Fermented milk containing active lactoferrin and preparation method thereof

Abstract

The invention provides fermented milk containing active lactoferrin and a preparation method of the fermented milk. Fresh raw milk is subjected to degreasing separation, and skim milk and single cream are obtained; carrying out cation exchange chromatography separation and elution on the obtained skimmed milk to obtain a lactoferrin salt-containing solution and lactoferrin-free skimmed milk; the obtained lactoferrin salt-containing solution is separated through a nanofiltration membrane, a permeate is a discarded salt-containing solution, and an intercepted lactoferrin solution is collected; mixing the obtained lactoferrin-free skimmed milk with the obtained single cream, heating, homogenizing, sterilizing to obtain cow milk, and cooling for later use; and finally, filtering and sterilizing the obtained lactoferrin solution through a microfiltration membrane, then adding the lactoferrin solution into cooled cow milk, adding a leavening agent, carrying out heat preservation fermentation to the end point, and turning over a vat for cooling to obtain the fermented milk containing the active lactoferrin. Through a special process, the prepared fermented milk contains lactoferrin with the same quality and the same activity in raw milk, and more beneficial effects are added to a healthy fermented milk product.

Description

Technical Field

[0001] The invention relates to the technical field of dairy products, in particular to fermented 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] Fermented dairy products are made by adding lactic acid bacteria to milk. During the process, more substances beneficial to human health and a sweet and sour taste are produced, which are loved by more and more consumers. However, the raw milk used in fermented milk needs to be processed at a higher temperature than pasteurization, so most of the immune components such as active lactoferrin will be lost. Currently, there is a lack of fermented milk products on the market that contain active lactoferrin in raw milk. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a fermented milk and a preparation method thereof, which are used to solve the problem that the fermented milk in the prior art does not contain active lactoferrin or the content is extremely low.

[0005] To achieve the above object and other related objects, the present invention provides a method for preparing fermented milk containing active lactoferrin, comprising the following steps:

[0006] (1) skimming and separating fresh raw milk to obtain skimmed milk and cream;

[0007] (2) separating the skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin-containing salt solution and lactoferrin-free skim milk;

[0008] (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected;

[0009] (4) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), heating and homogenizing, sterilizing to obtain milk, and cooling for later use;

[0010] (5) The lactoferrin solution obtained in step (3) is filtered and sterilized through a microfiltration membrane, and then added to the cow's milk cooled in step (4), and a starter is added. The fermentation is kept warm until the end point, and the fermented milk is turned over and cooled to obtain fermented milk containing active lactoferrin.

[0011] Preferably, the fermentation agent in step (5) is a lactic acid bacteria species.

[0012] The present invention also provides fermented milk containing active lactoferrin prepared by the above-mentioned preparation method.

[0013] As described above, the fermented milk containing active lactoferrin and the preparation method thereof of the present invention have the following beneficial effects:

[0014] The method for preparing fermented milk containing active lactoferrin of the present invention comprises the following steps: firstly, active lactoferrin is extracted from raw cow milk, and then active lactoferrin is added to lactoferrin-free skim milk and cream after mixing, homogenizing and sterilizing, and then a starter is added for fermentation. The fermented milk prepared by the method of the present invention not only contains more than 75% of active lactoferrin in raw milk, but also has a sodium content far lower than that of raw cow milk, and is currently a fermented milk product with the most complete and optimal nutritional retention. DETAILED DESCRIPTION

[0015] 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.

[0016] 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.

[0017] 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.

[0018] The first aspect of the present invention provides a method for preparing fermented milk containing active lactoferrin, comprising the following steps:

[0019] (1) skimming and separating fresh raw milk to obtain skimmed milk and cream;

[0020] (2) separating the skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin-containing salt solution and lactoferrin-free skim milk;

[0021] (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected;

[0022] (4) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), heating and homogenizing, sterilizing to obtain milk, and cooling for later use;

[0023] (5) The lactoferrin solution obtained in step (3) is filtered and sterilized through a microfiltration membrane, and then added to the cow's milk cooled in step (4), and a starter is added. The fermentation is kept warm until the end point, and the fermented milk is turned over and cooled to obtain fermented milk containing active lactoferrin.

[0024] 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 ≤4h, ≤3h, ≤2h or ≤1h and a protein content of ≥3.5%, ≥4%, ≥4.5%, or ≥5%.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In some embodiments of the present invention, the salt solution used for the salt solution elution in step (2) is a NaCl solution.

[0030] 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.

[0031] The saline solution is used for elution 2 to 3 times. In a preferred embodiment of the present invention, the elution is performed twice.

[0032] 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 1.6 nm, 1.6 to 1.8 nm or 1.8 to 2 nm.

[0033] 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.

[0034] In some embodiments of the present invention, the homogenization temperature in step (4) is 60-65°C, for example, 60-62°C, 62-64°C or 64-65°C.

