solutions with and without blueberry anthocyanins were measured in a scanning range of 240-400 nm.

[0120] 6. Data processing and analysis

[0121] In this example, Origin 2018 software was used to draw the graphs; Statistix 9.0 software was used to perform statistical analysis of the data using LSD (Least significance difference), with a significance level of p < 0.05. All experiments were performed three times and expressed as mean ± standard deviation.

[0122] 1. Basic ingredients of raw materials

[0123] Table 1: Basic ingredients of raw materials

[0124] Components protein Fat Moisture Ash content(%) 89.52±0.21 0.52±0.02 4.64±0.13 5.32±0.11

[0125] From Table 1, we can see that the protein content of pea protein is 89.52±0.21%, the moisture content is 4.64±0.13%, the ash content is 5.32±0.11%, and it contains a small amount of fat.

[0126] 2. Effects of cryo-ball milling modification on the physicochemical and functional properties of pea protein

[0127] (1) Influence of color

[0128] Table 2: Effect of cryo-milling on the color of pea protein

[0129] Sample name L* a* b* W PPI 0 <![CDATA[84.44±0.02 a ]]> <![CDATA[3.82±0.02 a ]]> <![CDATA[17.75±0.02 a ]]> <![CDATA[31.19±0.04 a ]]> PPI 10 <![CDATA[87.30±0.03 b ]]> <![CDATA[2.83±0.02 b ]]> <![CDATA[15.21±0.02 b ]]> <![CDATA[41.67±0.12 b ]]> PPI 20 <![CDATA[88.96±0.02 c ]]> <![CDATA[2.55±0.02 c ]]> <![CDATA[14.20±0.02 c ]]> <![CDATA[46.36±0.06 c ]]> PPI 30 <![CDATA[89.05±0.04 d ]]> <![CDATA[2.27±0.02 d ]]> <![CDATA[13.34±0.02 d ]]> <![CDATA[49.03±0.10 d ]]> PPI 40 <![CDATA[89.63±0.03 e ]]> <![CDATA[2.06±0.02 e ]]> <![CDATA[12.54±0.03 e ]]> <![CDATA[52.01±0.19 e ]]>

[0130] Note: L*, a*, b* and W in the table represent brightness, red-green, yellow-blue and white respectively. Data with different letters in the same column indicate significant differences (p<0.05), the same below.

[0131] Table 2 shows the effect of frozen ball milling on the color of pea protein powder. As can be seen from the table, with the extension of ball milling time, the L* value and W value of pea protein increased significantly, and b* decreased significantly (P<0.05). Compared with PPI 0, the L* value of PPI 40 increased from 84.44±0.02 to 89.63±0.03, the W value increased from 31.19±0.04 to 52.01±0.19, and the b* value representing yellowness decreased from 17.75±0.02 to 12.54±0.03. Ball milling makes pea protein show a trend of lighter color and increased brightness. Tang Caidie et al. used ball milling to process bamboo shoot powder, and Liu et al. used ball milling to process wheat gluten protein, and obtained similar experimental results. There are two possible reasons for this phenomenon: the violent collision during ball milling partially degrades dark substances such as lutein, causing the color to lighten; ball milling reduces the particle size of the protein, increases the specific surface area, and more light is reflected to the sample surface, thereby increasing the whiteness of the sample. The improvement in whiteness and brightness largely reflects the quality of the material and the milling accuracy, which can reduce the demand for whitening additives in industrial production.

[0132] (2) Influence of particle size distribution

[0133] Figure 1The particle size distribution diagram of pea protein after cryo-ball milling. As can be seen from the figure, cryo-ball milling significantly reduces the particle size of pea protein, and as the ball milling time increases, the particle size distribution gradually moves to the left (the side with smaller particle size). The particle size distribution span of pea protein that has not been treated with cryo-ball milling is large, distributed in the range of 0-500μm, while the particle size distribution of pea protein that has been treated with cryo-ball milling is more concentrated. Under a scanning electron microscope, it can be observed that the particle size of pea protein becomes smaller after 10 minutes of ball milling, and the particle size gradually tends to be consistent, which is consistent with the results here. Compared with PPI0, the D of PPI40 0.1 , D 0.5 , D 0.9 They decreased from 38.03±0.12μm, 106.93±0.63μm, 240.12±1.78μm to 11.41±0.04μm, 29.96±0.08μm, and 58.39±0.88μm, respectively, indicating that the particle size is gradually decreasing. Liang Yingjie et al. ball-milled pumpkin seed protein and found that the particle size distribution of pumpkin seed protein would gradually move to the small particle size with the increase of ball milling time, and the span value of the particle size distribution would decrease with the increase of ball milling time, which is consistent with the experimental results of this study. However, when the ball milling time exceeds 20min, the reduction in protein particle size is no longer as significant as before, which may be because more energy needs to be accumulated to further reduce the particle size. In addition, when the ball milling time exceeds 20min, the particle size change of the protein is no longer significant, and the solubility is no longer increased, which indicates that the change in solubility may be related to the particle size. Particle size reduction can break down large protein aggregates and promote the interaction between protein and water, thereby improving the solubility of pea protein.

[0134] Table 3: Effect of cryo-milling on particle size distribution of pea protein

[0135] Sample name D(4,3) / μm D(3,2) / μm D(0.1) / μm D(0.5) / μm D(0.9) / μm PPI 0 <![CDATA[125.01±0.74 a ]]> <![CDATA[60.52±0.13 a ]]> <![CDATA[38.03±0.12 a ]]> <![CDATA[106.93±0.63 a ]]> <![CDATA[240.12±1.78 a ]]> PPI 10 <![CDATA[59.14±2.26 b ]]> <![CDATA[27.87±1.00 b ]]> <![CDATA[16.18±1.14 b ]]> <![CDATA[42.09±2.06 b ]]> <![CDATA[119.70±8.95 b ]]> PPI 20 <![CDATA[47.84±0.48 c ]]> <![CDATA[28.74±0.25 b ]]> <![CDATA[18.37±0.24 c ]]> <![CDATA[44.59±0.22 c ]]> <![CDATA[82.38±0.91 c <!-- 9 -->]]> PPI 30 <![CDATA[35.48±1.57 d ]]> <![CDATA[19.06±1.39 c ]]> <![CDATA[10.36±0.72 d ]]> <![CDATA[26.05±1.35 d ]]> <![CDATA[58.28±3.96 d ]]> PPI 40 <![CDATA[32.90±0.39 d ]]> <![CDATA[20.72±0.08 d ]]> <![CDATA[11.41±0.04 d ]]> <![CDATA[29.96±0.08 e ]]> <![CDATA[58.39±0.88 d ]]>

