Lactoferrin gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of dairy products, and particularly relates to lactoferrin gel as well as a preparation method and application thereof. The preparation raw materials comprise lactoferrin, sodium alginate, an acidifying agent, an aqueous medium and a calcium ion slow-release system and / or chitosan, wherein the calcium ion slow-release system comprises inorganic calcium salt and sodium citrate; the pH value of the lactoferrin gel is less than or equal to the pH value of a lactoferrin isoelectric point. The lactoferrin in the lactoferrin gel is uniform in distribution, compact in structure and small in aperture, and the embedding rate of the lactoferrin can reach 96.19%.

Description

Technical Field

[0001] The invention belongs to the technical field of dairy product preparation, and particularly relates to a lactoferrin gel and a preparation method and application thereof. Background Art

[0002] Lactoferrin (LF) is an iron-binding glycoprotein belonging to the transferrin family. It is widely present in human milk and various secretions, especially in breast milk. It is an important immune function protein in colostrum. Because of its antibacterial, antioxidant, anti-inflammatory, anti-cancer, antiviral and immunomodulatory physiological activities, it is often used as a supplement in infant formula, nutritional supplements or food and cosmetics.

[0003] However, lactoferrin may be inactivated or degraded under specific pH and temperature conditions. In addition, salt ion concentration, buffer environment and the iron saturation of lactoferrin will also affect its stability. These instability factors limit the application of lactoferrin under certain conditions, and the relevant conditions must be strictly controlled during storage, processing and use, which to a certain extent limits the use of lactoferrin.

[0004] In the prior art, lactoferrin is generally prepared into different gel forms (such as microparticles, capsules, liposomes, microspheres, nanoparticles or gels) by encapsulation. Among them, the gel form is one of the main encapsulation forms of lactoferrin because of its advantages such as stable structure, good biocompatibility, excellent controlled release performance, easy preparation and wide application.

[0005] Therefore, developing a lactoferrin gel is of great significance for the application of lactoferrin. Summary of the invention

[0006] The present invention develops a lactoferrin gel, which is actually prepared by the combined action of sodium alginate and an acidifier and a calcium ion sustained-release system and / or chitosan. The lactoferrin in the gel has a high embedding rate, a compact structure and a small pore size.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] On one hand, the present invention provides a lactoferrin gel, wherein the raw materials for preparing the lactoferrin gel include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system and / or chitosan, wherein the calcium ion sustained-release system includes an inorganic calcium salt and sodium citrate; and the pH value of the lactoferrin gel is ≤ the isoelectric point pH of the lactoferrin.

[0009] Preferably, the pH value of the lactoferrin gel is ≤5.2.

[0010] More preferably, the pH value of the lactoferrin gel is ≤4.5.

[0011] Preferably, the raw materials for preparing the above-mentioned lactoferrin gel meet one or more combinations of the following conditions: (i) the mass fraction of lactoferrin is 1%-5%; (ii) the molar concentration of the inorganic calcium salt is 6mmol / L-18mmol / L; (iii) the molar concentration of sodium citrate is 10mmol / L-30mmol / L; (iv) the mass fraction of sodium alginate is 0.1%-0.5%; (v) the mass fraction of chitosan is 0.05%-0.15%; (vi) the mass fraction of the acidifier is 2%-3%; (vii) the acidifier is one or more of D-gluconolactone, L-malic acid or bacterial strains; (viii) the inorganic calcium salt is one or more of calcium chloride, calcium carbonate or calcium sulfate.

[0012] More preferably, the raw materials for preparing the lactoferrin gel satisfy one or more combinations of the following conditions: (a) the mass fraction of lactoferrin is 3%; (b) the molar concentration of inorganic calcium salt is 9 mmol / L; (c) the molar concentration of sodium citrate is 15 mmol / L; (d) the mass fraction of sodium alginate is 0.3%; (e) the mass fraction of chitosan is 0.15%; (f) the mass fraction of acidifier is 2.5%.

[0013] Another aspect of the present invention provides a method for preparing the lactoferrin gel of the present invention.

[0014] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, and a calcium ion sustained-release system, the preparation method includes:

[0015] (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution;

[0016] (2) mixing the sodium alginate aqueous solution and the lactoferrin solution to obtain a mixed solution;

[0017] (3) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel;

[0018] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium and chitosan, the preparation method includes:

[0019] (1) mixing a lactoferrin aqueous solution, a sodium alginate aqueous solution and a lactoferrin solution to obtain a mixed solution;

[0020] (2) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel;

[0021] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system, and chitosan, the preparation method includes:

[0022] (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution;

[0023] (2) mixing the sodium alginate aqueous solution, the chitosan aqueous dispersion and the lactoferrin solution to obtain a mixed solution;

[0024] (3) adding an acidifier to the mixture and fermenting it until the pH value is ≤ the isoelectric point pH of lactoferrin to obtain lactoferrin gel.

