CN115039830B

CN115039830B - Preparation method of shellac co-folded soybean protein isolate cold gel

Info

Publication number: CN115039830B
Application number: CN202210675477.4A
Authority: CN
Inventors: 吴卫成; 郭阳; 张治国; 章检明; 刘大群; 张程程; 张俊; 蔡静
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2024-05-17
Anticipated expiration: 2042-06-15
Also published as: CN115039830A

Abstract

The invention discloses a preparation method of shellac co-folded soybean protein isolate cold gel, which comprises the following steps: preparing a soybean protein isolate aqueous solution with the mass concentration of 11-13%; preparing shellac water solution with mass concentration of 11-13% and pH of 8+ -0.5; mixing the soybean protein isolate aqueous solution with the shellac aqueous solution, firstly adjusting the pH value of the obtained mixed solution to be 12+/-0.5, and then continuously stirring for reaction; after the reaction is finished, firstly adjusting the pH value of the obtained reaction solution to 8.5-9, and then centrifuging to remove gas to obtain a gel pre-solution; and (3) placing the gel pre-solution at the temperature of 4+/-1 ℃ for 12-15 hours to obtain the shellac co-folded soybean protein isolate cold gel. The shellac co-folded soybean protein isolate cold gel prepared by the invention has good texture characteristics and water retention capacity, and can be used as a delivery system for embedding active ingredients.

Description

Preparation method of shellac co-folded soybean protein isolate cold gel

Technical Field

The invention belongs to the technical field of preparation of soybean protein isolate cold gel, and relates to a preparation method of shellac modified soybean protein isolate gel.

Background

Soy protein isolates widely function in the food industry to increase protein content and form matrices with water, fat, spices, other ingredients of the food, based on their gel properties. Aggregation is a critical step in the formation of a gel from proteins, soy protein isolate gels can form a gel network structure by thermally induced covalent and serial non-covalent bonds, but are not suitable for loading or entrapment of heat sensitive bioactive components. Another type of protein gel is often induced by protein isoelectric point (acid-induced) gel or salt ions, such as calcium chloride and sodium chloride, and ferrous and magnesium ions can also be used to prepare protein cold gels with higher nutritional value and function. The main advantage of protein cryogels is that they are based on the denaturing exposure of different functional groups within the protein molecule, while at the same time interactions with other bioactive substances or polypeptide chains, such as hydrogen bonding, electrostatic and hydrophobic interactions, occur, which can be used to design targeted delivery systems. In addition, the cold gel can better control the shape, structure and texture of the formed gel.

The pH shift technology is a simple, convenient and low-cost method for changing the spatial structure and function of protein. The protein is fully unfolded in an extremely alkaline environment, so that the conformation of the globular protein is changed, a 'fused globular' structure is generated, and then the pH value is adjusted to be neutral so that the protein refolding. In addition to improving the functional properties of the protein itself after refolding, co-folding with another component during this process can be used to develop edible materials with new structures or functions.

Shellac is a natural polymer composed of wood eleostearic acid and cyclic terpene acid, and is linked through ester bonds to serve as a hydrophobic part and a hydrophilic part respectively; the carboxyl in the molecular structure does not participate in the esterification reaction of the cyclic terpene acid, so that the shellac is endowed with weak acidity, and therefore, the shellac is soluble in alkaline solution but not acidic solution and has pH responsiveness. Shellac can interact with hydrophilic polymers based on non-covalent interactions. Shellac has received increasing attention in the development of new food products due to its unique chemical properties, including the construction of food delivery systems, food foaming agents or oleogels, emulsifiers.

At present, nanometer particles or protein films with active ingredient loading function are prepared by utilizing the co-folding of shellac and protein so as to endow products with shellac intestinal phase compatibility or moisture resistance, but the invention of forming protein cold gel by utilizing the co-folding reaction between shellac and protein is not known.

Disclosure of Invention

The invention aims to provide a method for preparing soybean protein isolate cold gel based on a co-folding principle.

