Liquid milk containing active lactoferrin and preparation method thereof

Abstract

The invention provides liquid milk containing active lactoferrin and a preparation method of the liquid milk. Fresh raw milk is subjected to degreasing separation to obtain first skimmed milk; performing cation exchange chromatography separation and elution on the first skimmed milk to obtain a lactoferrin salt-containing solution; separating the salt-containing lactoferrin solution through a nanofiltration membrane, and collecting an intercepted lactoferrin solution; adding the lactoferrin solution into raw milk, and performing degreasing separation to obtain second single cream and high-content active lactoferrin skimmed milk; filtering and sterilizing the high-content active lactoferrin skimmed milk through a microfiltration membrane; sterilizing the second single cream, cooling for the first time, adding the second single cream into the sterilized high-content active lactoferrin skimmed milk, heating, homogenizing, and cooling for the second time to obtain the high-content active lactoferrin skimmed milk; the content of active lactoferrin in the obtained liquid milk is 1.7-2 times that of pasteurized milk with the same protein content, and the liquid milk can be stored for 19-21 days at the temperature of 2-6 DEG C.

Description

Technical Field

[0001] The invention relates to the technical field of liquid milk, in particular to liquid milk containing active lactoferrin and a preparation method thereof. Background Art

[0002] Milk not only contains basic nutrients, but also is rich in a variety of immune active proteins, which play a unique role in enhancing the body's immunity and resisting diseases. The main immune active proteins include lactoferrin, immunoglobulins, lactoperoxidase and lysozyme. Lactoferrin is an iron-binding glycoprotein with multiple physiological functions such as antibacterial activity, participation in iron metabolism, anti-cancer, antiviral and protection of intestinal health. It is often widely used in food health care and biomedicine in the form of nutritional enhancers and drug carriers. As a functional protein with multiple biological functions and physicochemical properties, it has strong antibacterial, antiviral and antifungal activities, and can inhibit the growth of pathogenic microorganisms by competing with them for iron ions.

[0003] Currently, liquid milk products on the market are divided into three categories: pasteurized milk, ultra-instantaneous sterilized milk, and ultra-high temperature sterilized milk. As the degree of heat increases, the content of active lactoferrin in the three types of products decreases sharply, and there is almost no active lactoferrin in ultra-high temperature sterilized milk. At the same time, most methods for extracting and enriching active lactoferrin in cow's milk cannot be used for industrial production. There is no specific implementation plan for reference in the methods currently disclosed, and there is no method for strengthening active lactoferrin in low-temperature cow's milk.

[0004] Therefore, it is extremely important to develop a low-temperature milk with a high content of active lactoferrin. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a liquid milk containing active lactoferrin and a preparation method thereof, so as to solve the problems in the prior art that there is no active lactoferrin in room temperature liquid milk, a small amount of lactoferrin in low temperature liquid milk, and most of the natural active lactoferrin in raw milk is lost and inactivated.

[0006] To achieve the above object and other related objects, the present invention provides a method for preparing liquid milk containing active lactoferrin, comprising the following steps:

[0007] (1) skimming and separating fresh raw milk to obtain first skimmed milk and first cream;

[0008] (2) separating the first skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin salt solution;

[0009] (3) separating the lactoferrin salt solution obtained in step (2) through a nanofiltration membrane, and collecting the retained lactoferrin solution;

[0010] (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1), performing skimming and separation, and obtaining a second cream and skimmed milk with a high content of active lactoferrin;

[0011] (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a microfiltration membrane for sterilization;

[0012] (6) sterilizing and cooling the second cream obtained in step (4), adding it to the skim milk with high active lactoferrin content sterilized in step (5), heating and homogenizing, and cooling it for a second time to obtain skim milk with high active lactoferrin content.

[0013] The present invention also provides liquid milk containing active lactoferrin prepared by the above-mentioned preparation method.

[0014] As described above, the liquid milk containing active lactoferrin and the preparation method thereof of the present invention have the following beneficial effects:

[0015] The preparation method of liquid milk containing active lactoferrin of the present invention comprises the following steps: firstly extracting active lactoferrin from raw cow milk, then adding active lactoferrin into the raw cow milk and performing defatting separation again, filtering and sterilizing high-content active lactoferrin skimmed milk through a microfiltration membrane, sterilizing cream separately, and then mixing the skimmed milk with high-content active lactoferrin. The present invention adopts non-thermal sterilization technology of skimmed milk, retains active lactoferrin and other heat-sensitive components in milk to the greatest extent, and makes low-temperature cow milk richer in nutrients.