[0025] Preferably, in the above preparation method, when the preparation raw materials include lactoferrin, sodium alginate, an acidulant and an aqueous medium as well as a calcium ion sustained-release system and chitosan, in step (2), sodium alginate aqueous solution and chitosan aqueous dispersion are sequentially added to the lactoferrin solution and mixed to obtain a mixed solution.

[0026] In another aspect, the present invention provides a lactoferrin powder, which is prepared from the lactoferrin gel of the present invention.

[0027] In another aspect, the present invention provides a dairy product, which comprises the lactoferrin gel of the present invention or the lactoferrin powder of the present invention.

[0028] Preferably, the above-mentioned dairy products include sterilized milk, sterilized milk, fermented milk, milk flakes, milk powder, cheese or lactose.

[0029] The beneficial effects of the present invention include: the lactoferrin in the lactoferrin gel provided by the present invention is evenly distributed, compact in structure, small in pore size, and the lactoferrin embedding rate can reach 96.19%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a lactoferrin solution situation diagram of Comparative Example 1 and Comparative Example 2;

[0031] Figure 2 The pH changes of the solutions before and after fermentation in different groups;

[0032] Figure 3 The storage modulus G' of rheological analysis for different groups;

[0033] Figure 4 The loss modulus G" of rheological analysis for different groups;

[0034] Figure 5 LF-Alg / Ca 2+ Microscopic image of group gel solid;

[0035] Figure 6 0.05% CS-LF-Alg / Ca 2+ Microscopic image of group gel solid;

[0036] Figure 7 0.10% CS-LF-Alg / Ca 2+ Microscopic image of group gel solid;

[0037] Figure 8 0.15% CS-LF-Alg / Ca 2+ Microscopic image of group gel solid;

[0038] Fig. 9 0.20% CS-LF-Alg / Ca 2+ Microscopic image of group gel solid;

[0039] Fig.10 This is the solid microscopic image of the gel of 0.15% CS-LF-Alg group;

[0040] Fig.11 This is the microscopic picture of the 0.15% CS-LF-Alg / CaCl2 system;

[0041] Fig.12 This is the solid microscopic image of 0.15% CS-LF-Alg / CC group. DETAILED DESCRIPTION

[0042] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0043] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0044] The embodiment of the present invention provides a lactoferrin gel, wherein the raw materials for preparing the lactoferrin gel include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system and / or chitosan, wherein the calcium ion sustained-release system includes an inorganic calcium salt and sodium citrate; and the pH value of the lactoferrin gel is ≤ the isoelectric point pH of the lactoferrin.

[0045] It should be noted that, in the present invention, the raw materials may include lactoferrin, sodium alginate, an acidulant, an aqueous medium and a calcium ion sustained release system, or the raw materials may include lactoferrin, sodium alginate, an acidulant, an aqueous medium and chitosan, or lactoferrin, sodium alginate, an acidulant, an aqueous medium and a calcium ion sustained release system and chitosan; the lactoferrin gel prepared in the above three situations has an excellent embedding rate; wherein, when the calcium ion sustained release system and chitosan are contained at the same time, the embedding rate is better; in addition, if only chitosan is used to embed lactoferrin (without sodium alginate and the calcium ion sustained release system), chitosan will cause the lactoferrin to be adsorbed to form a precipitate, and gel cannot be formed, thereby affecting the embedding rate of lactoferrin.

[0046] It should also be noted that in the present invention, when the calcium ion sustained-release system and chitosan are contained at the same time, the presence of inorganic calcium salt and sodium citrate can make the system slowly release calcium ions (Ca 2+ ), when the pH value is greater than the isoelectric point of lactoferrin (for example, greater than 6.0), chitosan is uncharged, and Ca 2+ Positively charged, lactoferrin and sodium alginate are negatively charged, Ca 2+ It coordinates with the -COO- of sodium alginate to form an "egg box" structure. A very small amount of lactoferrin and Ca 2+ After adding chitosan, chitosan is dispersed in the mixed system of lactoferrin and sodium alginate; when the acidifier is added, as the fermentation proceeds, the pH of the system slowly decreases to below the isoelectric point pH of lactoferrin (for example, 4.5), chitosan and lactoferrin are positively charged, and the Ca bound to lactoferrin 2+ It cross-links with sodium alginate to form calcium caseinate, which in turn forms a calcium cross-linking system. The positively charged chitosan is cross-linked with lactoferrin and Ca 2+ Electrostatic repulsion occurs, causing Ca 2+ The cross-linking system of lactoferrin and sodium alginate is more stable and less lactoferrin precipitates.

