Lysozyme condensed microdroplet as well as preparation method and application thereof

Abstract

The invention discloses lysozyme condensed microdroplets as well as a preparation method and application thereof. The lysozyme condensed microdroplet comprises egg white source lysozyme, food protein, edible inorganic salt and water, wherein the food protein comprises at least one of beta-conglycinin and beta-lactoglobulin. The preparation method of the lysozyme condensed microdroplet comprises the following steps: 1) preparing an egg white source lysozyme dispersion liquid and a food protein dispersion liquid; and 2) mixing the two dispersion liquids, and stirring until liquid-liquid phase separation occurs. The lysozyme condensed microdroplet is good in thermodynamic stability and long-term storage stability, the lysozyme is high in mobility in the condensed microdroplet and high in accessibility to a substrate, has long-acting antibacterial and preservative effects, is suitable for being used as a food additive, and is simple in preparation method, green and environment-friendly in process, wide in raw material source, low in production cost, low in energy consumption and suitable for industrial production. The method is suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a lysozyme agglomerated microdroplet and a preparation method and application thereof. Background Art

[0002] Lysozyme is a glycoside hydrolase that hydrolyzes the 1,4-β-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues in bacterial cell wall peptidoglycan, resulting in excellent antimicrobial activity. Egg white-derived lysozyme, derived from food raw materials, is abundant, easily extracted, readily digested and degraded by the human body, and highly safe, making it widely used in food preservation. However, complex food processing environments (e.g., high-temperature processing, weakly alkaline food processing systems) and storage processes can adversely affect the structure of lysozyme, thereby reducing its antimicrobial activity. This has made it difficult for lysozyme to replace chemically synthesized preservatives in the food industry.

[0003] At present, mainly by immobilizing lysozyme to improve its structural stability, and then maintain its activity, for example: CN 103041377A discloses a method for loading lysozyme using chitosan / γ-polyglutamic acid composite nanoparticles;CN 116410958A discloses a method for preparing lysozyme inclusion particles using macroporous resin AB-8 as a carrier and glutaraldehyde as a cross-linking agent;3) Pan et al. disclose a method (Journal of the American Chemical Society, 2018, 150, 47, 16032-16036) for immobilizing lysozyme using metal organic framework (MOF) material ZIF-8. However, due to the fact that lysozyme will not only be distributed on the surface of chitosan / γ-polyglutamic acid composite nanoparticles, macroporous resin AB-8 and ZIF-8, but will also enter their interior, the accessibility of lysozyme to the substrate will be reduced, and these carriers have irreversibility and need to add organic reagents during preparation and processing, and these factors will lead to structural changes and decreased activity of lysozyme.

[0004] Therefore, it is of great significance to develop a lysozyme product with good thermodynamic stability, good long-term storage stability, and long-lasting antibacterial and preservative effects. Summary of the Invention

[0005] One of the purposes of the present invention is to overcome the problems of poor stability and low activity of existing lysozyme products, and to provide lysozyme agglomerated droplets with good thermodynamic stability and long-term storage stability, strong mobility of lysozyme in agglomerated droplets, high accessibility to substrates, and long-lasting antibacterial and antiseptic effects.

[0006] The second object of the present invention is to provide a method for preparing lysozyme agglomerated droplets with simple operation, green and environmentally friendly process, wide source of raw materials, low production cost and low energy consumption.

[0007] A third object of the present invention is to provide a food comprising the above-mentioned lysozyme agglomerated droplets.

[0008] The technical solution adopted by the present invention is:

[0009] A lysozyme agglomerate droplet comprises lysozyme from egg white, food protein, edible inorganic salt and water; the food protein comprises at least one of beta-conglycinin and beta-lactoglobulin.

[0010] Preferably, the mass percentage of hen egg white-derived lysozyme in the lysozyme agglomerated droplets is 6% to 40%.

[0011] Preferably, the mass percentage of food protein in the lysozyme agglomerated droplets is 6% to 40%.

[0012] Preferably, the mass percentage of the edible inorganic salt in the lysozyme agglomerated droplets is 0.1% to 0.3%.