[0136] (3) Effect of solubility

[0137] The solubility of protein is affected by many factors, such as the interaction between protein molecules, particle size, charge distribution, etc. The solubility of protein plays an important role in promoting its emulsification, foaming and gelation capabilities. Figure 2It can be seen that the solubility of natural untreated pea protein is poor, with a solubility of only about 3%. After 10 minutes of frozen ball milling, the solubility of pea protein increased from 3.08±0.12% to 11.33±0.09%, and the solubility of pea protein after 20 minutes of treatment reached 12.37±0.21%. This is because the pea protein molecules stretch under the impact of ball milling, and large particles of pea protein are crushed into small particles, increasing the contact area between protein and water. The exposure of polar groups and hydrophobic groups inside the protein changes the charge on the surface of the protein molecules, and the hydration is enhanced, thereby increasing the solubility. When the ball milling time exceeds 20 minutes, the solubility no longer increases but shows a downward trend. The reason for this phenomenon may be that the particle size of pea protein no longer changes significantly after the ball milling time exceeds 20 minutes, and it cannot provide more contact sites with water; at the same time, excessive ball milling completely destroys the structure of pea protein, and a large number of hydrophobic residues are exposed, resulting in enhanced hydrophobic interactions of proteins and the formation of insoluble aggregates. Frederik et al. also found that ball milling can change the extraction rate of oat protein in water and salt water extracts, but the maximum protein extraction rate can only be 45%. In general, frozen ball milling improves the solubility of pea protein.

[0138] (4) Influence of Zeta potential value

[0139] Zeta potential can reflect the aggregation stability of a solution to a certain extent. Protein particles with high Zeta potential are more likely to be stabilized by strong electrostatic repulsion, while protein particles with low Zeta potential tend to aggregate because attractive interactions outweigh repulsive interactions. Figure 3 The Zeta potential diagram of pea protein after ball milling is shown. As can be seen from the figure, pea protein carries a negative charge, the Zeta potential value of untreated pea protein is -18.35±0.95, and the absolute value of the Zeta potential of pea protein treated with ball milling is higher than that of untreated. This is because ball milling reduces the particle size of the protein sample, which in turn causes changes in the structure of the protein, and non-covalent interactions such as hydrogen bonds between polar amino acid residues and hydrophobic interactions between non-polar residues are destroyed, resulting in an increase in the net surface charge of the protein. However, when the ball milling time exceeds 20 minutes, the absolute value of the Zeta potential of pea protein shows a downward trend. Gao et al. used high-intensity ultrasound treatment of 100-400W to treat commercial pea protein and found that the Zeta potential showed the same trend of first increasing and then decreasing. A possible explanation for this phenomenon is that excessive ball milling exposes the hydrophobic residues inside the protein, enhances hydrophobic interactions, and produces soluble aggregates, resulting in reduced repulsion and surface charge.

[0140] (5) Influence of emulsification activity and emulsification stability

[0141] Emulsifying activity depends largely on the diffusion ability of proteins at the oil-water interface, which indicates the rapid adsorption of proteins to the oil-water interface during emulsion formation. Figure 4 It can be seen that compared with untreated pea protein, ball milling treatment increased the emulsification activity of pea protein, and it showed a trend of increasing first and then decreasing. The emulsification activity of untreated pea protein was 7.71±0.03m 2 With the increase of ball milling time, the emulsifying activity of pea protein increased significantly to 14.71±0.09 m / s at 20 min. 2 , and then decreased to 12.95±0.04m at 40min 2 . The emulsification stability of pea protein also showed a similar trend, increasing from 18.69±0.31% of the untreated to 21.04±0.17%. From the analysis of Zeta potential, the emulsification activity and emulsification stability of pea protein showed the same trend of increasing first and then decreasing as the Zeta potential value. This phenomenon is reasonable because the higher surface net charge of protein contributes to the stability of the emulsion and is conducive to its stable dispersion at the interface. Studies have shown that milling can increase the hydrophobicity of protein and enhance its emulsification properties. The reason may be that milling causes the hydrophobic groups inside the protein to be exposed, and the increased hydrophobicity can enhance the adsorption capacity of oil. When ball milling is performed for 20 to 40 minutes, the emulsification activity of pea protein shows a downward trend, which is consistent with the results obtained by Zhang et al. using ball milling to treat egg yolk phospholipids, but the decrease in the emulsification activity of egg yolk phospholipids did not show a significant difference. This may be because the pea protein molecules are more flexible, resulting in the inability to form a stable interfacial film at the oil-water interface.

[0142] (6) Influence of surface hydrophobicity

[0143] from Figure 5 It can be seen that the surface hydrophobicity of pea protein significantly increased with the extension of the freezing ball milling time in the first 20 minutes of freezing ball milling. This is consistent with the research results of Liu et al., who found that the surface hydrophobicity of soy protein isolate increased with the increase of ball milling time. This result may be that the pea protein was damaged by impact and friction during the ball milling process, and the spatial conformation changed, exposing the internal hydrophobic groups on the molecular surface, causing the surface hydrophobicity to increase. When the freezing ball milling time exceeds 20 minutes, the surface hydrophobicity of pea protein shows a downward trend, but it is not statistically significant. This result may have the following two reasons: the hydrophobic groups of the protein may have all migrated to the molecular surface; the aggregation of hydrophobic residues causes the binding site of the ANS probe to be buried.

[0144] (7) Influence of free thiol content

[0145] Disulfide bonds are the force that maintains the secondary and tertiary structures of protein molecules. Free sulfhydryl groups (-SH) and disulfide bonds can be mutually converted in protein molecules under certain conditions. Therefore, changes in protein conformation can be indicated by changes in the free sulfhydryl content. Figure 6 It can be seen that the free thiol content of pea protein increases first and then decreases after ball milling. The free thiol content of pea protein without ball milling is 5.37±0.04μmol / g. After 10min of ball milling, the free thiol content increases to 5.49±0.03μmol / g, and then gradually decreases (p<0.05). The free thiol content decreases to 5.42±0.03μmol / g, 4.88±0.04μmol / g and 4.81±0.02μmol / g when the ball milling time is 20min, 30min and 40min, respectively. Li et al. found that ball milling increases the free thiol content of ovalbumin because the collision, shear force and friction force during the grinding process destroy the disulfide bonds, resulting in the exposure of free thiol groups previously located in the core of the protein molecule. This can explain the increase in the free thiol content of pea protein before 20min of ball milling. However, after the ball milling time exceeded 20 minutes, the free thiol content of pea protein began to gradually decrease, which was exactly the opposite of our results. This may be because during the ball milling process, the free thiol groups (free thiol groups on the surface of protein molecules) came into contact with oxygen in the air, causing the free thiol groups to be oxidized into disulfide bonds and the content to decrease.