In order to solve the technical problems, the invention provides a preparation method of shellac co-folded soy protein isolate cold gel (namely, a method for constructing soy protein cold gel by using shellac co-folded soy protein), which comprises the following steps:

1) Preparing soybean protein isolate aqueous solution with the mass concentration of 11-13% (w/w);

2) Preparing shellac water solution with the mass concentration of 11-13% (w/w) and the pH value of 8+/-0.5;

3) Isolated proteins according to soy: shellac=1.3 to 2.5: the mass ratio of 1 is that of the components,

Mixing the soybean protein isolate solution obtained in the step 1) with the shellac aqueous solution obtained in the step 2), firstly adjusting the pH value of the obtained mixed solution to be 12+/-0.5, and then continuously stirring and reacting for 2+/-0.5 hours;

after the reaction is finished (after the set reaction time is reached), firstly adjusting the pH value of the obtained reaction solution (solution after the callback reaction) to 8.5-9, and centrifuging to remove gas (4800 g for 15 min) to obtain gel pre-solution;

description: dialyzing part of the pre-solution in a 3kDa ultrafiltration centrifuge tube, and using the obtained co-folded substance after freeze-drying for characterization of protein properties;

4) And (3) placing the gel pre-solution at the temperature of 4+/-1 ℃ for 12-15 hours to obtain the shellac co-folded soy protein isolate cold gel (namely, the co-folded soy protein isolate cold gel).

As an improvement of the preparation method of the shellac co-folded isolated soy protein cold gel of the invention:

the step 1) is as follows:

Adding water (purified water) to 11-13 g of isolated soy protein to 100g, stirring for 1.5-2.5 h by a magnetic stirrer, and then storing for 12-15 h at 0-4 ℃ to completely hydrate the isolated soy protein, so as to form an aqueous solution of the isolated soy protein with the mass concentration of 11-13%;

The step 2) is as follows:

Adding water (purified water) to 11-13 g of shellac to 100g, regulating the pH value to 8+/-0.5, and continuously magnetically stirring at 45-55 ℃ until the shellac is completely dissolved to obtain the shellac aqueous solution with the mass concentration of 11-13% and the pH value of 8+/-0.5.

As a further improvement of the process for preparing a shellac co-folded soy protein isolate cold gel of the present invention, in step 3):

Regulating the pH value of the mixed solution to 12+/-0.5 by using NaOH with the mass concentration of 2%;

The pH of the resulting reaction solution was adjusted to 8.5 to 9 with 1M HCl solution.

Further improvements in the preparation method of shellac co-folded isolated soy protein cold gel of the present invention:

the step 1) is as follows:

adding water to 12g of isolated soy protein to 100g, stirring for 2h by a magnetic stirrer, and storing at 0-4 ℃ for 12-15 h to form a soy protein isolate aqueous solution with the mass concentration of 12%;

The step 2) is as follows:

adding water to 12g of shellac to 100g, regulating the pH value to 8, and continuously magnetically stirring at 50 ℃ until the shellac is completely dissolved to obtain shellac water solution with the mass concentration of 12% and the pH value of 8;

the step 3) is as follows:

According to the soy protein: shellac = 2.1:1, mixing a soybean protein aqueous solution and a shellac solution, regulating the pH value of the blend to 12, and continuously stirring at 25 ℃ for reaction for 2 hours; then, firstly adjusting the pH value of the obtained reaction solution (solution after reaction) to 9, and then centrifuging to obtain a gel pre-solution;

The step 4) is as follows:

And standing and preserving the gel pre-solution at 4 ℃ for 15 hours to obtain the shellac co-folded soybean protein isolate cold gel (soybean protein shellac co-folded cold gel).

The invention successfully prepares the protein cold gel with the function of reducing the in-vitro hydrolysis rate of the isolated soy protein by taking the principle that the isolated soy protein and the shellac are co-unfolded under an alkaline environment and then the pH environment is adjusted to induce the co-folding. The method is characterized by the change of the primary structure, the spatial conformation and the thermal stability of the combined protein, and the gel characteristics of the formed protein cold gel, such as hardness, rheological property and the like, are evaluated, and the microstructure of the cold gel is observed through a scanning electron microscope.

