Preparation method and application of chitosan-casein peptide nanocomposite

Abstract

The invention discloses a chitosan-casein peptide nano-particle and a preparation method and application thereof, and belongs to the technical field of food manufacturing, chitosan and casein peptide are combined into a nano-composite with the particle size of 10-1000 nm through electrostatic interaction in a non-covalent binding mode, and the nano-composite is used for preparing the chitosan-casein peptide nano-particle. A more stable oil-in-water emulsion can be prepared by utilizing the nano composite; the invention also provides a preparation method and application of the chitosan-casein peptide nanocomposite, and a preparation method of the oil-in-water emulsion. The nano-composite prepared by the invention has relatively strong oxidation resistance, foamability and foaming stability when the ratio of chitosan to casein peptide is 1: 1, and an oil-in-water emulsion prepared from the nano-composite has good storage stability, pH stability and ion stability. The preparation method of the nanocomposite is simple and low in cost, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a chitosan-casein peptide nanocomposite, belonging to the technical field of food processing. Background Art

[0002] Nanocomposites are a new type of material composed of two or more materials with different properties. These materials combine with each other at the nanoscale to form composite materials with unique properties and functions. Nanocomposites have attracted much attention from researchers in the food industry in recent years due to their unique physical and chemical properties and wide application potential. These materials are not only widely available and inexpensive, but also non-toxic, biocompatible, and biodegradable, which makes them show good application prospects in many fields. Especially in the food industry, nanocomposites are used as food additives, preservatives, and processing aids, which effectively improve the quality and safety of food.

[0003] Chitosan (CS) has no toxic side effects, no sensitization and mutagenicity, and has antibacterial and anti-inflammatory effects, inhibits tumor cells, and reduces lipids and cholesterol. The characteristic of chitosan is its strong hydrophilicity, which needs to be balanced with hydrophobic groups to achieve the desired purpose. Casein has a variety of physiological activities and is considered to be the main source of excellent bioactive peptides. Casein peptide (CHP) is a natural bioactive peptide obtained by enzymatic hydrolysis of casein with hydrolytic enzymes. After enzymatic hydrolysis, casein can provide more hydrophobic groups, which is conducive to combining with chitosan with strong hydrophilicity.

[0004] The traditional method of combining chitosan with casein to prepare a complex uses toxic reagents, and the toxicity is difficult to control. However, the preparation method of the nanocomposite does not use toxic reagents, has no toxic side effects, and has great application potential in the food industry due to its unique physical, chemical and biological properties. The application of nanocomposites in the food industry includes but is not limited to the development of food packaging, food additives, nutritional supplements and functional foods.

[0005] Chitosan-casein peptide nanocomplexes exhibit improved functional properties compared to chitosan or casein peptide alone. Such nanocomplexes can be used as food additives to improve the texture, taste and shelf life of food while providing additional nutritional and health benefits.

[0006] Although studies have explored the separate applications of chitosan and casein peptides, there are few studies on combining the two to form nanocomposites and applying them in oil-in-water emulsions. Oil-in-water emulsions have great potential in improving emulsion stability. With the in-depth study of the emulsifier market, the application prospects of oil-in-water emulsions in the food industry are even broader.

[0007] Therefore, the present invention aims to provide a chitosan-casein peptide nanocomposite and a preparation method and application thereof. The oil-in-water emulsion prepared using the nanocomposite has higher stability and is more resistant to storage.

[0008] At present, the application of nanocomposite prepared by casein and chitosan in oil-in-water emulsion is less, for example, patent CN101530765A discloses a casein / chitosan composite biological microsphere and its preparation method. Specifically, natural casein and chitosan are used as raw materials, and glutaraldehyde and formaldehyde are used as cross-linking agents to carry out polymerization reaction to obtain. The composite biological microsphere of the present invention presents a porous honeycomb structure on the surface, has a uniform morphology, is nontoxic, has good biocompatibility, and can be used as a carrier of enzymes or other bioactive molecules. However, the application of the complex formed by casein and chitosan in oil-in-water emulsion is not disclosed in this technology.