[0047] It should also be noted that in the present invention, inorganic calcium ions such as inorganic calcium salts or calcium citrate cannot be directly used to provide calcium ions; specifically, if inorganic calcium salts are directly used, when the pH value is greater than the isoelectric point pH of lactoferrin (for example, greater than 6.0), inorganic calcium ions (such as calcium chloride) are directly used, and the calcium ions are quickly released in the solution, combined with lactoferrin and sodium alginate, and spin to form a solution, resulting in the pH being reduced to below 4.5, and a gel of calcium-crosslinked lactoferrin and sodium alginate cannot be formed, and chitosan cannot show positive charge to lactoferrin. The electrostatic repulsion of ferritin will prevent the formation of an embedding system for lactoferrin. In addition, if calcium citrate is used directly, when the pH value is greater than the isoelectric point pH of lactoferrin (for example, greater than 6.0), calcium citrate is used directly. Because calcium citrate is slightly soluble in water, as the pH value decreases, even if a small amount of calcium ions are electrolyzed, the amount of free calcium ions in the entire system cannot meet the requirement of forming a calcium-crosslinked gel of lactoferrin and sodium alginate as the pH value decreases, and chitosan cannot exhibit the electrostatic repulsion of positive charge on lactoferrin.

[0048] In some specific examples, the pH value of the lactoferrin gel is ≤5.2, and more preferably the pH value is ≤4.5. For example, the pH value can be ≤4, ≤3, such as 5, 4.7, 4.5, 4.3, 4, 3.5 or 3, etc.; specifically, when the pH value is ≤4.5, the positive and negative ions in the system are basically balanced, and the gel system is more stable.

[0049] In some specific examples, the raw materials for preparing the lactoferrin gel meet one or more combinations of the following conditions:

[0050] (i) the lactoferrin mass fraction is 1%-5%, such as 2%, 3% or 4%; specifically, in the present invention, when the lactoferrin content is too high, the amount of calcium ions and sodium alginate will be relatively low, and a stable calcium-crosslinked gel of lactoferrin and sodium alginate cannot be formed, thereby reducing the embedding rate of lactoferrin;

[0051] (ii) the molar concentration of the inorganic calcium salt is 6mmol / L-18mmol / L, such as 7mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L or 17mmol / L. Specifically, if the concentration of calcium chloride is too high, the high content of calcium ions in the solution combines with lactoferrin and sodium alginate and spins to form a solution, resulting in the pH value being reduced to below 4.5, and a calcium-crosslinked gel of lactoferrin and sodium alginate cannot be formed, thereby reducing the embedding rate of lactoferrin;

[0052] (iii) the molar concentration of sodium citrate is 10mmol / L-30mmol / L, such as 12mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 18mmol / L, 20mmol / L, 25mmol / L, 27mmol / L or 28mmol / L. Specifically, if the concentration of sodium citrate is too high, calcium ions in the solution will be complexed with citrate, resulting in a low calcium ion concentration in the solution, and a calcium-crosslinked gel of lactoferrin and sodium alginate cannot be formed during the acidification process, thereby reducing the embedding rate of lactoferrin;

[0053] (iv) the mass fraction of sodium alginate is 0.1%-0.5%, such as 0.2%, 0.3% or 0.4%;

[0054] (v) the mass fraction of chitosan is 0.05%-0.15%, such as 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11% or 0.12%, etc.;

[0055] (vi) the mass fraction of the acidifier is 2.5%, such as 2.3%, 2.5%, 2.7% or 2.9%;

[0056] (vii) the acidulant is one or more of D-gluconolactone, L-malic acid or bacterial species; specifically, the purpose of the acidulant is to reduce the pH of the system to a pH lower than the isoelectric point of lactoferrin, thereby improving the stability of the gel; in addition, the bacterial species is other fermentation bacteria that can slowly reduce the pH value of the system, such as lactic acid bacteria;

[0057] (viii) The inorganic calcium salt is one or more of calcium chloride, calcium carbonate or calcium sulfate.

[0058] It should be noted that, in the present invention, the preparation of raw materials preferably satisfies at least one of the above conditions, and more preferably satisfies all of the above conditions simultaneously.

[0059] In some specific examples, the raw materials for preparing the above-mentioned lactoferrin gel meet one or more combinations of the following conditions: (a) the mass fraction of lactoferrin is 3%; (b) the molar concentration of inorganic calcium salt is 9 mmol / L; (c) the molar concentration of sodium citrate is 15 mmol / L; (d) the mass fraction of sodium alginate is 0.3%; (e) the mass fraction of chitosan is 0.15%; (f) the mass fraction of acidifier is 2.5%.