[0016] The invention provides a continuous process for producing liquid milk with high content of active lactoferrin. The process adopts a method for extracting active lactoferrin from raw milk to prepare low-temperature milk with high content of active lactoferrin. DETAILED DESCRIPTION

[0017] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0018] When a numerical range is disclosed herein, the above range is deemed to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges included therein. For example, a specified range from "1 to 10" should be deemed to include any and all sub-ranges between a minimum of 1 and a maximum of 10. Exemplary sub-ranges of ranges 1 to 10 include, but are not limited to, 1 to 6.1, 3.5 to 7.8, 5.5 to 10, and the like.

[0019] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; and, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0020] The first aspect of the present invention provides a method for preparing liquid milk containing active lactoferrin, comprising the following steps:

[0021] (1) skimming and separating fresh raw milk to obtain first skimmed milk and first cream;

[0022] (2) separating the first skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin salt solution;

[0023] (3) separating the lactoferrin salt solution obtained in step (2) through a nanofiltration membrane, and collecting the retained lactoferrin solution;

[0024] (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1), performing skimming and separation, and obtaining a second cream and skimmed milk with a high content of active lactoferrin;

[0025] (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a microfiltration membrane for sterilization;

[0026] (6) sterilizing and cooling the second cream obtained in step (4), adding it to the skim milk with high active lactoferrin content sterilized in step (5), heating and homogenizing, and cooling it for a second time to obtain skim milk with high active lactoferrin content.

[0027] In some embodiments of the present invention, the fresh raw cow's milk in step (1) is freshly produced raw cow's milk with a refrigerated storage time of ≤24h, ≤20h, ≤15h, ≤10h, ≤5h, optimally ≤4h, ≤3h, ≤2h or ≤1h, and a protein content of ≥3.5%, ≥4%, ≥4.5%, ≥5%.

[0028] In some embodiments of the present invention, the fat content of the skim milk in step (1) is less than 0.2%, for example, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.05%, etc.

[0029] In some embodiments of the present invention, the cation exchange chromatography separation in step (2) is performed using a cation exchange resin column, utilizing the difference in binding strength between the ion exchanger and different ions to achieve the purpose of separating the desired substance.

[0030] The temperature of the skim milk during the chromatographic separation process is ≤45°C, for example, ≤45°C, ≤40°C, ≤35°C, ≤30°C, ≤25°C, ≤20°C, etc.

[0031] The skim milk stays in the cation exchange resin column for 1 to 2 minutes, for example, 1 to 1.2 minutes, 1.2 to 1.4 minutes, 1.4 to 1.6 minutes, 1.6 to 1.8 minutes or 1.8 to 2 minutes.

[0032] In some embodiments of the present invention, the salt solution in step (2) is a NaCl solution.

[0033] The concentration of the NaCl solution is 0.9 to 1.2 mol / L, for example, 0.9 to 1.0 mol / L, 1.0 to 1.1 mol / L or 1.1 to 1.2 mol / L.

[0034] The saline solution is used for elution 2 to 3 times. In a preferred embodiment of the present invention, the elution is performed twice.

[0035] In some embodiments of the present invention, the pore size of the nanofiltration membrane in step (3) is 1 to 2 nm, for example, 1 to 1.2 nm, 1.2 to 1.4 nm, 1.4 to 1.5 nm, 1.5 to 1.6 nm, 1.6 to 1.8 nm or 1.8 to 2 nm.

[0036] In some embodiments of the present invention, the sodium content in the lactoferrin solution in step (3) is less than 5 mg / 100 mL, for example, less than 5 mg / 100 mL, less than 4 mg / 100 mL, less than 3 mg / 100 mL, less than 2 mg / 100 mL, less than 1 mg / 100 mL, etc.

[0037] In some embodiments of the present invention, the pore size of the microfiltration membrane in step (5) is 0.2-0.4 μm, for example, 0.2-0.25 μm, 0.25-0.3 μm, 0.3-0.35 μm or 0.35-0.4 μm.

[0038] In some embodiments of the present invention, the sterilization temperature in step (6) is 137-139°C, for example, 137-138°C or 138-139°C.

[0039] In some embodiments of the present invention, the sterilization time in step (6) is 4 to 6 seconds, for example 4 to 5 seconds or 5 to 6 seconds.

[0040] In some embodiments of the present invention, the primary cooling in step (6) is cooling to 25-35°C, for example, 25-27°C, 27-29°C, 29-30°C, 30-32°C, 32-34°C or 34-35°C.

