Preparation method of high-emulsibility casein

Abstract

The invention provides a preparation method of high-emulsibility casein, and belongs to the technical field of protein deep processing. The preparation method comprises the following steps: preparing surface succinyl chitosan oligosaccharide by taking succinyl anhydride and chitosan oligosaccharide as raw materials by adopting a grafting technology, and then mixing the succinyl chitosan oligosaccharide with casein by adopting a pH driving technology to prepare casein-succinyl chitosan oligosaccharide nanoparticles; according to the casein-succinyl chitosan oligosaccharide nano-particles obtained by the invention, the emulsibility of casein is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of protein deep processing, and in particular relates to a method for preparing highly emulsifiable casein. Background Art

[0002] Chitosan oligosaccharide is a new resource food with a wide range of applications in the food industry. It is often used in food preservation, food ingredients, and the development of functional foods. Adding some chitosan oligosaccharides to food can maintain the water content of the food, thereby increasing the water retention and water regulation of the food. It can also improve the structure of the food, making the food more stable during storage, thereby extending the shelf life of the food, and can exert the biological activity of chitosan oligosaccharides, making the food healthier. Chitosan oligosaccharides cannot be decomposed and digested by enzymes in the intestines. It can exist in the intestines for a long time in the form of prebiotics and play a role. In addition, it can balance the number of various flora in the intestines and promote the growth and reproduction of beneficial bacteria in the intestines, thereby maintaining intestinal health.

[0003] Casein is the main protein in cow's milk, accounting for 80% of milk protein. Casein is soluble in alkaline aqueous solutions, but insoluble in neutral and acidic aqueous solutions. By adding acid or rennet to cow's milk, the pH of the milk is lowered to reach the isoelectric point of casein or the surface charge of casein is changed to achieve the purpose of precipitating and separating casein from milk. In the food industry, casein is also used as a thickener and emulsifier in the food processing process, affecting the texture, stability, sensory and nutritional properties of food and dairy products. However, casein has poor stability near the isoelectric point, is easy to aggregate under environmental pressure (pH, ionic strength and heat treatment), and has reduced emulsifying properties. Therefore, obtaining a highly emulsifying casein is an effective way to solve the low performance of casein. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing highly emulsifiable casein. The present invention adopts grafting technology to prepare surface succinyl chitosan oligosaccharide, and prepares casein-succinyl chitosan oligosaccharide nanoparticles by pH-driven technology. The casein-succinyl chitosan oligosaccharide nanoparticles obtained by the present invention significantly improve the emulsification of casein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing highly emulsifiable casein, comprising the following steps:

[0007] (1) dissolving succinic anhydride in ethanol and adding hydrochloric acid dropwise to obtain a succinic anhydride solution; the mass volume ratio of succinic anhydride to ethanol is 1 g: 80-120 mL, the mass volume ratio of succinic anhydride to hydrochloric acid is 1 g: 2-4 mL; the concentration of the hydrochloric acid is 1-3 mol / L;

[0008] (2) mixing the succinic anhydride solution with chitosan oligosaccharide, reacting at 30-50° C. for 2-6 hours, filtering, washing, and drying to obtain succinyl chitosan oligosaccharide; the mass ratio of chitosan oligosaccharide to succinic anhydride is 1:0.5-1.5;

[0009] (3) dissolving casein in water and adjusting the pH to 11-13 to obtain a casein solution; the mass volume ratio of casein to water is 1 g: 80-120 mL;

[0010] (4) dissolving succinyl chitosan oligosaccharide in water, heating at 80-110° C. for 10-30 min, adjusting the pH to 11-13, and obtaining a succinyl chitosan oligosaccharide solution; the mass volume ratio of the succinyl chitosan oligosaccharide to water is 1 g: 80-120 mL;

[0011] (5) Mix the casein solution and the succinyl chitosan oligosaccharide solution, react for 30 to 90 minutes, adjust the pH to 4 to 7, centrifuge, and collect the precipitate to obtain highly emulsifiable casein.

[0012] Preferably, the concentration of the ethanol is 80% to 100%.

[0013] Preferably, the washing is performed with anhydrous ethanol for 3 to 8 times.

[0014] Preferably, the drying temperature is 35-55° C., and the drying time is 2-4 hours.