[0060] It should be noted that the mass fractions of lactoferrin, inorganic calcium salt, sodium citrate, sodium alginate, chitosan and acidulant in the present invention preferably meet the above conditions, and the prepared lactoferrin gel has a tighter structure, a smaller pore size, a more uniform distribution of lactoferrin, and a higher lactoferrin embedding rate; especially when the raw materials are prepared and all the above conditions are met, the embedding rate of the prepared lactoferrin gel can reach 96.19%, and the structure is tight.

[0061] It should also be noted that the mass fraction in the present invention refers to the mass proportion of the raw material in the entire raw material system. For example, a lactoferrin mass fraction of 1%-5% means that the mass of lactoferrin is 1%-5% of the mass of all raw materials added together. Of course, the raw materials here also include aqueous medium, which acts as a solvent. Among the preparation raw materials in the present invention, in addition to the above-mentioned lactoferrin, inorganic calcium salt, sodium citrate, sodium alginate, chitosan and acidulant, the rest is aqueous medium. The amount of aqueous medium varies with the amount of each raw material, so that the percentage of all raw materials added together can meet 100%.

[0062] The present invention also provides a method for preparing the lactoferrin gel of the present invention, as follows:

[0063] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, and a calcium ion sustained-release system, the preparation method includes:

[0064] (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution;

[0065] (2) mixing the sodium alginate aqueous solution and the lactoferrin solution to obtain a mixed solution;

[0066] (3) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel;

[0067] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium and chitosan, the preparation method includes:

[0068] (1) mixing a lactoferrin aqueous solution, a sodium alginate aqueous solution and a lactoferrin solution to obtain a mixed solution;

[0069] (2) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel;

[0070] When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system, and chitosan, the preparation method includes:

[0071] (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution;

[0072] (2) mixing the sodium alginate aqueous solution, the chitosan aqueous dispersion and the lactoferrin solution to obtain a mixed solution;

[0073] (3) adding an acidifier to the mixed solution and fermenting it until the pH value is ≤ the isoelectric point pH of lactoferrin to obtain lactoferrin gel.

[0074] In some specific examples, in the above preparation method, when the preparation raw materials include lactoferrin, sodium alginate, an acidulant and an aqueous medium, a calcium ion sustained-release system and chitosan, in step (2), sodium alginate aqueous solution and chitosan aqueous dispersion are sequentially added to the lactoferrin solution to mix to obtain a mixed solution.

[0075] It should be noted that when sodium alginate is added to the lactoferrin solution containing the calcium ion sustained-release system, the sodium alginate aqueous solution, the chitosan aqueous dispersion and the lactoferrin solution can be mixed evenly, which is more conducive to forming a calcium-crosslinked lactoferrin and sodium alginate gel system as the pH value decreases, thereby improving the embedding rate of lactoferrin; if the chitosan aqueous dispersion is added first, when the pH value is greater than 5.2, it may cause electrostatic adsorption between lactoferrin and chitosan, thereby causing lactoferrin precipitation, thereby affecting the embedding of lactoferrin; if the chitosan aqueous dispersion is added to the sodium alginate aqueous solution first, it may cause electrostatic adsorption between sodium alginate and chitosan, thereby causing lactoferrin to be in the supernatant, thereby affecting the embedding of lactoferrin.

[0076] The embodiment of the present invention further provides a lactoferrin powder, which is prepared from the lactoferrin gel of the present invention.

[0077] It should be noted that, in order to facilitate transportation and use, the lactoferrin gel of the present invention can be prepared into freeze-dried powder, and the method for preparing the freeze-dried powder is a method well known in the art.

[0078] The embodiment of the present invention further provides a dairy product, which includes the lactoferrin gel of the present invention or the lactoferrin powder of the present invention.

[0079] It should be noted that the lactoferrin gel or the lactoferrin powder of the present invention can be added to dairy products as a lactoferrin supplement to prepare different dairy products, such as sterilized milk, sterilized milk, fermented milk, milk tablets, milk powder, cheese or lactose.

[0080] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0081] 1. Preparation of Lactoferrin Gel

[0082] Example 1 to Example 5

[0083] (1) Preparation of sodium alginate solution: 1.2 g of sodium alginate was added to 118.8 g of deionized water and stirred at 70°C for 1 h until the sodium alginate was completely dissolved to form a sodium alginate solution with a mass fraction of 1.0%. Finally, the solution was hydrated in a refrigerator at 2°C-6°C for 12 h.

[0084] (2) Preparation of chitosan dispersion: 0.6 g of chitosan was added to 59.4 g of water and stirred at 70°C for 1 h until the chitosan swelled and dispersed to form a chitosan dispersion with a mass fraction of 1.0%. Finally, the dispersion was placed in a refrigerator at 2°C-6°C for 12 h.