The invention utilizes shellac to be unfolded together with protein in an extremely alkaline environment, then adjusts the pH value of the solution, and carries out a co-folding reaction to prepare the protein cold gel, the cold gel has good mechanical property, and the in-vitro digestion characteristic of the soybean protein isolate is obviously reduced, so that the protein cold gel is beneficial to the construction of a delivery system serving as an active ingredient. The cold gel can carry thermosensitive nutrients or functional active substances, and widens the application of the protein gel.

In conclusion, the soybean protein isolate cold gel is prepared by co-folding shellac and the soybean protein isolate at normal temperature, the preparation process is mild, the reaction is carried out at room temperature, no organic reagent is added, and the preparation method is environment-friendly.

The shellac co-folded soybean protein isolate cold gel prepared by the method has good texture characteristics (hardness is 23.57 g) and water retention (more than 90%) and can be used as a delivery system for embedding active ingredients, and meanwhile, researches show that the shellac co-folded soybean protein isolate cold gel effectively reduces the in vitro digestion degree (intestinal digestion hydrolysis degree DH is 15% lower than that of the soybean protein isolate) of the soybean protein isolate and is suitable for delivering sensitive active ingredients.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a co-folded electrophoresis chart of soybean protein isolate/shellac mass ratio 1,1.3,1.5,1.7,2.1,2.5 and blank comparative example-control:

(A) The reduction electrophoresis-electrophoresis sample is added with beta-mercaptoethanol;

(B) The non-reducing electrophoresis-electrophoresis sample is not added with beta-mercaptoethanol;

Comparative example 1 (SS 1), comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5), blank comparative example (control);

(C) The resulting co-fold electrophoresis patterns of different co-folding pH values (lanes 1-4 are in turn the reduction electrophoresis of comparative example 6, comparative example 7, comparative example 4, comparative example 5, lanes 5-8 are the non-reduction electrophoresis);

FIG. 2 is an infrared spectrum of a soy protein isolate/shellac co-fold;

(A) Infrared spectra of blank comparative example co-fold (control), soy Protein Isolate (SPI), comparative example 8 co-fold (CSmix), example 1 co-fold (SS 2.1), example 3 co-fold (SS 2.5), and Shellac (Shellac);

(B) Infrared spectra of different soy protein isolate/Shellac mass ratios of cofolds—comparative example 8 (CSmix), comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5) and Shellac (Shellac);

(C) Different co-folding pH- -blank control, example 1 (SS 2.1-9), comparative example 4 (SS 2.1-7), comparative example 5 (SS 2.1-8), example 2 (SS 1.7-9), comparative example 6 (SS 1.7-7), comparative example 7 (SS 1.7-8);

FIG. 3 shows fluorescence spectra of the resulting co-folds for each case;

(A) Cofolds for different soy protein isolate/shellac mass ratios- -comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5), blank comparative example (control) and Soy Protein Isolate (SPI).

(B) Cofolds of different cofolding pH-comparative example 6 (SS 1.7-7), comparative example 7 (SS 1.7-8), example 2 (SS 1.7-9), comparative example 4 (SS 2.1-7), comparative example 5 (SS 2.1-8) and example 1 (SS 2.1-9);

FIG. 4 shows the effect of different mass ratios of pH9 co-folded soy protein isolate/shellac- -comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5), on the condensed gum texture (A), water retention and swelling ratio (B);

FIG. 5 is a graph showing the effect of different mass ratios of pH 9 co-folded soy protein isolate/shellac- -comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5), on the loss modulus G 'and storage modulus G' of a cryogel under a change in strain;

FIG. 6 is an electron microscope scan of a cold gel at pH 9 with different soy protein isolates/shellac mass ratios co-folded;

In fig. 6: A-F was 1.3 (comparative example 2), 1.5 (comparative example 3), 1.7 (example 2), 2.1 (example 1), 2.5 (example 3) for a mass ratio of 500 μm, respectively, and the gel microstructure of the comparative example (control) was blank; a-f are the corresponding microstructures at 100 μm;

FIG. 7 in vitro simulated digestion of pH 9 cofolded soy protein isolate/shellac cold gel- -blank comparative example (conrol), comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1), example 3 (SS 2.5), variation in degree of hydrolysis.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

in the present invention, the stirring speed was 500.+ -.50 rpm.