[0041] In some embodiments of the present invention, the temperature increase in step (6) is to 60-65°C, for example, 60-62°C, 62-64°C or 64-65°C.

[0042] In some embodiments of the present invention, the homogenization pressure in step (6) is 180-250 bar, for example, 180-190 bar, 190-200 bar, 200-210 bar, 210-220 bar, 220-230 bar, 230-240 bar or 240-250 bar.

[0043] In some embodiments of the present invention, the secondary cooling in step (6) is cooling to 2-6°C, for example, 2-3°C, 3-4°C, 4-5°C or 5-6°C.

[0044] A second aspect of the present invention provides liquid milk containing active lactoferrin prepared by the preparation method as described above.

[0045] In some embodiments of the present invention, the content of active lactoferrin in the liquid milk product containing active lactoferrin is 1.55 times or more of that in raw cow's milk, for example, 1.55 to 1.6 times, 1.6 to 1.65 times, 1.65 to 1.7 times, 1.7 to 1.75 times, 1.75 to 1.8 times, 1.8 to 1.9 times, 2.0 times or more, etc.

[0046] In a preferred embodiment of the present invention, the content of active lactoferrin in the liquid milk is 18-24 mg / 100 mL, for example, 18-19 mg / 100 mL, 19-19.6 mg / 100 mL, 19.6-20 mg / 100 mL, 20-20.4 mg / 100 mL, 20.4-21 mg / 100 mL, 21-21.7 mg / 100 mL, 21.7-22 mg / 100 mL, 22-23 mg / 100 mL or 23-24 mg / 100 mL.

[0047] Example 1

[0048] Preparation of liquid milk containing active lactoferrin:

[0049] (1) skimming and separating freshly produced raw milk with a protein content of 3.5% and refrigerated storage for less than 24 hours to obtain a first skimmed milk and a first cream with a fat content of 0.02%;

[0050] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, the emulsion temperature is 28° C., the residence time of the emulsion in the resin column is 1 minute, and eluting with a NaCl solution with a concentration of 0.9 mol / L twice to obtain a lactoferrin saline solution;

[0051] (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane with a pore size of 1 nm, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected;

[0052] (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1) to perform skimming and separation to obtain a second cream and skimmed milk with a high content of active lactoferrin;

[0053] (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a ceramic microfiltration membrane with a pore size of 0.2 μm for sterilization;

[0054] (6) sterilizing the second cream obtained in step (4) at 137° C. for 4 seconds, cooling to 25° C., backfilling into the sterilized high-content active lactoferrin skim milk in step (5), heating to 60° C., homogenizing aseptically at 250 bar, cooling to 2° C., filling, and refrigerating.

[0055] Example 2

[0056] Preparation of liquid milk containing active lactoferrin:

[0057] (1) skimming and separating freshly produced raw milk with a protein content of 3.8% and refrigerated storage for less than 20 hours to obtain a first skimmed milk and a first cream with a fat content of 0.1%;

[0058] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, with the emulsion temperature at 35° C. and the emulsion residence time in the resin column being 2 minutes, and eluting twice with a 1.2 mol / L NaCl solution to obtain a lactoferrin saline solution;

[0059] (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane with a pore size of 1.5 nm, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected;

[0060] (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1) to perform skimming and separation to obtain a second cream and skimmed milk with a high content of active lactoferrin;

[0061] (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a ceramic microfiltration membrane with a pore size of 0.4 μm for sterilization;

[0062] (6) sterilizing the second cream obtained in step (4) at 139° C. for 6 s, cooling to 35° C., backfilling into the sterilized high-content active lactoferrin skim milk in step (5), heating to 65° C., homogenizing aseptically at 180 bar, cooling to 6° C., filling, and refrigerating.

[0063] Example 3

[0064] Preparation of liquid milk containing active lactoferrin:

[0065] (1) skimming and separating freshly produced raw milk with a protein content of 4.0% and refrigerated storage for less than 12 hours to obtain a first skimmed milk and a first cream with a fat content of 0.12%;

[0066] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, with the emulsion temperature at 38° C. and the emulsion residence time in the resin column being 2 minutes, and eluting twice with a 1.0 mol / L NaCl solution to obtain a lactoferrin saline solution;

[0067] (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane with a pore size of 2 nm, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected;

[0068] (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1) to perform skimming and separation to obtain a second cream and skimmed milk with a high content of active lactoferrin;

[0069] (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a ceramic microfiltration membrane with a pore size of 0.2 μm for sterilization;

[0070] (6) sterilizing the second cream obtained in step (4) at 137° C. for 4 s, cooling to 35° C., backfilling into the sterilized high-content active lactoferrin skim milk in step (5), heating to 65° C., homogenizing aseptically at 200 bar, cooling to 4° C., filling, and refrigerating.