[0035] In some embodiments of the present invention, the homogenization pressure in step (4) 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.

[0036] In some embodiments of the present invention, the sterilization temperature in step (4) is 95-97°C, for example, 95-96°C or 96-97°C.

[0037] In some embodiments of the present invention, the sterilization time in step (4) is 5 to 7 minutes, for example, 5 to 6 minutes or 6 to 7 minutes.

[0038] In some embodiments of the present invention, the cooling in step (4) is cooling to 40-43°C, for example, 40-41°C, 41-42°C or 42-43°C.

[0039] In some embodiments of the present invention, one or more of a stabilizer, a thickener, and white sugar are added to the lactoferrin-free skim milk and the cream after mixing in step (4).

[0040] Wherein, the stabilizer is selected from one or more of agar, pectin and gelatin.

[0041] The thickener is selected from one or more of hydroxypropyl distarch phosphate and physical starch.

[0042] In the present invention, if other ingredients are added, the total amount of skim milk and cream accounts for more than 80% w / w. And when other ingredients are added, the total mass must be 100%.

[0043] 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.

[0044] In some embodiments of the present invention, the fermentation agent described in step (5) is a lactic acid bacteria species. In a preferred embodiment of the present invention, the fermentation agent is selected from one or more of Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus (Lactobacillus GG), Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis, and Leuconostoc mesenteroides. In a more preferred embodiment of the present invention, the fermentation agent is Streptococcus thermophilus, Lactobacillus bulgaricus.

[0045] In some embodiments of the present invention, the amount of the leavening agent added in step (5) is 0.001-0.01wt% of the total amount of the raw materials. For example, it is 0.001-0.002wt%, 0.002-0.004wt%, 0.004-0.006wt%, 0.006-0.008wt% or 0.008-0.01wt%. The raw materials are skim milk and cream. If one or more of a stabilizer, a thickener and white granulated sugar are added during the preparation process, the raw materials also include one or more of a stabilizer, a thickener and white granulated sugar.

[0046] In some embodiments of the present invention, the temperature of the insulation in step (5) is 40-43°C, for example, 40-41°C, 41-42°C or 42-43°C.

[0047] In some embodiments of the present invention, the temperature of the tank turning over and cooling in step (5) is 2-6°C, for example, 2-3°C, 3-4°C, 4-5°C or 5-6°C.

[0048] In some embodiments of the present invention, the end point of the fermentation in step (5) is 70-75°T, for example, 70-71°T, 71-72°T, 72-73°T, 73-74°T or 74-75°T.

[0049] The second aspect of the present invention provides a fermented milk containing active lactoferrin prepared by the preparation method as described above.

[0050] In some embodiments of the present invention, the content of active lactoferrin in the fermented milk is 10-12 mg / 100 mL, for example, 10-10.5 mg / 100 mL, 10.5-10.7 mg / 100 mL, 10.7-11 mg / 100 mL, 11-11.25 mg / 100 mL, 11.25-11.3 mg / 100 mL, 11.3-11.5 mg / 100 mL or 11.5-12 mg / 100 mL.

[0051] The fermented milk obtained by the preparation method of the present invention can retain more than 80% of the active lactoferrin in the raw milk.

[0052] In the following embodiments:

[0053] White sugar is commercially available;

[0054] Stabilizer: purchased from Danisco China Co., Ltd.

[0055] Thickener: purchased from Danisco China Co., Ltd.

[0056] Starter culture: purchased from Danisco China Ltd.

[0057] Example 1

[0058] Preparation of fermented milk containing active lactoferrin:

[0059] (1) freshly produced raw milk with a protein content of 4.0% and refrigerated storage for 1 hour is skimmed and separated to obtain skimmed milk and cream with a fat content of 0.05%;

[0060] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, with the emulsion temperature at 25° 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-containing saline solution and lactoferrin-free skim milk;

[0061] (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;

[0062] (4) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), heating the mixture to 65° C., homogenizing the mixture at 180 bar, sterilizing the mixture at 95° C. for 7 min, and obtaining milk, which was then cooled to 40° C.;

[0063] (5) The lactoferrin solution obtained in step (3) is sterilized by filtering through a microfiltration membrane with a pore size of 0.2 μm, and then added to the cow's milk cooled to 40° C. in step (4); 0.002% of the lactic acid bacteria YO-MIX 883 starter is added, and the fermentation is kept warm to the end point of 70° F., and the tank is turned over and cooled to 2° C., filled, and refrigerated for storage.