[0085] (3) Preparation of lactoferrin solution: 9 g of lactoferrin powder was added to 91 g of deionized water and stirred at 30° C. for 30 min until the lactoferrin was completely dissolved. Then, 20 g of a mixed solution of anhydrous calcium chloride and sodium citrate dihydrate (prepared by adding water to 0.30 g of anhydrous calcium chloride and 1.337 g of sodium citrate dihydrate) was added and stirred for 5 min to obtain a lactoferrin solution with a mass fraction of 7.5%. The solution was ready for use without hydration.

[0086] (4) Preparation of lactoferrin gel: The sodium alginate solution and lactoferrin solution prepared above were mixed, deionized water was added, and the mixture was stirred for 2 min. The chitosan solution prepared above was then added and stirred for 5 min. Finally, the freshly prepared D-gluconolactone (GDL) solution (mass fraction 25%) was quickly added and stirred for 20 s. Finally, the mixture was fermented at a constant temperature of 42° C. until the pH value was lower than 4.5 (approximately 3 h) to prepare the lactoferrin gel system.

[0087] The addition amounts of sodium alginate solution, lactoferrin solution, chitosan solution and deionized water are shown in Table 1 below.

[0088] Table 1 Addition amount of different raw materials in lactoferrin gel system

[0089]

[0090]

[0091] In Table 1 above, in the raw materials for preparing the lactoferrin gel solution, the mass fraction of lactoferrin is 3%, the molar concentration of calcium chloride is 9 mmol / L, the molar concentration of sodium citrate is 15 mmol / L, the mass fraction of sodium alginate is 0.3%, the mass fractions of chitosan are 0%, 0.05%, 0.10%, 0.15%, 0.20%, respectively, the mass fraction of GDL is 2.5% and the balance is water.

[0092] Example 6

[0093] The difference between Comparative Example 6 and Example 3 is that: the preparation method of the lactoferrin solution is the same as that of Example 3, and a lactoferrin gel solution (0.15% CS-LF-Alg) is prepared;

[0094] In Example 6, the preparation method of lactoferrin does not include the step of adding calcium chloride and sodium citrate, and specifically comprises: adding 9 g of lactoferrin powder to 111 g of deionized water, and stirring at a constant temperature of 30° C. for 30 min until the lactoferrin is completely dissolved to prepare a lactoferrin solution.

[0095] Comparative Example 1

[0096] The difference between Comparative Example 1 and Example 3 is that the preparation method of the lactoferrin solution is the same as that of Example 3, and a lactoferrin gel (calcium chloride) solution (0.15% CS-LF-Alg / CaCl2) is prepared;

[0097] Among them, in Comparative Example 1, the preparation method of lactoferrin is: 9g lactoferrin powder is added to 91g deionized water, and stirred at a constant temperature of 30°C for 30min until the lactoferrin is completely dissolved, and then a mixed solution prepared by 20g anhydrous calcium chloride (prepared by adding 0.30g anhydrous calcium chloride to water) is added to prepare a lactoferrin solution (0.15% CS-LF-Alg / CaCl2).

[0098] The lactoferrin solution prepared produced a precipitate and did not form a gel state, such as Figure 1 As shown, Figure 1 The solution on the left.

[0099] Comparative Example 2

[0100] The difference between Comparative Example 2 and Example 3 is that: the preparation method of the lactoferrin solution is the same as that of Example 3, and a lactoferrin gel (calcium citrate) solution (0.15% CS-LF-Alg / CC) is prepared;

[0101] Among them, in Comparative Example 2, the preparation method of lactoferrin is: 9g lactoferrin powder is added to 91g deionized water, and stirred at a constant temperature of 30°C for 30min until the lactoferrin is completely dissolved, and then a mixed solution prepared with 20g anhydrous calcium citrate (prepared by adding water to 0.27g anhydrous calcium citrate) is used to prepare a lactoferrin solution (0.15% CS-LF-Alg / CC).

[0102] The lactoferrin solution prepared produced a precipitate and did not form a gel state, such as Figure 1 As shown, Figure 1 The solution on the right.

[0103] 2. Test

[0104] (I) Changes in pH value of solution before and after fermentation

[0105] The pH changes of the solutions before and after fermentation in the examples and comparative examples were monitored respectively (the pH of the system was measured using an electronic pH meter (Sevencompact multi-parameter tester S220, Mettler-Toledo Instruments (Shanghai) Co., Ltd.), before adding GDL, after adding GDL and stirring for 20 seconds, and after fermentation for 3 hours, and the results were as follows: Figure 2 As shown in Figure 2, after adding glucono-δ-lactone (GDL), as the fermentation proceeded, the pH value of the lactoferrin gel system decreased at a relatively slow rate to below the isoelectric point of lactoferrin pH 4.5-5.2, indicating that the lactoferrin gel system achieved the chelation of lactoferrin Ca 2+ Release of Ca 2+ The conditions can provide sufficient time for lactoferrin molecules and sodium alginate to form a gel structure.