Example 1, a method of preparing a soy protein isolate/shellac cold gel (i.e., shellac cofolded soy protein isolate cold gel) followed in sequence by the steps of:

1) Adding 88g of purified water into 12g of isolated soy protein, stirring for 2 hours by a magnetic stirrer, standing and preserving in a refrigerator at the temperature of 4 ℃ for 12-15 hours, and hydrating to completely hydrolyze the isolated soy protein to obtain an isolated soy protein aqueous solution with the mass concentration of 12%;

2) 88g of purified water was added to 12g of shellac and the pH was adjusted to 8 (with 2% NaOH solution) and magnetic stirring was continued at 50℃until shellac was dissolved (stirring time of about 4 hours) to give an aqueous shellac solution having a mass concentration of 12% and a pH of 8.

3) Isolated proteins according to soy: shellac=2.1 (i.e., mass ratio of soy protein isolate: shellac=2.1:1), mixing the aqueous soy protein isolate solution and the aqueous shellac solution, adjusting the pH of the blend to 12 (adjusted with NaOH solution having a concentration of 2%), and continuing the electric stirring reaction at room temperature for 2 hours to achieve complete development of the soy protein isolate and shellac; after the reaction is finished (after the set reaction time is reached), firstly, regulating (adjusting back) the pH of the solution after the reaction to 9 by using 1M HCl solution, and then centrifuging for 15min (the purpose of centrifugation is to degas) by 4800g to obtain gel pre-solution;

4) And placing the gel pre-solution in a refrigerator with the temperature of 4 ℃ for standing and preserving for 15 hours to obtain the soybean protein isolate shellac co-folding cold gel.

And (3) removing unreacted shellac or salt ions by ultrafiltration centrifugation (repeated centrifugation is carried out for 2 times by using an ultrafiltration centrifuge tube with molecular weight cutoff of 3kDa and the rotating speed is 4000 Xg for centrifugation for 15 minutes) of the gel pre-solution obtained in the step (3), collecting the solution in the inner tube of the ultrafiltration centrifuge tube after centrifugation, and determining the property of the modified protein by using the obtained co-folded substance after freeze drying.

The measurement method of the soybean protein isolate shellac co-folded substance and the property of the co-folded gel is as follows:

The following one to three were measured for the co-fold:

1. sodium dodecyl sulfate-polypropylene gel electrophoresis:

Experiments are carried out according to SDS-polyacrylamide gel electrophoresis in the annex V of Chinese pharmacopoeia, and reduction electrophoresis and non-reduction electrophoresis are carried out to obtain the product with the addition of 0.15mL of beta-mercaptoethanol and the product without the addition of beta-mercaptoethanol:

The concentration of the concentrated gel and the concentration of the separating gel are 5% and 10%, respectively, and a Marker with the molecular weight distribution in the range of 14.4-250 kDa is selected for carrying out SDS-PAGE electrophoresis experiments, wherein beta-mercaptoethanol is added into a co-folded sample for carrying out the reduction electrophoresis experiments, and beta-mercaptoethanol is not added for carrying out the non-reduction electrophoresis experiments.

2. Determination of the Fourier Infrared Spectrometry of the Co-folded Material

The co-fold was analyzed for infrared spectroscopy using a fourier infrared spectrometer. The scan parameters were as follows: the wavelength range is 600-3500cm ^-1, the resolution is 4cm ^-1, and 32 scans are performed for each sample.

3. Determination of fluorescence spectra of co-folds

A1 mg/mL concentration of the co-folded solution was prepared using a phosphate buffer solution (0.1 mol/L, pH 7). The chromophores such as tryptophan in protein molecules are used as probes, the excitation wavelength is 295nm, the emission wavelength is 300-450nm, and the excitation and emission slit widths are 2.5nm respectively.