[0009] Patent CN117941823B discloses a carboxymethyl chitosan-casein complex, a preparation method and application thereof. Specifically, the present invention provides a carboxymethyl chitosan-casein complex, a preparation method and application thereof, and belongs to the field of food manufacturing technology. The carboxymethyl chitosan-casein complex provided by the present invention is composed of the following components: casein, carboxymethyl chitosan and an acidity regulator, wherein the mass ratio of casein to carboxymethyl chitosan is 5:0.1-0.4. The carboxymethyl chitosan-casein complex can be used to prepare a more stable, flavorful and color-enhanced complete nutritional milk. The present invention also provides a method for preparing the above-mentioned carboxymethyl chitosan-casein complex and a process for preparing complete nutritional milk. However, the technology does not disclose the application of the chitosan and casein complex in an oil-in-water emulsion.

[0010] In summary, the prior art has not yet prepared chitosan-casein peptide nanocomposites that improve the stability of oil-in-water emulsions. Summary of the invention

[0011] In view of this, in view of the problem that the prior art is still unable to prepare a chitosan-casein peptide nanocomposite that improves the stability of an oil-in-water emulsion, the purpose of the present invention is to provide a chitosan-casein peptide nanocomposite and a preparation method and application thereof. The method combines chitosan and casein peptide into a nanocomposite by non-covalent binding and utilizing electrostatic interaction to prepare a dispersion with a particle size of 10-1000 nanometers.

[0012] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a chitosan-casein peptide nanocomposite and a preparation method thereof, comprising the following steps:

[0013] S1. Dilute acetic acid with water.

[0014] S2. Add chitosan to the solution treated in S1, stir until the chitosan is completely dissolved and the solution is clear and transparent, and adjust the pH to 5.5.

[0015] S3. Place casein powder in a beaker and keep the pH value of the solution at a certain level.

[0016] S4, taking the required volume of casein solution obtained in step S3, adjusting the pH to 7.0, adding trypsin according to the enzyme addition amount designed in the experiment, placing in a 50°C constant temperature water bath for enzymolysis, stirring continuously during the enzymolysis process, inactivating the enzyme after 3 hours, cooling, and then centrifuging the enzymolysis solution, collecting the supernatant to obtain the casein peptide after enzymolysis, and freeze-drying.

[0017] S5. Dissolve the casein peptide powder obtained in step S4 in water and adjust the pH to 7.0.

[0018] S6. During the stirring process, the chitosan solution obtained in step S2 is added to the casein peptide solution obtained in step S5, the pH is adjusted to 5.5, and the mixture is stirred until a transparent white solution is formed to obtain a chitosan-casein peptide dispersion. The suspension is freeze-dried in a vacuum freeze dryer for 48 hours to prepare a chitosan-casein nanocomposite.

[0019] Preferably, in step S1, the mass ratio of the acetic acid to the water is 1:99.

[0020] More preferably, in step S1, 2.5 mL of acetic acid and 247.5 mL of water are taken.

[0021] Wherein, in step S2, 0.5 g of chitosan is taken to prepare 0.2% chitosan.

[0022] Wherein, in step S3, the casein is 12 g, the water is 40 mL, the NaOH concentration is 1 mol, the volume is 3 mL, and the pH is maintained at 7.0.

[0023] Wherein, in step S4, the enzyme substrate ratio is 5%, the enzymatic hydrolysis condition is 50°C constant temperature water bath for 3 hours, the enzyme inactivation condition is heating at 100°C for 5 minutes, and the centrifugal condition is 4000 rpm for 10 minutes.

[0024] More preferably, the mass of trypsin in step S4 is 0.6 g.

[0025] Wherein, the mass of casein peptide taken in step S5 is 0.2 g.

[0026] Wherein, in step S6, the volume ratio of chitosan to casein peptide is 0:1, 1:1, 2:1, and the slow stirring time is 1 hour.