[0013] Preferably, the edible inorganic salt is at least one of sodium chloride and potassium chloride.

[0014] Preferably, the diameter of the lysozyme agglomerated droplets is 1 μm to 4 μm.

[0015] A method for preparing the above-mentioned lysozyme coagulation droplets comprises the following steps:

[0016] 1) dispersing hen egg white-derived lysozyme and food protein separately in water, adding edible inorganic salts and adjusting the pH to 6-7 to obtain hen egg white-derived lysozyme dispersion and food protein dispersion;

[0017] 2) The egg white-derived lysozyme dispersion and the food protein dispersion are mixed and stirred until liquid-liquid phase separation occurs, thereby obtaining lysozyme agglomerated droplets.

[0018] Preferably, the mass percentage of hen egg white-derived lysozyme in the hen egg white-derived lysozyme dispersion in step 1) is 0.1% to 2.0%.

[0019] Preferably, the mass percentage of edible inorganic salts in the egg white-derived lysozyme dispersion in step 1) is 0.1% to 0.3%.

[0020] Preferably, the mass percentage of food protein in the food protein dispersion in step 1) is 0.1% to 2.0%.

[0021] Preferably, the mass percentage of edible inorganic salts in the food protein dispersion in step 1) is 0.1% to 0.3%.

[0022] Preferably, the dispersion method in step 1) is mechanical stirring.

[0023] Preferably, the mechanical stirring is carried out at a stirring rate of 200 rpm to 300 rpm and a temperature of 10° C. to 40° C., and the stirring time is 1 hour to 3 hours.

[0024] Preferably, in step 2), the mass ratio of the egg white-derived lysozyme dispersion to the food protein dispersion is 1:0.25-4.

[0025] Preferably, the stirring in step 2) is carried out at a stirring rate of 100 rpm to 200 rpm and a temperature of 10° C. to 40° C., and the stirring time is 1 min to 5 min.

[0026] A food comprising the above-mentioned lysozyme agglomerated droplets.

[0027] The beneficial effects of the present invention are as follows: the lysozyme agglomerated droplets of the present invention have good thermodynamic stability and long-term storage stability, the lysozyme has strong mobility in the agglomerated droplets, high accessibility to the substrate, and has a long-lasting antibacterial and antiseptic effect, and is suitable as a food additive. Moreover, the preparation method thereof is simple, the process is green and environmentally friendly, the raw material source is wide, the production cost is low, and the energy consumption is low, and it is suitable for large-scale industrial production and application.

[0028] Specifically:

[0029] 1) The lysozyme coacervate droplets of the present invention have a dynamic structure and semi-permeability. Lysozyme is mobile and accessible to substrates in the coacervate droplets, which helps the lysozyme exert its antibacterial effect.

[0030] 2) The lysozyme coacervate microdroplets of the present invention contain food protein microspheres, which can self-assemble to form a molecularly crowded environment (without the introduction of chemical cross-linking agents or modifiers, which is environmentally friendly). This environmental crowding effect can increase the denaturation temperature related to the thermodynamic stability of lysozyme by about 5°C, and can maintain the enzymatic activity of lysozyme (the bactericidal efficacy against Gram-positive bacteria) for 10 days at 25°C without any decrease;

[0031] 3) The food protein in the lysozyme agglomerate droplets of the present invention is at least one of β-conglycinin and β-lactoglobulin, which are the main components of soy protein, a byproduct of soybean oil processing, and whey protein, a byproduct of cheese processing, respectively. These two food proteins are abundant in resources and low in cost, thereby reducing the production cost of the lysozyme agglomerate droplets;

[0032] 4) The preparation process of the lysozyme agglomerated droplets of the present invention uses only water as a solvent, is green, non-toxic and environmentally friendly, and has a simple preparation process, low energy consumption, and does not require large-scale equipment, making it suitable for large-scale industrial production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a microscope image of the lysozyme aggregate droplets in Example 1.

[0034] Figure 2 This is a laser confocal microscopy image of the lysozyme aggregated droplets in Example 1.

[0035] Figure 3 Graphs showing the results of lysozyme activity tests on the lysozyme agglomerated droplets in Examples 1 to 3, the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2, and the lysozyme dispersion in Comparative Example 3.