[0146] (8) Influence of endogenous fluorescence spectrum

[0147] When the conformation of a protein changes, its fluorescence spectrum changes. These conformational changes alter the local molecular environment of the tryptophan, tyrosine, and phenylalanine groups, thus affecting the intensity and maximum absorption wavelength of the fluorescence. Figure 7 It can be seen that the frozen ball milling treatment reduced the fluorescence intensity of pea protein, and the fluorescence absorption band was blue-shifted but not obvious. Li et al. used six physical modification methods, including cold plasma, ball milling, ultrafine grinding, ultrasound, wet ball milling and high-pressure micro-injection, to reduce the fluorescence intensity of walnut protein, which may be related to the changes in the tertiary structure of the protein caused by the physical method. When the maximum absorption wavelength is blue-shifted, it indicates that the tryptophan residue is buried inside and is in a more hydrophobic state. This shows that the frozen ball milling treatment changes the overall spatial conformation of pea protein, causing the polar groups inside the protein to be exposed to the aqueous solution to varying degrees, and the polarity of the environment changes. With the increase of ball milling time, the fluorescence intensity of pea protein shows a trend of increasing first and then decreasing, which may be related to the fact that the ball milling treatment changes the aggregation state of the protein. The aggregation of the protein will change the local microenvironment of the chromogenic group and thus affect the fluorescence intensity.

[0148] (9) Influence of secondary structure

[0149] In the Fourier transform infrared spectrum, the amide I band (1600-1700 cm -1 ) is the absorption peak caused by C=O stretching vibration, and the second-order derivative is usually used to calculate the area of ​​each sub-peak to analyze and predict the secondary structure content of the protein. As can be seen from Table 4, the β-fold and β-turn of untreated pea protein account for the main part of the secondary structure, accounting for 41.81±0.16% and 28.46±0.26%, respectively, and the α-helix accounts for only 12.27±0.15. With the increase of ball milling time, the β-fold content of pea protein decreases from 41.81±0.16% to 37.81±0.21% at most, the β-turn content increases from 28.46±0.26% to 34.03±0.32% at most, and the α-helix content shows a trend of first decreasing and then increasing, which indicates that most of the β-fold is converted into β-turn. This result shows that ball milling makes the structure of pea protein more stretchable and improves the flexibility of pea protein molecules. β-folds mainly exist in the folding area inside the protein, and their stability is mainly maintained by hydrogen bonds between polypeptide chains. The decrease in β-fold content indicates that the spatial structure of the protein has changed, and the internal hydrophobic groups are exposed, resulting in an increase in the surface hydrophobicity of the protein, which is consistent with the experimental results in the previous article. The stability of the α-helix depends on the hydrogen bonds within the polypeptide chain. The high shear force, high friction force and high collision force used in the ball milling process may cause the hydrogen bonds between the peptide chains to be destroyed, which is consistent with the research results of Ding et al. The secondary structure of the protein depends not only on the sequence of amino acids, but also on the interaction between different parts of the molecule. After ball milling, the spatial structure of pea protein is destroyed, the peptide chain is unfolded, and various polar amino acid residues are exposed on the surface of the protein. Under the interaction force between molecules, various amino acid residues are rearranged to form a new protein conformation.

[0150] Table 4: Effect of cryo-milling on the secondary structure of pea protein

[0151] Sample name α-helix (%) β-fold (%) β-turn angle (%) Irregular curl (%) PPI 0 <![CDATA[12.27±0.15 a ]]> <![CDATA[41.81±0.16 a ]]> <![CDATA[28.46±0.26 a ]]> <![CDATA[17.47±0.25 a ]]> PPI 10 <![CDATA[11.34±0.10 b ]]> <![CDATA[40.23±0.08 b ]]> <![CDATA[31.74±0.06 b ]]> <![CDATA[16.69±0.08 b ]]> PPI 20 <![CDATA[10.75±0.09 c ]]> <![CDATA[39.98±0.42 b ]]> <![CDATA[32.64±0.11 c ]]> <![CDATA[16.64±0.11 b ]]> PPI 30 <![CDATA[10.94±0.09 c ]]> <![CDATA[37.81±0.21 c ]]> <![CDATA[33.80±0.12 d ]]> <![CDATA[17.46±0.12 ac ]]> PPI 40 <![CDATA[12.16±0.02 ad ]]> <![CDATA[38.29±0.21 d ]]> <![CDATA[34.03±0.32 d ]]> <![CDATA[15.52±0.16 d ]]>

[0152] (10) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0153] Pea proteins include legumin (11S), a hexameric protein consisting of L-α acidic subunits (8-40 kDa) and L-β basic subunits (19-22 kDa), and vicilin (V), a 7S trimeric protein consisting of 51, 70, and 19-12.5 kDa heterogeneous subunits. Figure 8As can be seen in the figure, there is no significant change in the main bands of the ball-milled pea protein samples (channels 3-6) and the untreated pea protein (channel 2), indicating that the ball-milling treatment did not affect the subunit composition and structure of pea protein. Physical modification improves the physicochemical properties of proteins by changing the particle size and spatial conformation, and does not produce new substances, so no additional bands will be detected.

[0154] (11) Scanning electron microscopy

[0155] Fig. 9 The figure shows the morphology of pea protein at a magnification of 500 times. It can be seen from the figure that the untreated pea protein is spherical, with an uneven surface, uneven particle size, but a tight structure. The microstructure of pea protein after cryo-milling has changed significantly, and the overall structure has been destroyed. After 10 minutes of ball milling, the pea protein changed from a spherical shape to a flat shape, and some spheres broke. As the ball milling time increased, the flat pea protein was also destroyed and fragmented, and the particle size gradually became consistent. When the ball milling time reached 30 minutes, the spherical structure of pea protein completely disappeared. As the ball milling time continued to increase, some fragmented pea proteins showed mutual aggregation. In general, the cryo-milling treatment destroyed the structure of pea protein, reduced the particle size of pea protein, and made the particle size more uniform.

[0156] 3. Effects of frozen ball-milled modified pea protein on the gelatinization and digestibility of rice starch

[0157] (1) Paste attributes

[0158] Table 5 shows the effect of pea protein with different ball milling treatment time on the gelatinization properties of rice starch. Compared with single rice starch, the addition of untreated pea protein reduced the peak viscosity of rice starch from 3848.33±17.21cp to 3435.33±26.56cp, the valley viscosity from 2388.00±5.57cp to 1991.00±5.00cp, the final viscosity from 4209.67±9.02cp to 3525.67±9.61cp, and the recovery value from 1726.00±6.56cp to 1603.00±6.25cp. This shows that pea protein significantly affects (p<0.05) the gelatinization properties of rice starch.