[0027] More preferably, and as an example of the present invention, the system of chitosan and casein peptide is 30 mL, that is, 0 mL:30 mL, 15 mL:15 mL, 20 mL:10 mL, respectively.

[0028] On the other hand, the present invention provides an oil-in-water emulsion system prepared by using the chitosan-casein peptide nanocomposite prepared in the above steps as a raw material.

[0029] S1. Mix soybean oil and the nanocomposite obtained above at a volume ratio of 1:9.

[0030] S2. The mixed solution was homogenized at 15000 rpm for 2 min, and then subjected to ultrasonic treatment at 500 W for 5 min to prepare an oil-in-water emulsion system.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Provided are a chitosan-casein peptide nanocomposite and a preparation method and application thereof. The chitosan-casein peptide nanocomposite has superior properties such as strong antioxidant ability, good foaming property, and high stability. The preparation method has the advantages of simple operation, no toxic side effects, and low cost.

[0033] (2) Provided is an oil-in-water emulsion system prepared using chitosan-casein peptide nanocomplexes, which has good storage stability, pH stability and ion stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 is the ABTS free radical scavenging rate of nanocomposites made of different chitosan and casein ratios;

[0036] Figure 2 is the DPPH radical scavenging rate of nanocomposites made of different chitosan and casein ratios;

[0037] Figure 3 is the foaming properties of nanocomposites made with different chitosan and casein ratios;

[0038] Figure 4 is the foaming stability of nanocomposites made with different chitosan and casein ratios;

[0039] Figure 5The storage stability of oil-in-water emulsions made of different proportions of the complex;

[0040] Figure 6 It is the pH stability of oil-in-water emulsions made of different proportions of the complex;

[0041] Figure 7 It is the ionic stability of water-in-oil emulsions made of complexes in different proportions. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0043] The present invention is further described below by way of specific embodiments. Unless otherwise specified, the instruments, reagents and methods used in the embodiments of the present invention can be obtained through commercial channels.

[0044] Example 1 Preparation of chitosan-casein peptide complex

[0045] 1. Preparation of acetic acid solution:

[0046] Take 2.5 mL of acetic acid, add it into 247.5 mL of water, stir evenly, and obtain an acetic acid aqueous solution.

[0047] 2. Preparation of chitosan solution:

[0048] Add 0.5 g of chitosan to 250 mL of the above acetic acid solution, stir until the chitosan is completely dissolved and the solution is clear and transparent, and adjust the pH to 5.5.

[0049] 3. Preparation of casein solution:

[0050] Add 12g of casein powder to 40mL of water, then add 3mL of 1mol / L NaOH solution, soak overnight and stir to keep the pH at 7.0.

[0051] 4. Casein enzymatic hydrolysis:

[0052] Add 0.6 g of trypsin to the casein solution and perform enzymolysis in a constant temperature water bath at 50°C for 3 h.

[0053] 5. Inactivate enzymes and collect casein peptides:

[0054] After enzymatic hydrolysis, the enzyme was inactivated by heating at 100°C for 5 minutes. After cooling, the enzymatic hydrolyzate was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected to obtain the enzymatically hydrolyzed casein peptides.

[0055] 6. Freeze-dried casein peptides:

[0056] The collected casein peptide supernatant was freeze-dried for 48 hours to obtain casein peptide powder.

[0057] 7. Preparation of chitosan-casein peptide complex:

[0058] The chitosan and casein peptide mass ratio was 0:1 for preparing the composite.

[0059] Example 2

[0060] On the basis of Example 1, the mass ratio of chitosan to casein peptide was changed to 1:1 to prepare the composite, and the rest remained unchanged.

[0061] Example 3

[0062] On the basis of Example 1, the mass ratio of chitosan to casein peptide was changed to 2:1 to prepare the composite, and the rest remained unchanged.