4. Determination of the texture characteristics of the soy protein isolate/shellac co-folded condensate

Gel strength of the gel formed at the co-folded pH 9 (i.e., the soy protein isolate shellac co-folded cold gel obtained in step 4) was determined using a Rapid TA, tengba texture Analyzer, P/50 probe. The gel was formed in glass beakers with a height and an inner diameter of 55mm and 35mm, respectively. The maximum penetration force, defined as the force required to break the gel, is measured and expressed as the gel hardness. The test speed was 0.5mm/s.

5. Determination of Water-holding Capacity and swelling Property of Soybean protein isolate/shellac Co-folded Cold gel

Water retention capacity: the pH9 cofolding gel (i.e., the soy protein isolate shellac cofolding cold gel from step 4) was placed in a 5mL centrifuge tube and centrifuged at 14000 Xg for 20min. The free water was carefully sucked up with a syringe, the residual water was wiped off with filter paper, and the Water Holding Capacity (WHC) was calculated as follows:

wherein-W1 is the total weight of gel before centrifugation and centrifuge tube-g

W2 is the total weight of gel and centrifuge tube after centrifugation-g

Swelling properties: 3g of the pH 9 cofolding gel was immersed in 80mL of purified water for 12 hours, after which the carefully dried gel was removed to indicate moisture, weighed, and the Swelling (SR) calculated as follows:

wherein-M ₁ is the original gel weight

M ₂ is the weight of the gel after swelling

6. Determination of rheological properties of Soy protein isolate/shellac cofolding Cold gel

The rheology of the pH9 cofolded cryogel was determined using a HAAKE MARS Siemens rheometer. Plates with a pitch of 1mm and a diameter of 40mm were selected, the strain test fixed frequency was 1Hz and the temperature was 25 ℃.

7. Observation of microstructure of soybean protein isolate/shellac co-folded cold gel

After freeze-drying of the pH9 cofolding cryogel, the gold spraying was observed with a Hitachi scanning electron microscope at an accelerating voltage of 3 kV.

8. Determination of degree of hydrolysis in vitro simulated digestion of soy protein isolate/shellac cofolding cryogel

All pH9 co-folded cryogels were adjusted to the same protein concentration and cut into 3mm by 3mm blocks. They were then mixed with an equal volume of simulated gastric fluid solution (2000U/mL pepsin) and the pH of the gastric phase simulated fluid was adjusted to 3. The mixture was digested in a water bath at 37℃and 170rpm for 2h. In the intestinal digestion stage, the pH of the gastric digest was adjusted to 7.0, mixed with an equal volume of simulated intestinal fluid solution (100U/mL pancreatin) and digestion was continued for 2h under the same conditions. The intestinal chyme was then centrifuged at 10,000Xg for 20min and the supernatant was assayed by the o-phthalaldehyde method to calculate the degree of hydrolysis of the protein hydrogel.

Example 2:

Soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 1.7; the remainder was identical to example 1.

Example 3:

Soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 2.5; the remainder was identical to example 1.

Comparative example 1:

soy protein isolate of example 1, step 3): the mass ratio of shellac is changed into 1; the remainder was identical to example 1.

Comparative example 2:

Soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 1.3; the remainder was identical to example 1.

Comparative example 3:

soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 1.5; the remainder was identical to example 1.

Blank comparative example, cancel shellac use:

Step 2) was omitted and the "shellac aqueous solution" in step 3) of example 1 was not added; the remainder was identical to example 1.

Comparative example 4:

changing the callback pH in step 3) of example 1 from 9 to 7; the remainder was identical to example 1.

Comparative example 5:

Changing the callback pH in step 3) of example 1 from 9 to 8; the remainder was identical to example 1.

Comparative example 6:

Soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 1.7, and the pH callback value is changed to 7; the remainder was identical to example 1.

Comparative example 7: soy protein isolate of example 1, step 3): the mass ratio of shellac is changed to 1.7, and the pH callback value is changed to 8; the remainder was identical to example 1.