[0015] Preferably, the pH is adjusted to 11-13 using a NaOH solution with a concentration of 1-3 mol / L.

[0016] Preferably, the volume ratio of the casein solution to the succinyl chitosan oligosaccharide solution is 1:0.5-1.5.

[0017] Preferably, the pH is adjusted to 4-7 using a phytic acid solution with a concentration of 1%-3%.

[0018] Preferably, the centrifugal speed is 3000-5000 r / min, and the centrifugal time is 5-15 min.

[0019] The present invention also provides the highly emulsifiable casein obtained by the preparation method.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for preparing highly emulsifiable casein, the present invention adopts a grafting technology, takes succinic anhydride and chitosan oligosaccharide as raw materials to prepare surface succinyl chitosan oligosaccharide, and then mixes the obtained succinyl chitosan oligosaccharide with casein through a pH-driven technology to prepare casein-succinyl chitosan oligosaccharide nanoparticles, and the casein-succinyl chitosan oligosaccharide nanoparticles obtained by the present invention significantly improve the emulsifying property of casein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the TEM image of highly emulsifiable casein nanoparticles;

[0022] Figure 2 The diagram is a diagram of the emulsification stability of casein-succinyl chitosan oligosaccharide nanoparticles; the left diagram is a diagram of the state of casein-succinyl chitosan oligosaccharide nanoparticles at day 0, and the right diagram is a diagram of the state of casein-succinyl chitosan oligosaccharide nanoparticles at day 7, with a scale of 20 μm;

[0023] Figure 3 This is a graph showing the emulsification ability of different samples. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing highly emulsifiable casein, comprising the following steps:

[0025] (1) dissolving succinic anhydride in ethanol and adding hydrochloric acid dropwise to obtain a succinic anhydride solution; the mass volume ratio of succinic anhydride to ethanol is 1 g: 80-120 mL, the mass volume ratio of succinic anhydride to hydrochloric acid is 1 g: 2-4 mL; the concentration of the hydrochloric acid is 1-3 mol / L;

[0026] (2) mixing the succinic anhydride solution with chitosan oligosaccharide, reacting at 30-50° C. for 2-6 hours, filtering, washing, and drying to obtain succinyl chitosan oligosaccharide; the mass ratio of chitosan oligosaccharide to succinic anhydride is 1:0.5-1.5;

[0027] (3) dissolving casein in water and adjusting the pH to 11-13 to obtain a casein solution; the mass volume ratio of casein to water is 1 g: 80-120 mL;

[0028] (4) dissolving succinyl chitosan oligosaccharide in water, heating at 80-110° C. for 10-30 min, adjusting the pH to 11-13, and obtaining a succinyl chitosan oligosaccharide solution; the mass volume ratio of the succinyl chitosan oligosaccharide to water is 1 g: 80-120 mL;

[0029] (5) Mix the casein solution and the succinyl chitosan oligosaccharide solution, react for 30 to 90 minutes, adjust the pH to 4 to 7, centrifuge, and collect the precipitate to obtain highly emulsifiable casein.

[0030] In the present invention, succinic anhydride is dissolved in ethanol, and hydrochloric acid is added dropwise to obtain a succinic anhydride solution. The succinic anhydride of the present invention is purchased from Shanghai Yubo Biotechnology Co., Ltd., and the mass volume ratio of succinic anhydride to ethanol is 1g:80-120mL, preferably 1g:90-110mL, and more preferably 1g:100mL; the volume concentration of ethanol is 80%-100%, preferably 90%-98%, and more preferably 95%; the mass volume ratio of succinic anhydride to hydrochloric acid is 1g:2-4mL, preferably 1g:2.5-3.5mL, and more preferably 1g:3mL; the concentration of hydrochloric acid is 1-3mol / L, preferably 1.5-2.5mo / L, and more preferably 2mol / L. In the present invention, stirring is performed during the process of adding hydrochloric acid dropwise. The purpose of adding hydrochloric acid dropwise in the present invention is to provide an acidic reaction environment while enabling succinic anhydride to be grafted onto chitosan oligosaccharides to obtain succinyl chitosan oligosaccharides.