[0063] Example 4

[0064] On the basis of Example 1-3, soybean oil and the prepared nanocomposite were homogenized at a volume ratio of 1:9 at a rotation speed of 15000 rpm for 2 minutes, and then ultrasonically treated at 500 W for 5 minutes to prepare an oil-in-water emulsion.

[0065] Example 5

[0066] Effect of different chitosan:casein peptide ratios on the antioxidant properties of the nanocomposites.

[0067] 1) ABTS free radical scavenging activity assay

[0068] Equal volumes of ABTS aqueous solution (7.4 mmol / L) and potassium persulfate aqueous solution (2.6 mmol / L) were mixed to form ABTS+ solution, and stored at room temperature in the dark for 12-16 h. The ABTS+ solution was diluted with deionized water and adjusted to an OD value of 0.70±0.02 at 734 nm. 0.1 mL of the nanocomposite (1 mg / mL) was mixed with 3.9 mL of ABTS+, and allowed to stand at room temperature in the dark for 5 min. The absorbance was measured at 734 nm, and deionized water was used instead of the nanocomposite as a blank control.

[0069]

[0070] A0 is the absorbance value of the blank group

[0071] A1 is the absorbance value of the nanocomposite

[0072] 2) DPPH free radical scavenging activity assay

[0073] Weigh DPPH and dissolve it in anhydrous ethanol to prepare a DPPH solution with a concentration of 0.2mmol / L, and store it in the dark for later use. Take 100uL of DPPH solution, add 100uL of nanocomposite solution, mix well, react at room temperature in the dark for 30min, and measure the absorbance of the nanocomposite at 517nm; at the same time, use anhydrous ethanol (100uL) instead of DPPH solution as a blank group, and use deionized water (100uL) mixed with DPPH solution as a control group, and measure the absorbance after mixing.

[0074]

[0075] A0 is the absorbance value of the nanocomposite

[0076] A1 is the absorbance value of the blank group

[0077] A2 is the absorbance value of the control group

[0078] 3) Based on Examples 1-4, the effects of different chitosan: casein peptide mass ratios on the antioxidant properties of the nanocomposite were studied. The specific experimental results are shown in Figure 1 and Figure 2 .

[0079] like Figure 1 and Figure 2 As shown in the figure, the free radical scavenging rate of casein peptide after complexing with chitosan in different ratios was significantly increased compared with that without adding chitosan, and the free radical scavenging rate was the highest when the ratio was 1:1. This indicates that the nanocomplex formed by chitosan and casein peptide has stronger antioxidant properties, and the antioxidant activity is strongest when the ratio is 1:1.

[0080] Example 6

[0081] Effect of different chitosan:casein peptide ratios on the foaming properties of nanocomposites.

[0082] 1) Determination of foaming property (OR) and foaming stability (FS)

[0083] Take 20mL of nanocomposite solution into a 50mL centrifuge tube, homogenize at 10000rpm for 2min, and immediately measure the total volume of liquid and foam in the centrifuge tube. After standing for 10min, record the total volume again. The calculation formula is as follows:

[0084]

[0085] v is the total volume of the solution,

[0086] v0 is the foam volume at 0 min when homogenization is completed

[0087] v 10 The foam volume 10 minutes after the average is completed

[0088] 2) Based on Examples 1-3, the effects of different chitosan: casein peptide mass ratios on the foaming properties of the nanocomposite were studied. Foaming properties generally refer to the ability to form and stabilize foam. Figure 3 and Figure 4 .

[0089] Depend on Figure 3 and Figure 4 It can be seen that the foaming property and foaming stability of chitosan-casein peptide nanocomposite combined with chitosan are significantly increased compared with casein peptide without chitosan (P<0.05), which may be due to the electrostatic interaction between chitosan and casein peptide, which can enhance the foam stability. And when the ratio of chitosan to casein peptide is 1:1, the foaming ability is the best.

[0090] Example 7

[0091] Effect of different ratios of complexes on the storage stability of prepared oil-in-water emulsions.