[0064] Example 2

[0065] (1) freshly produced raw milk with a protein content of 3.5% and refrigerated storage for 4 hours is skimmed and separated to obtain skimmed milk and cream with a fat content of 0.02%;

[0066] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, with the emulsion temperature at 45° C. and the emulsion residence time in the resin column being 1 minute, and eluting twice with a 0.9 mol / L NaCl solution to obtain a lactoferrin-containing saline solution and lactoferrin-free skim milk;

[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) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), heating the mixture to 60° C., homogenizing the mixture at 250 bar, and sterilizing the mixture at 97° C. for 5 min to obtain milk, which is then cooled to 43° C.;

[0069] (5) The lactoferrin solution obtained in step (3) is sterilized by filtering through a microfiltration membrane with a pore size of 0.4 μm, and then added to the cow's milk cooled to 43° C. in step (4); 0.01% of the lactic acid bacteria YF-L904 starter is added, and the fermentation is kept warm to the end point of 75° F., the tank is turned over and cooled to 6° C., filled, and refrigerated.

[0070] Example 3

[0071] (1) freshly produced raw milk with a protein content of 3.8% and refrigerated storage for 4 hours is skimmed and separated to obtain skimmed milk and cream with a fat content of 0.01%;

[0072] (2) separating the skim milk obtained in step (1) by cation exchange resin column chromatography, the emulsion temperature is 40° 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 salt solution and lactoferrin-free skim milk;

[0073] (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;

[0074] (4) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), and adding 0.01% pectin, 0.2% hydroxypropyl distarch phosphate, and 7% white sugar, heating the mixture to 50° C. and stirring for 10 to 20 minutes, heating the mixture to 60° C., homogenizing the mixture at 200 bar, and sterilizing the mixture at 95° C. for 5 minutes to obtain milk, which is then cooled to 43° C.; wherein the percentages of the pectin, hydroxypropyl distarch phosphate, and white sugar are the mass percentages of the total amount of the raw materials (i.e., pectin, hydroxypropyl distarch phosphate, white sugar, skim milk, and cream).

[0075] (5) The lactoferrin solution obtained in step (3) is sterilized by filtering through a microfiltration membrane with a pore size of 0.2 μm, and then added to the cow's milk cooled to 43° C. in step (4); 0.005% of the lactic acid bacteria YF-L904 starter is added, and the fermentation is kept warm to the end point of 75° F., the tank is turned over and cooled to 4° C., filled, and refrigerated.

[0076] Sample testing:

[0077] The samples produced by the processes of Examples 1 to 3 of the present invention were compared with raw cow's milk and commercially available fermented milk in terms of active lactoferrin (GB 5009.299-2024) and sodium (GB 5009.91-2017). The results are shown in Table 1 below.

[0078] Table 1

[0079]

[0080]

[0081] It can be seen that the fermented 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, further increasing the nutritional value of the fermented milk product.

[0082] In summary, the method for preparing fermented milk containing active lactoferrin of the present invention uses a special process to make the prepared fermented milk contain lactoferrin of approximately the same quality and activity as that in raw milk (i.e., raw cow's milk), thereby adding more beneficial effects to healthy fermented milk products.

[0083] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0084] 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 fermented milk containing active lactoferrin, characterized in that: The following steps are involved: (1) skimming and separating fresh raw milk to obtain skimmed milk and cream; (2) separating the skim milk obtained in step (1) by cation exchange chromatography and eluting with a salt solution to obtain a lactoferrin-containing salt solution and lactoferrin-free skim milk; (3) separating the lactoferrin saline solution obtained in step (2) through a nanofiltration membrane, wherein the permeate is the discarded saline solution, and the retained lactoferrin solution is collected; (4) mixing the lactoferrin-free skim milk obtained in step (2) with the cream obtained in step (1), heating and homogenizing, sterilizing to obtain milk, and cooling for later use; (5) The lactoferrin solution obtained in step (3) is filtered and sterilized through a microfiltration membrane, and then added to the cow's milk cooled in step (4), and a starter is added. The fermentation is kept warm until the end point, and the fermented milk is turned over and cooled to obtain fermented milk containing active lactoferrin.

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 ≤4h and a protein content of ≥3.5%; And / or, the fat content of the skim milk in step (1) 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 skim milk during the chromatographic separation process is ≤45°C; And / or, the 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 used for the salt solution elution in step (2) is NaCl solution.

6. 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.

7. The preparation method according to claim 1, characterized in that: The homogenization temperature in step (4) is 60-65°C; And / or, the homogenization pressure in step (4) is 180 to 250 bar; And / or, the sterilization temperature in step (4) is 95-97°C; And / or, the sterilization time in step (4) is 5 to 7 minutes; And / or, the cooling in step (4) is cooling to 40-43°C.

8. The preparation method according to claim 1, characterized in that: In step (4), after the lactoferrin-free skim milk and cream are mixed, one or more of a stabilizer, a thickener and white sugar are added.

9. 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; And / or, the fermentation agent in step (5) is a lactic acid bacteria species; and / or, the amount of the leavening agent added in step (5) is 0.001 to 0.01 wt % of the total amount of the raw materials; And / or, the temperature of the insulation in step (5) is 40-43° C.: And / or, the temperature of the tank turning over and cooling in step (5) is 2 to 6°C; And / or, the end point of the fermentation in step (5) is 70-75°T.

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

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