[0106] (II) Lactoferrin gel microstructure and rheological tests

[0107] (1) Gel rheology test

[0108] The lactoferrin gel solutions after fermentation in the examples (Comparative Example 1 and Comparative Example 2 did not form gel, so rheological analysis was not performed) were filtered out with filter paper to remove the supernatant to obtain gel solids, and rheological tests were performed using a rheometer (ThermoScientific, Mars IQ Air, New York, USA) (i.e., the gel samples were cooled to 4°C and then subjected to a frequency sweep experiment to record the changes in storage modulus (G′) and loss modulus (G″) with frequency, with the frequency set to 0.1-10 Hz and the strain set to 1.0%).

[0109] The test results are as follows Figure 3 and Figure 4 As shown in the results, the storage modulus G' is significantly higher than the loss modulus G", indicating that the entire frequency range is highly elastic and the gel system is a three-dimensional network structure; among them, 0.10% CS-LF-Alg / Ca 2+ , 0.15% CS-LF-Alg and 0.20% CS-LF-Alg / Ca 2+ Compared with LF-Alg / Ca 2+ 、0.05%CS-LF-Alg / Ca 2+ and 0.15% CS-LF-Alg / Ca 2+ The rheological difference of 0.10% CS-LF-Alg / Ca 2+ , 0.15% CS-LF-Alg and 0.20% CS-LF-Alg / Ca 2+Compared with other groups, the cross-linking effect in the cross-linking system of lactoferrin and sodium alginate in the group changed.

[0110] (2) Gel microstructure

[0111] The lactoferrin gel solution after fermentation in the embodiment and the comparative example was filtered out with filter paper to remove the supernatant to obtain a gel solid or a precipitate (the precipitate was observed in comparative example 1 and comparative example 2), and the microstructure of the gel solid was observed; Results Figures 5 to 12 As shown, the results show:

[0112] Figure 5 In LF-Alg / Ca 2+ In the absence of chitosan and when the pH value is greater than 6.0, there is electrostatic repulsion between lactoferrin and sodium alginate, resulting in a small amount of lactoferrin in the gel system and Ca 2+ Combined with a large amount of sodium alginate and Ca 2+ After GDL is added, as the fermentation proceeds, the pH of the system slowly decreases to below 4.5, and some positively charged lactoferrin and sodium alginate combine to form a composite system. However, some lactoferrin or sodium alginate still aggregates with each other, and the gel system is not tightly combined.

[0113] In 0.05% CS-LF-Alg / Ca 2+ 、0.10%CS-LF-Alg / Ca 2+ and 0.15% CS-LF-Alg / Ca 2+ In (such as Figure 6 , Figure 7 and Figure 8 As shown in the figure, when chitosan was added to the gel system, lactoferrin and sodium alginate showed obvious cross-linking, and the cross-linking effect gradually increased with the increase of chitosan concentration; when the pH was greater than 6.0, chitosan was uncharged, and Ca 2+ Positively charged, lactoferrin and sodium alginate are negatively charged, Ca 2+ It coordinates with the -COO- of sodium alginate to form an "egg box" structure. A very small amount of lactoferrin and Ca 2+ After adding chitosan, chitosan is dispersed in the mixed system of lactoferrin and sodium alginate; when GDL is added, as the fermentation proceeds, the pH of the system slowly decreases to below 4.5, chitosan and lactoferrin are positively charged, and the Ca2+ bound to lactoferrin 2+ It cross-links with sodium alginate to form calcium caseinate, which in turn forms a calcium cross-linking system. The positively charged chitosan is cross-linked with lactoferrin and Ca 2+ Electrostatic repulsion occurs, causing Ca 2+The cross-linking system of lactoferrin and sodium alginate is more stable, and lactoferrin precipitation is less; it is shown that 0.15% CS-LF-Alg / Ca 2+ Compared with other groups, the lactoferrin and sodium alginate in the group were evenly distributed, and the network structure was denser.

[0114] 0.15%CS-LF-Alg / Ca 2+ and 0.20% CS-LF-Alg / Ca 2+ Compared with (such as Figure 8 and Fig. 9 shown),

[0115] 0.15%CS-LF-Alg / Ca 2+ The spatial structure of 0.20% CS-LF-Alg / Ca 2+ The spatial structure of the pores is layered and the pore size is enlarged. The reason is that as the concentration of chitosan continues to increase, too much chitosan combines with sodium alginate, resulting in Ca 2+ , lactoferrin and sodium alginate cross-linking system was destroyed; it was shown that 0.20% CS-LF-Alg / Ca 2+ The network structure of the group is not dense enough.