[0036] Figure 4 Graphs showing the test results of lysozyme diffusion rates for the lysozyme agglomerated droplets in Examples 1 to 3, the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2, and the lysozyme dispersion in Comparative Example 3.

[0037] Figure 5 Graphs showing the lysozyme denaturation temperature test results for the lysozyme agglomerated droplets in Examples 1 to 3, the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2, and the lysozyme dispersion in Comparative Example 3. DETAILED DESCRIPTION

[0038] The present invention will be further explained and illustrated below with reference to specific embodiments.

[0039] Example 1:

[0040] A lysozyme agglomerated microdroplet, the preparation method of which is as follows:

[0041] 1) adding hen egg white-derived lysozyme to distilled water at a mass ratio of 0.1:99.9, adding 0.1 wt% NaCl, stirring at a stirring rate of 200 rpm and a temperature of 10° C. for 1 hour, and adjusting the pH value to 6 to obtain a hen egg white-derived lysozyme dispersion; adding β-conglycinin to distilled water at a mass ratio of 0.1:99.9, adding 0.1 wt% NaCl, stirring at a stirring rate of 200 rpm and a temperature of 10° C. for 1 hour, and adjusting the pH value to 6 to obtain a food protein dispersion;

[0042] 2) The egg white-derived lysozyme dispersion and the food protein dispersion were mixed at a mass ratio of 1:0.25, and then stirred at a stirring rate of 100 rpm and a temperature of 10°C for 1 min until liquid-liquid phase separation occurred, thereby obtaining lysozyme agglomerated droplets.

[0043] The microscopic image of the lysozyme aggregate droplets in this example is as follows: Figure 1 As shown, laser confocal microscopy images are Figure 2 (fluorescently labeled lysozyme) as shown.

[0044] Depend on Figure 1 and Figure 2 It can be seen that the size of the lysozyme agglomerated droplets is relatively uniform, with a diameter of 1 μm to 4 μm.

[0045] Example 2:

[0046] A lysozyme agglomerated microdroplet, the preparation method of which is as follows:

[0047] 1) adding hen egg white-derived lysozyme to distilled water at a mass ratio of 2:98, adding 0.3 wt % NaCl, stirring at a stirring rate of 300 rpm and a temperature of 30° C. for 3 hours, and adjusting the pH value to 7 to obtain a hen egg white-derived lysozyme dispersion; adding β-conglycinin to distilled water at a mass ratio of 2:98, adding 0.3 wt % NaCl, stirring at a stirring rate of 300 rpm and a temperature of 30° C. for 3 hours, and adjusting the pH value to 7 to obtain a food protein dispersion;

[0048] 2) The egg white-derived lysozyme dispersion and the food protein dispersion were mixed in a mass ratio of 1:4 and stirred at 200 rpm and 30°C for 5 min until liquid-liquid phase separation occurred, thereby obtaining lysozyme agglomerated droplets.

[0049] Example 3:

[0050] A lysozyme agglomerated microdroplet, the preparation method of which is as follows:

[0051] 1) adding hen egg white-derived lysozyme to distilled water at a mass ratio of 1:99, adding 0.2 wt % NaCl, stirring for 2 h at a stirring rate of 250 rpm and a temperature of 25° C., and adjusting the pH to 6.5 to obtain a hen egg white-derived lysozyme dispersion; adding β-lactoglobulin to distilled water at a mass ratio of 1:99, adding 0.2 wt % NaCl, stirring for 2 h at a stirring rate of 250 rpm and a temperature of 25° C., and adjusting the pH to 6.5 to obtain a food protein dispersion;

[0052] 2) The egg white-derived lysozyme dispersion and the food protein dispersion were mixed in a mass ratio of 1:2 and stirred at 150 rpm and 25°C for 3 min until liquid-liquid phase separation occurred, thereby obtaining lysozyme agglomerated droplets.