Comparative example 8: and (3) canceling the callback of the pH value in the step 3. The dosage of shellac is kept unchanged; the remainder was identical to example 1.

The experimental results of the above cases are as follows:

1. SDS-PAGE gel electrophoresis

The results of SDS-PAGE gel electrophoresis of the co-folded products obtained in examples 1 to 3, comparative examples 1 to 3 and blank comparative examples were shown in FIG. 1 (A) and FIG. 1 (B);

As can be seen from FIG. 1 (A), under reducing conditions (with beta-mercaptoethanol), the co-folds of examples 1-3, comparative examples 1-3 have a larger molecular weight protein accumulation at the top of the separation gel, but not in the blank comparative example. In contrast, as can be seen from FIG. 1 (B), in the non-reducing electrophoresis, a large molecular weight accumulation exists in the blank comparative example. The differences in the blank comparative examples under reducing and non-reducing conditions indicate the formation of S-S bonds in protein aggregates during the pH shift treatment. The high molecular weight aggregates of the co-folds are shown in both fig. 1 (a) and fig. 1 (B), illustrating that shellac is involved in the folding of the soy protein isolate, and that shellac cross-links with the protein under folding conditions at pH 9 to form macromolecular protein aggregates.

The co-folds obtained in comparative examples 4 to 7 were subjected to SDS-PAGE gel electrophoresis, and the results were as shown in FIG. 1 (C):

The effect of different folding pH values on cross-linking of the co-folds was significant, and in FIG. 1 (C) at pH 7 or 8, there was no accumulation of protein macromolecules in each of comparative examples 4-7 under reducing conditions, corresponding to the accumulation of macromolecular weight formed in non-reducing electrophoresis, indicating that copolymer aggregates formed below pH9 were only built up by inter-protein disulfide bonds, with no formation of covalent bonds.

2. Fourier infrared spectroscopy

Fig. 2 (a) shows: when the folding pH of the co-folded matter is 9, the blank comparative example (control) also comprises a characteristic peak at 983cm ^-1, and the absorbance value at 1600-1700cm ^-1 is increased compared with the soybean protein isolate, which indicates that the pH shift has an effect on the protein structure; after co-folding with shellac (examples 1 and 3), the absorbance at the same wavelength drops significantly. Example 1 compared to comparative example 8, with a characteristic peak at 1658cm ^-1, 1673cm ^-1 in comparative example 8, demonstrates that the isolated soy protein is not simply mixed with shellac, but rather the interaction in protein structure.

Fig. 2 (B) shows: blank control (control) increased absorbance at amide i over soy protein isolate; after co-folding with shellac, the absorbance was significantly reduced in examples 1-3 (SS 2.1, SS1.7, SS 2.5) and comparative examples 2,3 and the characteristic absorbance peak at 1650cm ^-1 red shifted to 1658cm ^-1,1538cm^-1 characteristic peak from red shifted to 1544-1552cm ^-1 (fig. 2B) compared to the blank comparative example (control), indicating a change in the secondary structure of the co-fold, the amide group of soy protein isolate-NH ₂ interacted with the carboxylate group of shellac. Wherein the higher concentrations of shellac in the comparative examples significantly reduced the beta-sheet content. Generally, the higher the beta-sheet content, the better the gel properties.

Fig. 2 (C) shows: in comparative example 4 (SS 2.1-7) and comparative example 5 (SS 2.1-8), the intensity of the absorption peak at 2927cm ^-1、2858cm^-1、1654cm^-1 was significantly higher than that of example 1 (SS 2.1-9), the characteristic absorption peak was blue-shifted from 2858cm ^-1 to 2856cm ^-1, and the characteristic absorption peak intensity in example 1 was significantly lower than that in example 2 (SS 1.7-9). The absorbance at 3290cm ^-1 was higher in example 2 than in comparative examples 6 (SS 1.7-7) and 7 (SS 1.7-8), indicating the formation of hydrogen bonds in the co-fold. As the pH decreases, the soy protein isolate and shellac co-fold, and changes in the IR spectrum indicate that changes in pH significantly affect the unfolding of the protein, as well as co-folding with shellac.