[0159] Peak viscosity can reflect the ability of starch granules to absorb water and swell. The lower the peak viscosity, the more inhibited the swelling of starch granules. Compared with single rice starch, the addition of pea protein with different ball milling times resulted in a decrease in the peak viscosity of the system. Among them, the peak viscosity of pea protein ball-milled for 20 minutes decreased by 523.66cp compared with pure rice starch, and decreased by 112.66cp compared with unmilled pea protein. Yang et al. added four endogenous proteins to barley starch and found that the peak viscosity of barley starch decreased, which is consistent with our experimental results, indicating that pea protein can inhibit the swelling of starch granules. The ball-milled pea protein-rice starch system has a lower peak viscosity than the untreated pea protein-starch system, which indicates that ball milling can enhance the ability of pea protein to inhibit the swelling of starch granules.

[0160] The attenuation value represents the difference between the peak viscosity and the valley viscosity, reflecting the ability of the starch slurry to resist shear at high temperatures. The smaller the attenuation value, the higher the stability. Compared with single rice starch, the attenuation value of pea protein with different ball milling treatment times decreased, but not significantly, indicating that pea protein has almost no effect on the gelatinization stability of rice starch. Zhang Zihao found that adding the same proportion of rice gluten to glutinous rice starch would increase the attenuation value of glutinous rice starch, but adding rice protein peptides had almost no effect on the disintegration value of glutinous rice starch, which may be related to the difference in protein.

[0161] The addition of pea protein with different ball milling treatment times resulted in a decrease in the retrogradation value of rice starch, which means that the addition of pea protein can inhibit the retrogradation of starch, which may be due to the inhibition of starch granule expansion and the decrease in starch paste viscosity. Amylose and pea protein interact by forming hydrogen bonds, hydrophobic forces and electrostatic adhesion, thereby reducing the number of amylose-amylose interactions and showing retrogradation inhibition. With the increase of ball milling treatment time, the retrogradation value of pea protein-rice starch complex decreased significantly. The retrogradation value of pea protein treated with ball milling for 10min, 20min, 30min and 40min was reduced by 43.00cp, 96.00cp, 95.00cp and 86.00cp respectively compared with the untreated pea protein. This may be because ball milling changes the spatial structure and particle size of pea protein, making it easier to interact with rice starch and inhibiting the gelatinization of rice starch.

[0162] In summary, pea proteins inhibited the gelatinization of rice starch. Their presence hindered the water absorption, swelling and disintegration of starch granules, while ball milling enhanced the gelatinization inhibition of starch by pea proteins.

[0163] Table 5: Effects of pea protein with different ball milling times on starch gelatinization properties

[0164]

[0165]

[0166] 3.3.2 Gel texture characteristics

[0167] As can be seen from Table 6, the addition of pea protein caused a significant change in rice starch gel (P<0.05). After adding pea protein, the elasticity and cohesion of rice starch gel were not significantly affected, but the hardness and adhesiveness decreased. The unmilled pea protein reduced the hardness of rice starch gel by 11.92%, while the pea protein ball-milled for 10min, 20min, 30min, and 40min reduced the hardness of rice starch gel by 16.16%, 19.60%, 20.81%, and 19.80%, respectively. Therefore, the ball-milled pea protein had a greater effect on the gel change of rice starch. The hardness of starch gel is highly correlated with the content of amylose. The higher the degree of cross-linking and entanglement between amylose molecules, the greater the hardness of starch gel. Ball milling treatment reduces the particle size of pea protein, improves solubility, and makes it more competitive with water, which hinders the aggregation and rearrangement of starch molecules, weakens the intermolecular forces of amylose, and reduces the hardness of gel. Xin et al. used ultrasonic treatment of rice protein to make it have smaller particle size and higher solubility, and applied it to composite gel. They found that ultrasonic treatment of rice protein reduced the hardness of the composite gel, which is consistent with our experimental results.

[0168] Table 6: Effect of pea protein with different ball milling time on starch gel texture

[0169] Sample name Hardness / N elasticity Cohesion Adhesiveness RS <![CDATA[4.95±0.06 a ]]> <![CDATA[0.99±0.01 a ]]> <![CDATA[0.52±0.01 a ]]> <![CDATA[2.57±0.03 a ]]> PPI0-RS <![CDATA[4.36±0.04 b ]]> <![CDATA[0.98±0.01 a ]]> <![CDATA[0.51±0.01 a ]]> <![CDATA[2.22±0.02 b ]]> PPI10-RS <![CDATA[4.15±0.08 c ]]> <![CDATA[0.98±0.01 a ]]> <![CDATA[0.51±0.01 a ]]> <![CDATA[2.12±0.04 c ]]> PPI20-RS <![CDATA[3.98±0.06 d ]]> <![CDATA[0.97±0.01 a ]]> <![CDATA[0.50±0.01 a ]]> <![CDATA[1.99±0.02 d ]]> PPI30-RS <![CDATA[3.92±0.07 e ]]> <![CDATA[0.98±0.01 a ]]> <![CDATA[0.51±0.01 a ]]> <![CDATA[2.00±0.03 d ]]> PPI40-RS <![CDATA[3.97±0.05 e ]]> <![CDATA[0.98±0.01 a ]]> <![CDATA[0.50±0.01 a ]]> <![CDATA[1.99±0.02 e ]]>

[0170] (3) Water absorption capacity, solubility and swelling capacity

[0171] Table 7: Effects of pea protein with different ball milling times on starch water absorption capacity, solubility and swelling capacity

[0172] Sample name Swelling index (g / g) Water absorption index (g / g) Solubility (%) RS <![CDATA[7.1825±0.0058 a ]]> <![CDATA[6.8377±0.0031 a ]]> <![CDATA[4.80±0.04 e ]]> PPI0-RS <![CDATA[6.9002±0.0109 b ]]> <![CDATA[6.5637±0.0112 b ]]> <![CDATA[4.88±0.02 b ]]> PPI10-RS <![CDATA[6.8070±0.0165 c ]]> <![CDATA[6.4716±0.0151 c ]]> <![CDATA[4.92±0.01 d ]]> PPI20-RS <![CDATA[6.7266±0.0047 d ]]> <![CDATA[6.3719±0.0064 d ]]> <![CDATA[5.27±0.04 c ]]> PPI30-RS <![CDATA[6.7477±0.0095 e ]]> <![CDATA[6.3736±0.0082 e ]]> <![CDATA[5.54±0.05 b ]]> PPI40-RS <![CDATA[6.7958±0.0075 c ]]> <![CDATA[6.4005±0.0055 e ]]> <![CDATA[5.82±0.03 a ]]>