[0116] 0.15%CS-LF-Alg / Ca 2+ Compared with 0.15% CS-LF-Alg ( Figure 8 and Fig.10 shown),

[0117] 0.15%CS-LF-Alg / Ca 2+ The spatial structure of 0.15% CS-LF-Alg is more compact, and the spatial structure pore size is larger; the reason is that compared with Ca 2+ Chitosan made the cross-linking between lactoferrin and sodium alginate worse; the network structure of the 0.15% CS-LF-Alg group was loose and the pore size was large.

[0118] 0.15%CS-LF-Alg / Ca 2+ Compared with 0.15% CS-LF-Alg / CaCl2 (such as Figure 8 and Fig.11 As shown), 0.15% CS-LF-Alg / Ca 2+ The spatial structure of the CS-LF-Alg / CaCl2 group has a cross-linked three-dimensional structure, which is a gel structure of calcium-cross-linked lactoferrin and sodium alginate, while the spatial structure of the 0.15% CS-LF-Alg / CaCl2 group is collapsed, indicating that there is no calcium cross-linking or the calcium cross-linking effect is extremely poor.

[0119] 0.15%CS-LF-Alg / Ca 2+Compared with 0.15% CS-LF-Alg / CC (e.g. Figure 8 and Fig.12 shown),

[0120] 0.15%CS-LF-Alg / Ca 2+ The spatial structure of the 0.15% CS-LF-Alg / CC group was more regular and dense, which was a gel structure of calcium-crosslinked lactoferrin and sodium alginate, while the spatial structure of the 0.15% CS-LF-Alg / CC group had larger gaps and was disordered, indicating that there was basically no calcium cross-linking effect.

[0121] (III) Lactoferrin content in lactoferrin gel

[0122] The lactoferrin gel solutions after fermentation in the embodiment and the comparative example were filtered using filter paper to obtain supernatants, and the lactoferrin content of the supernatants was tested (GB 5009.299 "National Food Safety Standard Determination of Lactoferrin in Foods" was used to test the lactoferrin content), and then the lactoferrin content of the gel solid (i.e., the embedding rate) was calculated. The results are shown in Table 2.

[0123] Table 2 Lactoferrin embedding in different lactoferrin gels

[0124] Group Retention time Embedding rate Lactoferrin standard solution (500ug / mL) 15.970 / <![CDATA[LF-Alg / Ca 2+ (Example 1)]]> 16.085 93.25％ <![CDATA[0.05%CS-LF-Alg / Ca 2+ (Example 1)]]> 16.122 93.60％ <![CDATA[0.10%CS-LF-Alg / Ca 2+ (Example 2)]]> 16.107 94.85％ <![CDATA[0.15%CS-LF-Alg / Ca 2+ (Example 3)]]> 16.127 96.19％ <![CDATA[0.20%CS-LF-Alg / Ca 2+ (Example 4)]]> 15.989 94.12％ 0.15% CS-LF-Alg (Example 5) 15.968 95.48％ <![CDATA[0.15% CS-LF-Alg / CaCl2 (Comparative Example 1)]]> 16.295 14.23％ 0.15% CS-LF-Alg / CC (Comparative Example 2) 16.295 13.10％

[0125] From Table 2 above, we can know that:

[0126] First, for the lactoferrin embedding rate, 0.05% CS-LF-Alg / Ca 2+ The embedding rate of lactoferrin in the LF-Alg / Ca group was higher than that in the LF-Alg / Ca group. 2+ group, indicating that adding chitosan is helpful for the embedding of lactoferrin; when the amount of chitosan added gradually increased to 0.15%, the embedding rate of lactoferrin reached the highest value of 96.19%, and the embedding rate of lactoferrin decreased slightly with the further increase of chitosan addition; 0.15% CS-LF-Alg / Ca 2+ The embedding rate of lactoferrin in the 0.15% CS-LF-Alg group was higher than that in the 0.15% CS-LF-Alg group, indicating that the cross-linking effect of calcium ions also helps to increase the embedding rate of lactoferrin. 2+ The embedding efficiency is better than 0.15% CS-LF-Alg and LF-Alg / Ca 2+ , indicating that the lactoferrin encapsulation rate of the gel containing both the calcium release system and chitosan is better than that of the gel containing only the calcium release system and chitosan.