3. Fluorescence spectrum analysis

FIG. 3A/B shows the effect of different mass ratios of soy protein isolate/shellac and co-fold pH on the fluorescence spectrum of the co-fold. The intensity of the characteristic absorption peak of the isolated soy protein increases at pH 12 to 9 and the maximum emission wavelength of the isolated soy protein is significantly red shifted, indicating that the protein conformation changes after pH 12 development and that co-folding at pH 9 results in a change in the structure of the protein fraction, i.e., exposure to polar solvents- -control). In addition, the shellac content significantly changed the tertiary structure and side chain amino acids of the isolated soy protein compared to the blank comparative example (control). The significant decrease in fluorescence intensity of examples 1, 2 and 3 suggests that the soy protein isolate interacts with shellac to form a complex that exhibits significant fluorescence quenching. Furthermore, λ _max of examples 1, 2 and 3 gradually red shifted from 351nm (comparative example 5) to 357nm, indicating that more side chains were exposed to the buffer solution. The red shift of the maximum absorption peak and the decrease in fluorescence intensity in example 1 compared to comparative examples 3 and 4 indicate that the effect of the co-folding pH on the tertiary structure of the protein is significant.

The fluorescence intensity of the proteins in example 1 is significantly lower than that of comparative examples 4, 5 and 2 compared with comparative examples 6, 7 in fig. 3B, showing that the co-folding pH has a significant effect on the change in protein conformation, and that co-folding under pH 9 results in complex formation and a decrease in fluorescence intensity upon binding to side chain amino acids.

4. Protein cold gel character, swelling and water holding capacity

The cryogels obtained in comparative example 2 (SS 1.3), comparative example 3 (SS 1.5), example 2 (SS 1.7), example 1 (SS 2.1) and example 3 (SS 2.5) were examined.

Thereby obtaining the influence of different mass ratios of the pH 9 co-folded soybean protein isolate/shellac on the condensed gum texture characteristics (A), the water holding capacity and the swelling rate (B); as described in fig. 4.

The gel hardness in example 1 is significantly higher than that in example 3, and significantly higher than that in comparative examples 2 and 3. Indicating that an increase in the isolated soy protein content has a positive effect on the firmness of the gel. When the pH value of the isolated soy protein is changed from 12 to 9, a cold gel which can be used for measuring hardness cannot be formed, and therefore, the co-folding of the isolated soy protein and shellac is a main reason for inducing the formation of the cold gel. The maximum gel hardness in example 1, peak 23.57.+ -. 1.09g, is related to the change in protein conformation, structure when the isolated soy protein and shellac are co-folded. Cohesiveness represents the stability of the internal structure of the gel, exhibiting a tendency to conform to changes in hardness.

The water retention is related to the texture and structure of the gel, and the gel retention of examples 1,2, 3 is significantly higher than the comparison 2 and 3 due to the increase in protein molecules involved in building the gel network. The better the swelling stability of the gel during its application, the more mechanical properties may be improved. The gel swelling ratio of comparative examples 2,3 is significantly higher than that of examples 1,2, 3, i.e., the gel swelling stability of comparative examples 2,3 is significantly lower than that of examples 1,2, 3, which is related to the internal structure of the gel, indicating that crosslinking between soy protein isolate and shellac results in a significant reduction in swelling. A stable network is formed in the gel, and the water absorption capacity of the gel is reduced. Illustrating that the soy protein isolate/shellac mass ratios of examples 1 and 2 are more suitable for preparing a cryogel.

5. Rheological determination of protein cryogels

Fig. 4 and 5 show the relationship between the changes in the cold gel hardness and rheology of examples 1-3 and comparative examples 2, 3. As can be seen from fig. 5, as the strain changes, the storage modulus (G') decreases, the loss modulus (G ") increases, and then crosses. When the strain changes beyond the crossover points, the internal structure of the gel will be destroyed and the gel will appear as a liquid. The elasticity and stiffness of the gel are reflected by the G 'before the intersection point, and the G' is higher than G "(G '> G") for all examples 1-3 and comparative examples 2 and 3, but the G' and G "are higher for examples 1 and 3 than for the other examples, indicating better gel performance.