[0173] The swelling degree can reflect the interaction between starch particles and water molecules. During the heating process, water molecules enter the starch, resulting in the leaching of amylose and the expansion of amylopectin molecules. As can be seen from Table 7, the addition of pea protein reduces the swelling index of rice starch. The swelling index of rice starch without pea protein is 7.1825±0.0058g / g. The addition of pea protein treated with ball milling for 0min, 10min, 20min, 30min, and 40min reduces the swelling index of rice starch to 6.9002±0.0109g / g, 6.8070±0.0165g / g, 6.7266±0.0047g / g, 6.7477±0.0095g / g, and 6.7958±0.0075g / g, respectively. This shows that pea protein affects the swelling capacity of starch. Liu et al. found that whey protein can significantly reduce the swelling capacity of potato starch, which is consistent with our experimental results. The reason may be that protein molecules compete with starch for water and inhibit the swelling of starch.

[0174] The water absorption capacity of starch is closely related to the swelling degree. As can be seen from Table 7, the effect of pea protein with different ball milling treatment times on the water absorption index of rice starch is the same as the effect on the swelling index, that is, pea protein can reduce the absorption of water molecules by rice starch. The hydrophilic groups of protein can bind to water molecules, while the hydrophobic groups repel other water molecules, resulting in some water molecules being unable to penetrate starch granules, causing the water absorption index of starch granules to decrease. The water absorption index of the pea protein-rice starch complex with ball milling treatment is lower than that of the pea protein-rice starch complex with untreated treatment. This may be because the ball milling treatment destroys the spatial structure of pea protein, resulting in a large number of internal hydrophobic groups being exposed, repelling water molecules, and making more water molecules unable to penetrate starch granules; when the ball milling time exceeds 30 minutes, the water absorption index of the pea protein-rice starch complex is improved, but it is not statistically significant (p>0.05).

[0175] Compared with single rice starch, the pea protein-rice starch composite system with pea protein added showed higher solubility. The longer the pea protein ball milling treatment time, the higher the solubility of the pea protein-rice starch composite system, and the solubility of PPI40-RS increased by up to 17.53%. Yang et al. found that adding cations can increase the solubility of potato starch, probably because cations can destroy or reduce the crystalline area of ​​starch, causing starch dissolution. Sun et al. found that the solubility of pea starch-peanut protein mixture increases with the addition ratio of peanut protein, because pea protein contains many charged amino acids, which may affect the formation of starch crystalline areas. Ball milling destroys the spatial structure of pea protein, exposing more charged amino acids, and thus showing greater solubility.

[0176] (4) Thermodynamic properties

[0177] The results of thermodynamic parameter determination of pea protein and rice starch mixture treated with different ball milling times are shown in Table 8. Compared with the control group, the gelatinization temperature (T0, T P and T C ) increased but not significantly, but the gelatinization enthalpy (ΔH) decreased significantly (p < 0.05). Adding pea protein treated with ball milling for 0min, 10min, 20min, 30min, and 40min reduced the gelatinization enthalpy of rice starch by 20.48%, 26.48%, 29.43%, 29.61%, and 29.25%, respectively. During the gelatinization process, the protein will surround the starch granules, limiting the water absorption and expansion of the starch, resulting in an increase in the gelatinization temperature. Yang et al. also found that whey protein isolate will form a wrap around corn starch, thereby increasing the anti-digestibility of starch. The following reasons may be responsible for the decrease in gelatinization enthalpy after adding pea protein: there is water competition between pea protein and rice starch, which affects the migration of water during starch gelatinization. The starch-water interaction in the starch-protein system is weak, so the gelatinization enthalpy (ΔH) decreases significantly; the addition of pea protein changes the arrangement structure of starch, and the weak electrostatic interaction between starch granules and protein leads to the depolymerization of starch granules or the loss of amorphous regions, which reduces the energy required for starch gelatinization; pea protein replaces part of the starch, changes the starch concentration, and reduces the energy required to destroy starch granules during gelatinization. ΔH shows a downward trend with the extension of ball milling treatment time. This is because ball milling changes the particle size of pea protein, reduces the particle size, and increases the specific surface area, so that pea protein can be wrapped on the surface of starch granules to a greater extent, the starch-protein interaction is stronger, and gelatinization is inhibited (ΔH) decreases. When the ball milling time exceeds 20 minutes, the particle size of pea protein no longer changes significantly. At the same time, the spatial structure is destroyed, the internal hydrophobic residues are exposed, the hydrophobic interaction is enhanced, and soluble aggregates are produced, resulting in reduced repulsion and surface charge. The interaction between starch and protein is highly affected by electrostatic interaction. Therefore, the ΔH change is no longer significant after the ball milling time exceeds 20 minutes.

[0178] Table 3-8 Effects of pea protein treated with different ball milling times on the thermodynamic properties of starch

[0179] sample <![CDATA[T0(℃)]]> Tp(℃) Tc(℃) ΔH(J / g) RS <![CDATA[58.62±0.02 a ]]> <![CDATA[63.02±0.01 a ]]> <![CDATA[66.96±0.01 a ]]> <![CDATA[11.18±0.04 a ]]> PPI0-RS <![CDATA[59.70±0.02 b ]]> <![CDATA[63.11±0.02 b ]]> <![CDATA[67.72±0.02 b ]]> <![CDATA[8.89±0.01 b ]]> PPI10-RS <![CDATA[60.03±0.02 c ]]> <![CDATA[63.16±0.03 c ]]> <![CDATA[67.54±0.03 c ]]> <![CDATA[8.22±0.02 c ]]> PPI20-RS <![CDATA[59.92±0.02 d ]]> <![CDATA[63.26±0.02 d ]]> <![CDATA[67.74±0.03 b ]]> <![CDATA[7.89±0.02 f ]]> PPI30-RS <![CDATA[59.81±0.01 e ]]> <![CDATA[63.15±0.03 bc ]]> <![CDATA[67.23±0.01 d ]]> <![CDATA[7.87±0.03 e ]]> PPI40-RS <![CDATA[59.83±0.01 e ]]> <![CDATA[63.14±0.02 bc ]]> <![CDATA[67.41±0.02 e ]]> <![CDATA[7.91±0.02 d ]]>

[0180] Note: T0 is the starting temperature, Tp is the peak temperature, Tc is the end temperature, and ΔH is the gelatinization enthalpy.