[0071] Sample testing:

[0072] The samples produced by the processes of Examples 1 to 3 of the present invention were compared with raw cow milk and three types of liquid milk, namely, pasteurized milk, ultra-instantaneous sterilized milk and ultra-high temperature sterilized milk, for three indicators: active lactoferrin (GB 5009.299-2024), sodium (GB5009.91-2017) and shelf life at 2 to 6°C. The results are shown in Table 1.

[0073] Table 1

[0074]

[0075] It can be seen that the liquid milk obtained by the preparation method of the present invention contains a relatively high content of active lactoferrin, while the sodium content meets the national standard for low sodium content, and the shelf life is better than the pasteurized milk with the highest nutritional retention currently available on the market.

[0076] In summary, the preparation method of liquid milk containing active lactoferrin of the present invention comprises the following steps: extracting active lactoferrin from raw cow milk through a special process, mixing the active lactoferrin with the raw cow milk and performing defatting separation again, then sterilizing the cream by UHT, filtering and sterilizing the skimmed milk with high active lactoferrin content by microfiltration membrane, and mixing the sterilized two, so that the content of active lactoferrin in the obtained liquid milk is 1.7 to 2 times that of pasteurized milk with the same total protein content (i.e., comparing the lactoferrin content therein when the total protein content is the same), and the shelf life is 2 to 6° C. and can be stored for 19 to 21 days.

[0077] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0078] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing liquid milk containing active lactoferrin, characterized in that: The following steps are involved: (1) skimming and separating fresh raw milk to obtain first skimmed milk and first cream; (2) separating the first skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin salt solution; (3) separating the lactoferrin salt solution obtained in step (2) through a nanofiltration membrane, and collecting the retained lactoferrin solution; (4) adding the lactoferrin solution obtained in step (3) to an amount of raw cow milk equal to that in step (1), performing skimming and separation, and obtaining a second cream and skimmed milk with a high content of active lactoferrin; (5) filtering the high-content active lactoferrin skimmed milk obtained in step (4) through a microfiltration membrane for sterilization; (6) sterilizing and cooling the second cream obtained in step (4), adding it to the skim milk with high active lactoferrin content sterilized in step (5), heating and homogenizing, and cooling it for a second time to obtain skim milk with high active lactoferrin content.

2. The preparation method according to claim 1, characterized in that: The fresh raw cow's milk in step (1) is freshly produced raw cow's milk with a refrigerated storage time of ≤24h and a protein content of ≥3.5%; And / or, in step (1), the fat content of the first skim milk is less than 0.2%.

3. The preparation method according to claim 1, characterized in that: The cation exchange chromatography separation in step (2) is performed using a cation exchange resin column.

4. The preparation method according to claim 3, characterized in that: The temperature of the first skim milk during the chromatographic separation process in step (2) is ≤45°C; And / or, in step (2), the first skim milk stays in the cation exchange resin column for 1 to 2 minutes.

5. The preparation method according to claim 1, characterized in that: The salt solution in step (2) is a NaCl solution.

6. The preparation method according to claim 5, characterized in that: The concentration of the NaCl solution in step (2) is 0.9-1.2 mol / L.

7. The preparation method according to claim 5, characterized in that: The pore size of the nanofiltration membrane in step (3) is 1 to 2 nm; And / or, the sodium content in the lactoferrin solution in step (3) is less than 5 mg / 100 mL.

8. The preparation method according to claim 1, characterized in that: The pore size of the microfiltration membrane in step (5) is 0.2 to 0.4 μm.

9. The preparation method according to claim 1, characterized in that: The sterilization temperature in step (6) is 137-139°C; And / or, the sterilization time in step (6) is 4 to 6 seconds; And / or, the primary cooling in step (6) is cooling to 25-35° C.; And / or, the heating in step (6) is to 60-65° C.; And / or, the homogenization pressure in step (6) is 180 to 250 bar; And / or, the secondary cooling in step (6) is cooling to 2-6°C.

10. Liquid milk containing active lactoferrin prepared according to the preparation method according to any one of claims 1 to 9.

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