[0092] 1) Storage stability determination

[0093] The particle size of the water-in-oil emulsion system was measured using a laser particle size analyzer on October 23, October 30, November 6, and November 13, with a measurement period of 28 days. The specific experimental results are shown in Figure 5 .

[0094] 2) If Figure 5 As shown, the particle size of the water-in-oil emulsion made of the complex with added chitosan is smaller than that of the emulsion without added chitosan, and when the chitosan: casein peptide ratio is 1:1, the particle size is the smallest, which means that the water-in-oil emulsion obtained by homogenizing the complex prepared in this ratio with oil has good storage stability.

[0095] Example 8

[0096] Effect of different ratios of complexes on pH stability of prepared oil-in-water emulsions.

[0097] 1) pH stability determination

[0098] The particle size of the water-in-oil emulsion made of nanocomposites with different proportions was measured by a laser particle size analyzer at pH 3, 5, 7, and 9. The specific experimental results are shown in Figure 6 .

[0099] 2) If Figure 6As shown in the figure, when chitosan is not added, the particle size of the casein emulsion is the largest at pH 4.7 or so. This is because the isoelectric point of casein is 4.7. When the pH is at this point, casein reaches the isoelectric point and aggregates and precipitates. When the pH increases, the particle size decreases because casein has the highest solubility under alkaline conditions. When the water-in-oil emulsion made with 1:1 and 2:1 ratio complexes has the highest solubility at pH 5.5 and the smallest particle size, that is, the optimal pH is 5.5, and the particle size of the emulsion made with the 1:1 ratio complex is generally smaller than 2:1, indicating that the water-in-oil emulsion has the strongest stability at pH 5.5, and the stability of the 1:1 ratio is greater than 2:1.

[0100] Example 9

[0101] 1) Determination of ion stability

[0102] The particle sizes of the water-in-oil emulsions made of different ratios of the complex were measured by a laser particle size analyzer at ion concentrations of 0, 50, 100, and 200 mM. The specific experimental results are shown in Figure 7 .

[0103] 2) If Figure 7 As shown, the particle size increases with the increase of ion concentration and decreases with the increase of chitosan content, that is, the emulsion made of the complex with the addition of chitosan has better ion stability than that without chitosan, and the ion stability is strongest when the ratio of chitosan: casein peptide is 1:1.

[0104] 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 technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chitosan-casein peptide nanocomposite, characterized in that: The preparation method comprises the following steps: 1) Dissolve chitosan in an acidic solution; 2) Dissolve casein in an alkaline solution; 3) using protease to enzymatically hydrolyze the protease to obtain an enzymatic solution, and then inactivating the enzyme; 4) The enzymatic hydrolysate is freeze-dried to obtain casein peptide powder; 5) casein peptide powder is dissolved in water to obtain casein peptide solution; 6) Mixing the casein peptide solution and the chitosan solution in an appropriate ratio to obtain a dispersion, and freeze-drying to obtain chitosan-casein peptide nanocomposite powder.

2. The chitosan-casein peptide nanocomposite according to claim 1, characterized in that: The acidic solution described in step 1 is an acetic acid solution, and the mass ratio of acetic acid to water is 1:

99.

3. The chitosan-casein peptide nanocomposite according to claim 1, characterized in that: The alkaline solution described in step 2 is a NaOH solution.

4. The chitosan-casein peptide nanocomposite according to claim 1, characterized in that: The protease described in step 3 is trypsin.

5. The chitosan-casein peptide nanocomposite according to claim 1, characterized in that: The volume ratio of chitosan to casein peptide in step 6 is 0:1, 1:1, and 2:

1.

6. Use of a chitosan-casein peptide nanocomposite in the preparation of an oil-in-water emulsion according to claim 1, characterized in that: The soybean oil and the chitosan-casein peptide nanocomplex are homogenized and ultrasonically mixed at a ratio of 1:9 to obtain an oil-in-water emulsion.

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