[0181] 3.3.5 Fourier transform infrared spectroscopy

[0182] Fourier transform infrared spectra of rice starch and pea protein-rice starch composites Fig.10 For starch samples, 3000-3800cm -1 The absorption peak at 2931 cm corresponds to the OH stretching vibration and is related to the intermolecular hydrogen bonding. -1 The position corresponds to the CH stretching vibration, 1644 cm -1 The peak at 1644 cm-1 corresponds to the O-bond stretching vibration of water molecules. Compared with RS, no new absorption peak was found in the sample with pea protein added, which indicates that there is no destruction or formation of chemical groups between pea protein and rice starch. However, the addition of pea protein resulted in a peak at 1644 cm-1. -1 and 3382cm -1 This indicates that pea protein may interact with starch through hydrogen bonds and affect the interaction between starch and water molecules. Wang Jing's research also proves that exogenous proteins and starch are combined through hydrogen bonds, electrostatic forces or hydrophobic interactions.

[0183] Table 9: Short-range ordered structural parameters of pea protein-starch treated with different ball milling

[0184]

[0185]

[0186] Table 9: Short-range ordered structural parameters of pea protein-starch treated with different ball milling

[0187] For sample 1200cm -1 -800cm -1 The infrared spectrum of the range was deconvoluted and recorded at 1047 cm -1 、1022cm -1 and 995cm -1 The short-range order of starch is usually related to the absorbance of 1022cm -1 and 995cm -1 The ratio is negatively correlated with 1047cm -1 and 1022cm -1 As can be seen from Table 9, after adding pea protein, 1047cm -1 and 1022cm -1 The ratio of 1022cm -1 and 995cm -1 The ratio of gradually increases, which indicates that the hydrogen bond strength and order degree of rice starch molecules decrease. Wu Tianhao et al. added ultrasonically treated soy protein isolate to corn starch and found that ultrasonic treatment reduced the particle size of soy protein isolate, inhibited the interaction between starch molecules, and caused the short-range order of starch particles to be destroyed, which is consistent with our experimental results.

[0188] 3.3.6 Scanning electron microscopy

[0189] The surface morphology of pea protein-rice starch composites observed by scanning electron microscopy Fig.12 As shown. Natural rice starch is angular and irregular in shape. After adding pea protein, the surface morphology of rice starch did not change significantly, but there was a tendency for rice starch and pea protein to aggregate with each other. The surface of pea protein is uneven, and rice starch can load its surface. The spherical structure of pea protein treated with ball milling for 10 minutes was broken, and more rice starch gathered at the broken end of the sphere. After ball milling for 20 minutes, pea protein was broken and fragmented, more rice starch and pea protein aggregated, and even some starch particles were embedded in the protein matrix. After ball milling for 30 minutes, pea protein was rearranged to form a new protein conformation under the intermolecular interaction force, and the aggregation between rice starch was weakened, but it was still higher than that of pea protein without ball milling. In vitro digestion also proved that ball-milled pea protein can better hinder water and enzymes from contacting starch than untreated pea protein, and inhibit starch gelatinization and digestion.

[0190] 3.3.7 In vitro digestion

[0191] The effect of pea protein on the in vitro digestibility of rice starch is shown in Table 10. Compared with RS, the RDS content of PPI0-RS, PPI10-RS, PPI20-RS, PPI30-RS, and PPI40-RS decreased by 3.29%, 6.43%, 9.07%, 8.85%, and 8.52%, respectively, and the SDS content increased by 1.41%, 2.83%, 5.15%, 5.17%, and 5.14%, respectively, and the RS content increased by 1.87%, 3.59%, 3.92%, 3.68%, and 3.37%, respectively, which indicates that pea protein can inhibit the gelatinization of starch. Existing research results have shown that protein can bind to starch through non-covalent bonds (hydrogen bonds, hydrophobic interactions, and electrostatic attraction), acting as a physical barrier to hinder the binding of starch to enzymes, thereby inhibiting the hydrolysis of starch. Compared with PPI0-RS, the RDS content of the pea protein-rice starch complex ball-milled within 20 minutes decreased with the increase of ball-milling time; the RDS content of the ball-milling time exceeding 20 minutes decreased slightly but was not statistically significant. The content of SDS and RS also no longer changed significantly after pea protein ball-milling for 20 minutes, which indicates that ball-milling for 20 minutes may be the key condition for modification. The reason may be that the particle size of pea protein changed greatly before ball-milling for 20 minutes, the particle size decreased, the specific surface area increased, and it was able to combine with more starch particles. At the same time, the exposure of the internal hydrophobic groups enhanced the interaction between protein and starch and hindered the interaction between starch and amylase. When the ball-milling time exceeded 20 minutes, the particle size change of pea protein was no longer as significant as before, and it was rearranged to form a new protein conformation under the interaction force between protein molecules, and the interaction with starch was slightly weakened.

[0192] Table 10: Effect of ball-milled modified pea protein on in vitro digestion of rice starch

[0193] sample RDS(%) SDS(%) RS(%) RS <![CDATA[69.51±0.08 a ]]> <![CDATA[16.32±0.17 a ]]> <![CDATA[14.17±0.10 a ]]> PPI0-RS <![CDATA[66.22±0.09 b ]]> <![CDATA[17.73±0.13 b ]]> <![CDATA[16.04±0.14 b ]]> PPI10-RS <![CDATA[63.08±0.25 c ]]> <![CDATA[19.15±0.19 c ]]> <![CDATA[17.76±0.07 c ]]> PPI20-RS <![CDATA[60.44±0.08 d ]]> <![CDATA[21.47±0.14 d ]]> <![CDATA[18.09±0.11 d ]]> PPI30-RS <![CDATA[60.66±0.05 d ]]> <![CDATA[21.49±0.16 d ]]> <![CDATA[17.85±0.13 cd ]]> PPI40-RS <![CDATA[60.99±0.10 d ]]> <![CDATA[21.46±0.07 d ]]> <![CDATA[17.54±0.08 cd ]]>

[0194] Note: RDS, SDS and RS stand for rapidly digestible starch, slowly digestible starch and resistant starch, respectively.