6. Microstructure of protein cryogels

The microstructure of the cryogel obtained by observing the example embodiment by scanning electron microscopy is shown in fig. 6. The cryogel has a three-dimensional porous structure, with small pores in the gel possibly contributing to the loading of the bioactive ingredient. The cold gels in examples 1,2, 3 exhibited a uniform, regular and compact porous structure, especially the smaller pore size of the gel in example 1. The blank comparative example (control) exhibited an irregularly porous disordered sheet structure (fig. 6F), indicating that soy protein isolate and shellac structure folding promoted formation of a cryogel.

The figure shows: comparative examples 2 and 3 have the disadvantage of poor integrity of the three-dimensional voids for examples 1 to 3.

7. Degree of hydrolysis of co-folded gel after in vitro digestion

The changes in the degree of hydrolysis of the cold gels obtained in examples 1 to 3 and comparative examples 2 to 3 in vitro simulated digestion are shown in FIG. 7. Examples 1,2,3 showed a DH significantly higher than the comparative blank control during intestinal digestion but not during gastric digestion. This is probably because the binding site of shellac is located at a hydrophobic position in the isolated soy protein, which spatially reduces the accessibility sites for digestive enzymes, and thus the stability of the isolated soy protein during digestion is enhanced. Another possible reason is that by co-folding with shellac, a conformational change of the isolated soy protein is induced, resulting in a compact structure that makes it difficult to hydrolyze by digestive enzymes. This shows that the cold gel obtained in example 1 can delay the degree of proteolysis during in vitro digestion.

In conclusion, the shellac co-folded isolated soy protein cold gel obtained by the invention is detected according to a conventional gel texture measurement and an in vitro simulated gastrointestinal digestion method, the hardness is 23.57g, the water retention is more than 90%, and the in vitro simulated gastrointestinal digestion hydrolysis degree is lower than that of isolated soy protein, so that the isolated soy protein cold gel has the application/function suitable for delivering sensitive active ingredients.

Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.

Claims

1. The preparation method of the shellac co-folded soybean protein isolate cold gel is characterized by comprising the following steps of:

1) Preparing a soybean protein isolate aqueous solution with the mass concentration of 11-13 percent:

Adding water to 11-13 g of isolated soy protein to 100g, stirring for 1.5-2.5 h by a magnetic stirrer, and then storing for 12-15 h at 0-4 ℃ to form an isolated soy protein aqueous solution with the mass concentration of 11-13%;

2) Preparing shellac water solution with mass concentration of 11-13% and pH of 8+/-0.5:

Adding water to 11-13 g of shellac to 100g, regulating the pH value to 8+/-0.5, and continuously magnetically stirring at 45-55 ℃ until the shellac is completely dissolved to obtain shellac water solution with the mass concentration of 11-13% and the pH value of 8+/-0.5;

After the reaction is finished, firstly adjusting the pH value of the obtained reaction solution to 8.5-9, and then centrifuging to remove gas to obtain a gel pre-solution;

4) And (3) placing the gel pre-solution at the temperature of 4+/-1 ℃ for 12-15 hours to obtain the shellac co-folded soybean protein isolate cold gel.

2. The method for preparing shellac co-folded isolated soy protein cold gel of claim 1, wherein in step 3):

3. The method for preparing shellac co-folded isolated soy protein cold gel of claim 2, wherein:

the step 1) is as follows:

The step 2) is as follows:

the step 3) is as follows:

According to the soy protein: shellac = 2.1:1, mixing a soybean protein aqueous solution and a shellac solution, regulating the pH value of the blend to 12, and continuously stirring at 25 ℃ for reaction for 2 hours; then, firstly adjusting the pH value of the obtained reaction solution to 9, and then centrifuging to obtain a gel pre-solution;

The step 4) is as follows:

And (3) standing and preserving the gel pre-solution at 4 ℃ for 15 hours to obtain the shellac co-folded soybean protein isolate cold gel.