[0031] In the present invention, succinic anhydride solution is mixed with chitosan oligosaccharide, reacted at 30-50°C for 2-6h, filtered, washed, and dried to obtain succinyl chitosan oligosaccharide. The mass ratio of chitosan oligosaccharide to succinic anhydride is 1:0.5-1.5, preferably 1:0.7-1.2, and more preferably 1:1; the reaction temperature is 30-50°C, preferably 35-45°C, and more preferably 40°C; the reaction time is 2-6h, preferably 3-5h, and more preferably 4h; the pore size of the filter paper is 0.22μm; the ethanol washing times are 3-8 times, preferably 4-7 times, and more preferably 5 times; the drying temperature is 35-55°C, preferably 40-50°C, and more preferably 45°C, and the drying time is 2-4h. Preferably 2.5-3.5h, more preferably 3h.

[0032] In the present invention, casein is dissolved in water, and the pH is adjusted to 11-13 to obtain a casein solution. The water is preferably deionized water; the mass volume ratio of casein to water is 1g:80-120mL, preferably 1g:90-110mL, and more preferably 1g:100mL; the concentration of the NaOH solution is 1-3mol / L, preferably 1.5-2.5mol / L, and more preferably 2mol / L.

[0033] In the present invention, succinyl chitosan oligosaccharide is dissolved in water, heated at 80-110°C for 10-30min, and the pH is adjusted to 11-13 to obtain a succinyl chitosan oligosaccharide solution. The water is preferably deionized water; the mass volume ratio of the succinic anhydride chitosan oligosaccharide to water is 1g:80-120mL, preferably 1g:90-110mL, and more preferably 1:100mL; the heating temperature is 80-110°C, preferably 90-105°C, and more preferably 100°C; the heating time is 10-30min, preferably 15-25min, and more preferably 20min; the concentration of the NaOH solution is 1-3mol / L, preferably 1.5-2.5mol / L, and more preferably 2mol / L.

[0034] In the present invention, casein solution and succinyl chitosan oligosaccharide solution are mixed, reacted for 30 to 90 minutes, pH adjusted to 4 to 6, centrifuged, and precipitated to obtain highly emulsifiable casein (casein-succinyl chitosan oligosaccharide nanoparticles). The volume ratio of the casein solution to the succinyl chitosan oligosaccharide solution is 1:0.5-1.5, preferably 1:0.7-1.2, and more preferably 1:1. The rotation speed of the stirring reaction is 250-450 r / min, preferably 300-400 r / min, and more preferably 350 r / min. The stirring reaction time is 30-90 min, preferably 45-75 min, and more preferably 60 min. The concentration of the phytic acid solution is 1%-3%, preferably 1.5%-2.5%, and more preferably 2%. The pH is adjusted to 4-7, preferably 5-6, and more preferably 5.5. The centrifugal rotation speed is 3000-5000 r / min, preferably 3500-4500 r / min, and more preferably 4000 r / min. The centrifugal time is 5-15 min, preferably 7-12 min, and more preferably 10 min.

[0035] The present invention also provides the highly emulsifiable casein obtained by the preparation method.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] (1) Dissolve 1 g of succinic anhydride in 100 mL of 95% ethanol, and add dropwise 3.0 mL of 2.0 mol / L hydrochloric acid while stirring to obtain a succinic anhydride solution;

[0039] (2) adding 1 g of chitosan oligosaccharide to the succinic anhydride solution obtained in step (1), mixing, shaking and reacting at 40° C. for 4 h, filtering with a 0.22 μm filter paper, collecting the retentate, washing with anhydrous ethanol 5 times, and drying in an oven at 45° C. for 3 h to obtain succinyl chitosan oligosaccharide;

[0040] (3) dissolving 1 g of casein in 100 mL of deionized water, and adjusting the pH to 12 with a 2 mol / L NaOH solution to obtain a casein solution;

[0041] (4) dissolving 1 g of succinyl chitosan oligosaccharide in 100 mL of deionized water, heating at 100° C. for 20 min, and adjusting the pH to 12 with a 2 mol / L NaOH solution to obtain a succinyl chitosan oligosaccharide solution;

[0042] (5) Mix equal volumes of casein solution and succinyl chitosan oligosaccharide solution, stir and react at 350 r / min for 60 min, adjust the pH of the mixture to 5.5 with 2% phytic acid solution, centrifuge at 4000 r / min for 10 min, and collect the precipitate to obtain highly emulsifiable casein (casein-succinyl chitosan oligosaccharide nanoparticles).