[0195] 4. Effect of frozen ball-milled modified pea protein on the color stability of blueberry anthocyanins

[0196] 3.4.1 Effects of heat treatment and light treatment on the color stability of blueberry anthocyanins

[0197] Table 11 shows the color changes of blueberry anthocyanins before and after heat treatment by frozen ball-milled modified pea protein at different concentrations. As can be seen from the table, after heat treatment, each group of samples showed an increase in L*, a decrease in C*, and a decrease in Hue. oThe trend of increasing ΔE values ​​is manifested as a lighter solution color, less saturation, and a yellowish hue, indicating that the solution faded significantly after heat treatment, which is consistent with Chen Xi's research results. In general, people can study the stability of color by the change in ΔE, and a lower ΔE value indicates better color stability. As can be seen from Table 3-11, the ΔE value of the blueberry anthocyanin solution without the addition of pea protein is higher, because anthocyanins undergo a series of reactions such as deglycosylation, water nucleophilic attack, cleavage and polymerization at high temperatures, which accelerates their degradation. As the concentration of pea protein increases, the ΔE value of the blueberry anthocyanin solution continues to decrease, which shows that pea protein can prevent the fading of the blueberry anthocyanin solution to a certain extent. At the same protein concentration, the ΔE value of PPI20-BAS is lower than that of BAS, PPI0-BAS and PPI40-BAS, which indicates that pea protein treated with ball milling for 20 minutes has the best color preservation effect on blueberry anthocyanins. The reason may be that ball milling reduces the particle size of pea protein, making the pea protein and blueberry anthocyanins more tightly bound; at the same time, the conformation of pea protein changes, which is conducive to better binding of pea protein and blueberry anthocyanins. Wang et al. combined preheated whey protein with rose anthocyanins and found that the two were more tightly bound. The reason is that preheating can reduce the particle size of whey protein. The smaller the particle size, the tighter the binding of anthocyanins, which is consistent with our experimental results. The color preservation effect of pea protein treated with ball milling for 40 minutes on blueberry anthocyanins is lower than that of pea protein treated with ball milling for 20 minutes. This may be because excessive ball milling causes pea protein to rearrange under the intermolecular interaction force to form a new stable protein conformation, and the interaction between pea protein and blueberry anthocyanins is weakened.

[0198] In the light treatment experiment, the pea protein ball-milled for 20 minutes also had a better stabilizing effect on the color of blueberry anthocyanins than the pea protein ball-milled for 40 minutes and the untreated pea protein. As can be seen from Table 12, the ΔE values ​​of blueberry anthocyanins without pea protein addition were higher than those of PPI0-BAS, PPI20-BAS and PPI40-BAS on the 1st, 3rd, 5th and 7th days, indicating that the color change was the largest. The ΔE value of PPI20-BAS was the lowest, indicating that the color change was the smallest. The ΔE values ​​of PPI20-BAS on the 1st, 3rd, 5th and 7th days were 52.48%, 44.27%, 41.39% and 39.99% lower than those of BAS, respectively, indicating that with the increase of light treatment, the protection of pea protein on the color stability of blueberry anthocyanins was weakened. The above results show that appropriate ball milling can enhance the interaction between pea protein and blueberry anthocyanins, thereby enhancing the color stability of blueberry anthocyanins.

[0199] Table 11: Effects of different concentrations of ball-milled modified pea protein on the color change of blueberry anthocyanins at 80°C

[0200]

[0201] Note: L* value represents the brightness of the solution (from 0 to 100 represents from black to white); C* value represents the saturation or intensity of the color; Hue° value represents the hue angle (0°: red; 90°: yellow; 180°: green; 270°: blue); ΔE is the color difference between the sample before heating and the sample after heating.

[0202] Table 12: Effect of ball-milled modified pea protein on the color stability of blueberry anthocyanins under light conditions

[0203]

[0204]

[0205] (2) Fluorescence quenching mechanism and binding constant

[0206] The intrinsic fluorescence of proteins is mainly caused by tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). At an excitation wavelength of 280nm, Trp and Tyr have maximum emission wavelengths, while Phe is difficult to observe. The intrinsic fluorescence spectrum of proteins can usually be used to explore the interactions and structural changes between proteins and other substances such as polyphenols. Fig.13 The fluorescence images of PPI0, PPI20 and PPI40 with different concentrations of blueberry anthocyanins at 25°C are shown. It can be seen from the figure that the fluorescence intensity of PPI0, PPI20 and PPI40 decreases with the increase of the concentration of blueberry anthocyanins, and a slight red shift occurs, which indicates that blueberry anthocyanins interact with pea protein, and the decrease in protein fluorescence intensity is due to the quenching effect of blueberry anthocyanins on pea protein.

[0207] To further understand the binding mechanism, the fluorescence data were analyzed using the Stern-Volmer equation, using [Q] as the independent variable and F0 / F as the dependent variable to fit a linear function equation. q Exceeding the limiting rate constant of the protein (2×10 10 M -1s-1 ), static quenching is the main quenching mechanism. Fig.14 The Stern-Volmer plots of PPI0, PPI20 and PPI20 at 25°C, 35°C and 45°C are shown respectively. Table 13 shows the calculated quenching constants (K SV and K q (K q =K SV / τ0=K SV ×10 8 )) values. As can be seen from the table, PPI0, PPI20 and PPI40 have a significant relationship with the K qThe values ​​are all greater than the limiting rate constant of protein (2×10 10 M -1s-1 ), showing static quenching, which indicates that pea protein and blueberry anthocyanins have a strong binding force. Linear regression was performed on lg[(F0-F) / F] against lg[Q], and the slope and intercept obtained were the number of binding sites (n) and binding constant (K) of the two molecules, respectively. s ). The binding constant (K s ) value can reflect the affinity between protein and quencher. The K s The value is the largest, indicating that the affinity of PPI20 to blueberry anthocyanins is higher than that of PPI0 and PPI40. In addition, all n values ​​are about 1, indicating that there is only one binding site between pea protein and blueberry anthocyanins.

[0208] Table 13: Quenching constants, binding constants and binding sites of pea protein and blueberry anthocyanin at different temperatures

[0209]

[0210]

[0211] (3) Synchronous fluorescence spectroscopy

[0212] Synchronous fluorescence spectroscopy uses a scanning monochromator to excite and emit at specific wavelengths to observe the details of structural changes in protein-ligand interactions. Spectra with excitation and emission wavelength intervals (Δλ) fixed at 15nm and 60nm can show the characteristics of Tyr and Trp residues, respectively. Fig.16 and Fig.17 The synchronous fluorescence spectra of PPI0-BAS, PPI20-BAS and PPI40-BAS at Δλ=15nm and Δλ=60nm are shown respectively. When Δλ=15nm, the fluorescence intensity of PPI0-BAS, PPI20-BAS and PPI40-BAS decreases with the concentration of blueberry anthocyanins, and the maximum emission wavelength red-shifts by 2nm, 4nm and 3nm, respectively, indicating that the microenvironment of the Tyr region has changed, the polarity of Tyr has increased, and it is exposed to a more hydrophilic microenvironment. At Δλ=60nm, as the concentration of blueberry anthocyanins increases, the fluorescence intensity decreases continuously, but the maximum emission wavelength does not change significantly. This shows that pea protein is less affected by Trp and is mainly affected by Tyr. These changes in synchronous fluorescence spectra indicate that pea protein interacts with blueberry anthocyanins, resulting in changes in the microenvironment of amino acid residues.