[0053] Comparative Example 1:

[0054] A lysozyme-food protein mixed dispersion, the preparation method of which is as follows:

[0055] 1) adding hen egg white-derived lysozyme to distilled water at a mass ratio of 1:99, adding 1 wt% NaCl, stirring at a stirring rate of 300 rpm and a temperature of 25° C. for 3 hours, and adjusting the pH value to 6 to obtain a hen egg white-derived lysozyme dispersion; adding β-lactoglobulin to distilled water at a mass ratio of 1:99, adding 1 wt% NaCl, stirring at a stirring rate of 300 rpm and a temperature of 25° C. for 3 hours, and adjusting the pH value to 6 to obtain a food protein dispersion;

[0056] 2) The egg white-derived lysozyme dispersion and the food protein dispersion were mixed in a mass ratio of 1:1, and then stirred at a stirring rate of 200 rpm and a temperature of 25°C for 3 minutes (no liquid-liquid phase separation occurred) to obtain a lysozyme-food protein mixed dispersion.

[0057] Comparative Example 2:

[0058] A lysozyme-food protein mixed dispersion, the preparation method of which is as follows:

[0059] 1) adding hen egg white-derived lysozyme to distilled water at a mass ratio of 1:99, adding 0.2 wt % NaCl, stirring for 3 h at a stirring rate of 300 rpm and a temperature of 25° C., and adjusting the pH value to 9 to obtain a hen egg white-derived lysozyme dispersion; adding β-lactoglobulin to distilled water at a mass ratio of 1:99, adding 0.2 wt % NaCl, stirring for 3 h at a stirring rate of 300 rpm and a temperature of 25° C., and adjusting the pH value to 9 to obtain a food protein dispersion;

[0060] 2) The egg white-derived lysozyme dispersion and the food protein dispersion were mixed in a mass ratio of 1:1, and then stirred at a stirring rate of 200 rpm and a temperature of 25°C for 3 minutes (no liquid-liquid phase separation occurred) to obtain a lysozyme-food protein mixed dispersion.

[0061] Comparative Example 3:

[0062] A lysozyme dispersion, the preparation method of which is as follows:

[0063] The egg white-derived lysozyme was added to distilled water at a mass ratio of 1:99, and the mixture was stirred at a stirring rate of 300 rpm and a temperature of 25° C. for 3 h to obtain a lysozyme dispersion.

[0064] Performance testing:

[0065] 1) Using Micrococcus lysodeikticus as a substrate, the lysozyme activity of the lysozyme aggregated droplets in Examples 1 to 3, the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2, and the lysozyme dispersion in Comparative Example 3 before and after storage at 25°C for 12 days was determined using a lysozyme activity kit. The test results are shown in FIG. Figure 3 shown.

[0066] Depend on Figure 3 It can be seen that: before storage, all samples had a complete enzyme activity of 40,000 U. After storage at 25°C for 12 days, the lysozyme activity of the lysozyme coagulated droplets in Examples 1 to 3 was still greater than 37,000 U, and the decrease in lysozyme activity was small. However, the lysozyme activity of the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2 and the lysozyme dispersion in Comparative Example 3 was less than 30,000 U, and the decrease in lysozyme activity was large. The reason is that the lysozyme coagulated droplets in Examples 1 to 3 all had molecular crowding, which led to a reduction in the available volume. At this time, the folded protein occupied less space. On the contrary, the unfolding of the protein would pay a higher entropy cost due to the crowding of the surrounding molecules. Therefore, the lysozyme maintained the folded state, while the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2 and the lysozyme dispersion in Comparative Example 3 lacked the crowded environment effect.

[0067] 2) Lysozyme was labeled with fluorescein isothiocyanate, and lysozyme agglomerated droplets, a lysozyme-food protein mixed dispersion, and a lysozyme dispersion were prepared, respectively, according to the methods of Examples 1 to 3 and Comparative Examples 1 to 3. Then, fluorescence was excited at a wavelength of 488 nm using a laser confocal microscope (40× objective lens). A target area with a radius of 1 μm was bleached using 100% laser intensity. The bleached images were collected for 5 frames until the fluorescence returned to normal. The diffusion rate of lysozyme (denoted as D) was then calculated; the diffusion rate calculation formula is as follows: D = r e 2 / 4τ D , where r e is the effective bleaching radius, τ D is the characteristic recovery time calculated by fitting the fluorescence intensity with an exponential function), and the test results are shown in Figure 4 shown.