[0043] Example 2

[0044] (1) dissolving 1 g of succinic anhydride in 80 mL of anhydrous ethanol, and adding dropwise 4.0 mL of 1.0 mol / L hydrochloric acid while stirring to obtain a succinic anhydride solution;

[0045] (2) adding 1 g of chitosan oligosaccharide to the succinic anhydride solution obtained in step (1), mixing, shaking and reacting at 50° C. for 2 h, filtering with a 0.22 μm filter paper, collecting the retentate, washing with anhydrous ethanol 5 times, and drying in an oven at 35° C. for 4 h to obtain succinyl chitosan oligosaccharide;

[0046] (3) dissolving 1 g of casein in 80 mL of deionized water, and adjusting the pH to 11 using a 1 mol / L NaOH solution to obtain a casein solution;

[0047] (4) dissolving 1 g of succinyl chitosan oligosaccharide in 80 mL of deionized water, heating at 80° C. for 30 min, and adjusting the pH to 11 with a 1 mol / L NaOH solution to obtain a succinyl chitosan oligosaccharide solution;

[0048] (5) 50 mL of casein solution was mixed with 25 mL of succinyl chitosan oligosaccharide solution, and the mixture was stirred at 250 r / min for 90 min. The pH of the mixture was adjusted to 7 with 1% phytic acid solution, and the mixture was centrifuged at 3000 r / min for 15 min. The precipitate was collected to obtain highly emulsifiable casein.

[0049] Example 3

[0050] (1) Dissolve 1 g of succinic anhydride in 120 mL of 80% ethanol, and add dropwise 2.0 mL of 3.0 mol / L hydrochloric acid while stirring to obtain a succinic anhydride solution;

[0051] (2) adding 1 g of chitosan oligosaccharide to the succinic anhydride solution obtained in step (1), mixing, shaking and reacting at 30° C. for 6 h, filtering with a 0.22 μm filter paper, collecting the retentate, washing with anhydrous ethanol 5 times, and drying in an oven at 55° C. for 2 h to obtain succinyl chitosan oligosaccharide;

[0052] (3) dissolving 1 g of casein in 120 mL of deionized water, and adjusting the pH to 13 using a 3 mol / L NaOH solution to obtain a casein solution;

[0053] (4) dissolving 1 g of succinyl chitosan oligosaccharide in 120 mL of deionized water, heating at 110° C. for 10 min, and adjusting the pH to 13 with a 3 mol / L NaOH solution to obtain a succinyl chitosan oligosaccharide solution;

[0054] (5) 50 mL of casein solution was mixed with 75 mL of succinyl chitosan oligosaccharide solution, and the mixture was stirred at 450 r / min for 30 min. The pH of the mixture was adjusted to 4 with 3% phytic acid solution, and the mixture was centrifuged at 5000 r / min for 5 min. The precipitate was collected to obtain highly emulsifiable casein.

[0055] Comparative Example 1

[0056] The method of Example 1 was used to obtain highly emulsifiable casein, except that succinic anhydride was replaced by maleic anhydride.

[0057] Comparative Example 2

[0058] The method of Example 1 is adopted to obtain highly emulsifiable casein, except that chitosan oligosaccharide is replaced by chitosan.

[0059] Comparative Example 3

[0060] The method of Example 1 is used to obtain highly emulsifiable casein, except that phytic acid is replaced by citric acid.

[0061] Experimental Example 1

[0062] The highly emulsifiable casein obtained in Example 1 was used as a sample, and TME and emulsification stability were tested. The results are shown in Figure 1 and Figure 2 .

[0063] Emulsion stability test: Take 20mL of the sample solution with a mass concentration of 60mg / mL (dissolve the sample in distilled water to obtain the sample solution), add 20mL of soybean salad oil, and high-speed shear at 8000r / min for 3min at room temperature to obtain the sample to be tested. Put the sample to be tested in a constant temperature bath at 25±1℃ for 7d, observe the stratification state of the sample, and observe the particle size and distribution before and after standing under a microscope.