[0213] (4) Infrared spectroscopy

[0214] from Fig.18It can be seen that the characteristic absorption bands of untreated pea protein can be observed at 3397.34 (amide A band, OH vibration or NH stretching vibration), 1650.44 (amide I band, C=O stretching vibration and NH vibration) and 1540.11 (amide II band, NH vibration and CN vibration). The amide A band and amide II band of ball-milled pea protein both shifted to higher wavenumbers. The secondary structure of ball-milled pea protein changed, and the specific results are shown in Table 4. After adding blueberry anthocyanidins, the amide A band of pea protein continued to move to higher wavenumbers. This result may be due to the incorporation of OH groups into polyphenol molecules, which produced stronger OH-stretching vibrations, indicating that pea protein interacted with blueberry anthocyanidins. After the addition of blueberry anthocyanidins to the ball-milled pea protein, the peak positions of the amide I band and amide II band changed further, indicating that blueberry anthocyanidins further changed the secondary structure of pea protein. The contents of α-helix and random coil decreased significantly, and the content of β-turn structure increased significantly, indicating the transformation of random structure into more regular structure and more stable protein structure, which may be the reason for the improved thermal stability of blueberry anthocyanins.

[0215] Table 14: Effect of adding blueberry anthocyanins on the secondary structure of pea protein

[0216]

[0217]

[0218] 3.4.5 UV absorption spectrum

[0219] Pea protein has a characteristic absorption peak at 260-280nm due to the combined effects of tyrosine (275nm), tryptophan (279nm) and phenylalanine (257nm). PPI20 and PPI40 have higher UV-visible light absorption intensity than PPI0, which may be due to the change in protein conformation caused by ball milling modification, exposing more hydrophobic amino acid residues on the surface of protein molecules. After adding blueberry anthocyanins, the peak intensity of PPI0, PPI20 and PPI40 at 270-280nm increased and slightly red-shifted, which may be due to the increase in the hydrophobicity of the microenvironment around the aromatic amino acid residues, further proving that pea protein interacts with blueberry anthocyanins.

[0220] This example studies the effect of frozen ball milling modification on the physicochemical properties and functional properties of pea protein, and adds the modified pea protein to rice starch to prepare a pea protein-rice starch composite. The main conclusions are as follows:

[0221] (1) Effects of cryo-ball milling modification on the physicochemical and functional properties of pea protein

[0222] The cryo-milling treatment changed the spatial structure of pea protein, significantly reduced the particle size, and changed the surface hydrophobicity and fluorescence intensity, which shows that cryo-milling is conducive to the unfolding of the internal structure of the protein, thereby improving the functional properties of pea protein. When the ball milling time is 20 minutes, the solubility of pea protein increases from 3.08±0.12% to 12.37±0.21%, the emulsification increases by 47.59%, and the emulsification stability increases by 11.17%, so the ball milling treatment for 20 minutes may be the most suitable modification condition.

[0223] (2) Effects of frozen ball-milled modified pea protein on the gelatinization and digestibility of rice starch

[0224] Compared with rice starch, the pea protein-rice starch composite with pea protein added had lower recovery value, gel hardness, gelatinization enthalpy and order degree; compared with the unmodified pea protein-rice starch composite system, the effect of the ball-milled pea protein-rice starch composite system was more significant (P<0.05), and the gelatinization enthalpy of the pea protein-rice starch composite modified by ball milling for 20 minutes decreased by 13.72%, and the mass fractions of slowly digestible starch and resistant starch increased by 3.74% and 2.05%, respectively. Before ball milling for 20 minutes, the effect of pea protein on rice starch changed greatly. When the ball milling time exceeded 20 minutes, the effect was no longer as obvious as before, which may be related to the change of pea protein particle size. Fourier transform infrared spectroscopy results showed that hydrogen bonding was the main driving force for the interaction between pea protein and rice starch. Scanning electron microscopy also showed that the conformation of the pea protein-rice starch composite treated by ball milling changed, which may be the main reason affecting the gelatinization characteristics and digestibility of rice starch.

[0225] (3) Effect of frozen ball-milled modified pea protein on the color stability of blueberry anthocyanins

[0226] Heat treatment and light treatment experiments proved that ball-milled pea protein had a certain protective effect on the color stability of blueberry anthocyanins, among which pea protein ball-milled for 20 minutes had the best effect. Fluorescence spectroscopy, infrared spectroscopy and ultraviolet spectroscopy verified the interaction between the two: pea protein and blueberry anthocyanins had a high affinity, belonging to static quenching, and the binding site was about 1; blueberry anthocyanins changed the secondary structure of pea protein, and the complex showed a more stable structure, while affecting the microenvironment of amino acid residues. The research results provide inspiration for the interaction between pea protein and anthocyanins, and provide direction for further practical application in food.

[0227] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for preparing a composite containing pea protein, characterized in that: The steps include: Step 1: placing the pea protein in a ball mill for ball milling. During the ball milling process, the liquid nitrogen is always turned on and the temperature is maintained between -20°C and -10°C. The ball milling time is 5 min to 50 min to obtain frozen ball milled modified pea protein. Step 2: Add water to dry rice starch to prepare starch milk, then add water to dissolve frozen ball-milled modified pea protein to obtain a frozen ball-milled modified pea protein solution, then add the frozen ball-milled modified pea protein solution to the starch milk, stir at room temperature to mix the starch milk and the frozen ball-milled modified pea protein solution evenly, and freeze-dry to obtain a pea protein-rice starch complex; alternatively, prepare the frozen ball-milled modified pea protein into a frozen ball-milled modified pea protein solution using PBS buffer, and then mix it with the blueberry anthocyanin solution, stir in the dark at room temperature to obtain a pea protein-blueberry anthocyanin complex.

2. The method for preparing a complex containing pea protein according to claim 1, characterized in that: During ball milling, the speed of the ball mill is set to 400-600r / min, and the rotation direction is alternated every 4-6 minutes.

3. The method for preparing a complex containing pea protein according to claim 1, characterized in that: The concentration of the starch milk is 3-8 g / 100 g, and the mass of the rice starch is 5-15% of the mass of the frozen ball-milled modified pea protein.

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