[0068] Depend on Figure 4 It can be seen that the lysozyme aggregate droplets in Examples 1 to 3 (diffusion rate of 31.6 μm 2 / s~33.5μm 2 / s), the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2 and the lysozyme dispersion in Comparative Example 3 all have high fluidity and good accessibility to the substrate.

[0069] 3) The lysozyme agglomerated droplets in Examples 1 to 3, the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2, and the lysozyme dispersion in Comparative Example 3 were freeze-dried, and 3 mg of the freeze-dried sample was weighed into a 40 μL standard aluminum crucible. 70 μL of distilled water was added and sealed, and the crucible was fully hydrated for 6 h. The denaturation temperature of the lysozyme was measured by differential scanning calorimetry at a heating rate of 5 ° C / min and a heating range of 30 ° C to 110 ° C. The test results are as follows: Figure 5 (An empty sealed crucible was used as a reference; the peak temperature was used to represent the denaturation temperature of lysozyme).

[0070] Depend on Figure 5 It can be seen that the denaturation temperature of lysozyme in the lysozyme agglomerated droplets in Examples 1 to 3 is 70.3°C to 70.8°C, while the denaturation temperatures of lysozyme in the lysozyme-food protein mixed dispersions in Comparative Examples 1 to 2 and the lysozyme dispersion in Comparative Example 3 are 65.5°C, 67.7°C and 65.3°C, respectively. This indicates that the present invention increases the energy required for protein folding through molecular crowding, thereby significantly improving the thermal denaturation temperature of lysozyme.

[0071] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A lysozyme agglomerated droplet, characterized in that: The composition includes egg white-derived lysozyme, food protein, edible inorganic salt and water; the food protein includes at least one of beta-conglycinin and beta-lactoglobulin.

2. The lysozyme agglomerated droplet according to claim 1, characterized in that: The mass percentage of the hen egg white-derived lysozyme in the lysozyme agglomerated microdroplets is 6% to 40%.

3. The lysozyme agglomerated droplet according to claim 1 or 2, characterized in that: The mass percentage of the food protein in the lysozyme agglomerated microdroplets is 6% to 40%.

4. The lysozyme agglomerated droplet according to claim 1 or 2, characterized in that: The mass percentage of the edible inorganic salt in the lysozyme agglomerated microdroplets is 0.1% to 0.3%.

5. The lysozyme agglomerated droplet according to claim 1 or 2, characterized in that: The edible inorganic salt is at least one of sodium chloride and potassium chloride.

6. The lysozyme agglomerated droplet according to claim 1 or 2, characterized in that: The diameter of the lysozyme agglomerated droplets is 1 μm to 4 μm.

7. A method for preparing lysozyme agglomerated droplets according to any one of claims 1 to 6, characterized in that: The following steps are involved: 1) dispersing hen egg white-derived lysozyme and food protein separately in water, adding edible inorganic salts and adjusting the pH to 6-7 to obtain hen egg white-derived lysozyme dispersion and food protein dispersion; 2) The egg white-derived lysozyme dispersion and the food protein dispersion are mixed and stirred until liquid-liquid phase separation occurs, thereby obtaining lysozyme agglomerated droplets.

8. The preparation method according to claim 7, characterized in that: The mass percentage of hen egg white-derived lysozyme in the hen egg white-derived lysozyme dispersion in step 1) is 0.1% to 2.0%; the mass percentage of food protein in the food protein dispersion in step 1) is 0.1% to 2.0%; and the mass ratio of the hen egg white-derived lysozyme dispersion to the food protein dispersion in step 2) is 1:0.25 to 4.

9. The preparation method according to claim 7 or 8, characterized in that: Step 2) The stirring is carried out at a stirring rate of 100 rpm to 200 rpm and a temperature of 10° C. to 40° C., and the stirring time is 1 min to 5 min.

10. A food, characterized in that The lysozyme agglomerated droplets are comprised of any one of claims 1 to 6.

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