[0064] Depend on Figure 1 and Figure 2 It can be seen that the casein-succinyl chitosan oligosaccharide nanoparticles obtained by the method of the present invention have a small particle size. After being placed for 7 days, no stratification phenomenon occurs. When observed under a microscope, it is found that the particle size does not change from that before standing, indicating that the casein-succinyl chitosan oligosaccharide nanoparticles obtained by the method of the present invention have good emulsification stability.

[0065] Experimental Example 2

[0066] The emulsification abilities of the products obtained in Examples 1 to 3, Comparative Examples 1 to 3, casein and the prior art (Patent 202111571129.4, control group) were compared. The results are shown in Figure 3 .

[0067] Emulsifying ability: Take 20mL of the sample solution with a mass concentration of 60mg / mL (dissolve the sample in distilled water to obtain the sample solution), add 20mL of soybean salad oil, and high-speed shear at 8000r / min for 3min at room temperature to obtain the sample to be tested. Take 30mL of the sample to be tested, place it in a 50mL stoppered flat-bottomed glass tube, store it in an ultra-low temperature refrigerator at -20℃ for 24h, then thaw it in a constant temperature water bath at 30℃ for 2h, and let it stand at room temperature at 25℃ for 1h. Measure the height of the whey layer and the total height of the emulsion of the sample, and calculate the emulsifying ability of the sample. The calculation formula is as follows:

[0068] Emulsifying capacity = 1 - (height of serum layer / total height of emulsion) * 100%.

[0069] The higher the whey layer is, the weaker the emulsifying ability of the emulsifier is. Figure 3 It can be seen that the emulsifying capacity of casein, the samples obtained in the prior art, and the samples of comparative examples 1 to 3 are significantly lower than the emulsifying capacity obtained in Examples 1 to 3 of the present application. It can be seen that the casein-succinyl chitosan oligosaccharide nanoparticles obtained by the present invention significantly improve the emulsifying property of casein and have good stability.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing highly emulsifiable casein, characterized in that: The following steps are involved: (1) dissolving succinic anhydride in ethanol and adding hydrochloric acid dropwise to obtain a succinic anhydride solution; the mass volume ratio of succinic anhydride to ethanol is 1 g: 80-120 mL, the mass volume ratio of succinic anhydride to hydrochloric acid is 1 g: 2-4 mL; the concentration of the hydrochloric acid is 1-3 mol / L; (2) mixing the succinic anhydride solution with chitosan oligosaccharide, reacting at 30-50° C. for 2-6 hours, filtering, washing, and drying to obtain succinyl chitosan oligosaccharide; the mass ratio of chitosan oligosaccharide to succinic anhydride is 1:0.5-1.5; (3) dissolving casein in water and adjusting the pH to 11-13 to obtain a casein solution; the mass volume ratio of casein to water is 1 g: 80-120 mL; (4) dissolving succinyl chitosan oligosaccharide in water, heating at 80-110° C. for 10-30 min, adjusting the pH to 11-13, and obtaining a succinyl chitosan oligosaccharide solution; the mass volume ratio of the succinyl chitosan oligosaccharide to water is 1 g: 80-120 mL; (5) Mix the casein solution and the succinyl chitosan oligosaccharide solution, react for 30 to 90 minutes, adjust the pH to 4 to 7, centrifuge, and collect the precipitate to obtain highly emulsifiable casein.

2. The preparation method according to claim 1, characterized in that: The concentration of the ethanol is 80% to 100%.

3. The preparation method according to claim 1, characterized in that: The washing is performed by washing with anhydrous ethanol for 3 to 8 times.

4. The preparation method according to claim 1, characterized in that: The drying temperature is 35-55° C., and the drying time is 2-4 hours.

5. The preparation method according to claim 1, characterized in that: The pH is adjusted to 11-13 using a NaOH solution with a concentration of 1-3 mol / L.

6. The preparation method according to claim 1, characterized in that: The volume ratio of the casein solution to the succinyl chitosan oligosaccharide solution is 1:0.5-1.

5.

7. The preparation method according to claim 1, characterized in that: The pH value is adjusted to 4-7 by using a phytic acid solution with a concentration of 1%-3%.

8. The preparation method according to claim 1, characterized in that: The centrifugal speed is 3000-5000 r / min, and the centrifugal time is 5-15 min.

9. Highly emulsifiable casein obtained by the production method according to any one of claims 1 to 8.

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