[0127] Second, for the retention time of lactoferrin during detection, LF-Alg / Ca 2+ 、0.05%CS-LF-Alg / Ca 2+ 、0.10%CS-LF-Alg / Ca 2+ and 0.15% CS-LF-Alg / Ca 2+ At around 16.1 min, 0.20% CS-LF-Alg / Ca 2 + The retention time of 0.15% CS-LF-Alg and 0.20% CS-LF-Alg / Ca 2+ With 0.05% CS-LF-Alg / Ca 2+ 、0.10%CS-LF-Alg / Ca 2+ 、0.15%CS-LF-Alg / Ca 2+ The cross-linking effect of 0.15% CS-LF-Alg / Ca 2+ At around 16.1 min, 0.15% CS-LF-Alg / CaCl2 and 0.15% CS-LF-Alg / CC were at around 16.3 min, indicating that there may be no cross-linking effect of calcium ions in the systems of 0.15% CS-LF-Alg / CaCl2 and 0.15% CS-LF-Alg / CC, resulting in excessive cations in the solution, which affect the detection results of lactoferrin.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A lactoferrin gel, characterized in that The preparation raw materials include lactoferrin, sodium alginate, acidulant, water medium, calcium ion sustained release system and / or chitosan, the calcium ion sustained release system includes inorganic calcium salt and sodium citrate; the pH value of lactoferrin gel is ≤ the isoelectric point pH of lactoferrin.

2. The lactoferrin gel according to claim 1, characterized in that The pH value of lactoferrin gel is ≤5.

2.

3. The lactoferrin gel according to claim 2, characterized in that The pH value of lactoferrin gel is ≤4.

5.

4. The lactoferrin gel according to any one of claims 1 to 3, characterized in that The raw materials for preparation meet one or more of the following conditions: (i) Lactoferrin mass fraction is 1%-5%; (ii) the molar concentration of calcium chloride is 6 mmol / L to 18 mmol / L; (iii) the molar concentration of sodium citrate is 10 mmol / L to 30 mmol / L; (iv) the mass fraction of sodium alginate is 0.1%-0.5%; (v) the mass fraction of chitosan is 0.05%-0.15%; (vi) the mass fraction of the acidifier is 2%-3%; (vii) the acidulant is one or more of D-gluconolactone, L-malic acid or bacterial species; (viii) The inorganic calcium salt is one or more of calcium chloride, calcium carbonate or calcium sulfate.

5. The lactoferrin gel according to claim 4, characterized in that The raw materials for preparation meet one or more of the following conditions: (a) Lactoferrin mass fraction is 3%; (b) the molar concentration of the inorganic calcium salt is 9 mmol / L; (c) the molar concentration of sodium citrate is 15 mmol / L; (d) the mass fraction of sodium alginate is 0.3%; (e) The mass fraction of chitosan is 0.15%; (f) The mass fraction of the acidifier is 2.5%.

6. The method for preparing lactoferrin gel according to any one of claims 1 to 5, characterized in that: When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, and a calcium ion sustained-release system, the preparation method includes: (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution; (2) mixing the sodium alginate aqueous solution and the lactoferrin solution to obtain a mixed solution; (3) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel; When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium and chitosan, the preparation method includes: (1) mixing a lactoferrin aqueous solution, a sodium alginate aqueous solution and a lactoferrin solution to obtain a mixed solution; (2) adding an acidifier to the mixed solution and fermenting it until the pH value is less than or equal to the isoelectric point pH of lactoferrin to obtain lactoferrin gel; When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system, and chitosan, the preparation method includes: (1) mixing an inorganic calcium salt aqueous solution and a sodium citrate aqueous solution to form an inorganic calcium salt-sodium citrate aqueous solution; mixing the inorganic calcium salt-sodium citrate aqueous solution and a lactoferrin aqueous solution to form a lactoferrin solution; (2) mixing the sodium alginate aqueous solution, the chitosan aqueous dispersion and the lactoferrin solution to obtain a mixed solution; (3) adding an acidifier to the mixed solution and fermenting it until the pH value is ≤ the isoelectric point pH of lactoferrin to obtain lactoferrin gel.

7. The preparation method according to claim 6, characterized in that: When the raw materials include lactoferrin, sodium alginate, an acidulant, an aqueous medium, a calcium ion sustained-release system and chitosan, in step (2), sodium alginate aqueous solution and chitosan aqueous dispersion are sequentially added to the lactoferrin solution and mixed to obtain a mixed solution.

8. Lactoferrin powder, characterized in that Prepared from the lactoferrin gel according to any one of claims 1 to 5.

9. A dairy product, characterized in that The invention comprises the lactoferrin gel according to any one of claims 1 to 5 or the lactoferrin powder according to claim 8.

10. The dairy product according to claim 9, characterized in that Dairy products include pasteurized milk, sterilized milk, fermented milk, milk flakes, milk powder, cheese or lactose.

Images