Liquid milk as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food, and particularly discloses liquid milk as well as a preparation method and application thereof. According to the method for preparing the liquid milk, lysine and a to-be-treated casein micelle solution separated from animal milk are mixed, incubated and then separated, an incubated casein micelle solution is obtained, and the incubated casein micelle solution is mixed with other components of the liquid milk; other components of the liquid milk comprise components except the casein micelle solution to be treated in animal milk. According to the method disclosed by the invention, plasmin components influencing the stability of the product are reduced when liquid milk is prepared from animal milk, the problems of bitter wrapping, precipitation and the like of the product in the shelf life due to incomplete inactivation of plasmin caused by direct thermal sterilization are solved, effective retention of each active component is further preferably realized, and the efficacy of the product is improved.

Description

Technical Field

[0001] The present invention relates to the field of food technology, in particular to liquid milk and a preparation method and application thereof. Background Art

[0002] Animal milk has become an important part of people's diet. It is considered to be an important source of natural active ingredients with high nutritional value. The multifunctional properties of active proteins in animal milk have been widely characterized, and their functional properties depend on the intact molecular structure and natural active state.

[0003] Animal milk contains plasmin, which breaks down casein into smaller compounds, resulting in a bitter taste and the destruction of the casein structure. Especially at room temperature, its activity increases with storage time, significantly affecting the shelf life of dairy products stored at room temperature.

[0004] The existing method for solving this problem is to inactivate plasmin by protein retention tube in the heat processing process, but it has the following defects: 1. The inactivation is not thorough and the residual activity of plasmin is relatively high; 2. While the heat processing inactivates plasmin, it also denatures and inactivates some active substances in the milk, thereby losing the inherent nutritional value of the milk.

[0005] In addition, in the modern dairy industry, most of the heat treatment is used to kill pathogenic microorganisms in milk and inactivate related enzymes to extend the shelf life of products. The degree of denaturation of active proteins in animal milk is closely related to the intensity of heat treatment. The higher the intensity of heat treatment, the less active protein content, and some active proteins are completely inactivated. Therefore, how to retain a variety of native active proteins in animal milk is of great significance for meeting consumers' functional needs for animal milk, and on the other hand, it is also of great significance for increasing the added value and development and utilization of animal milk.

[0006] The research progress and solutions for the above problems are as follows: ① Pasteurized milk retains some active proteins such as lactalbumin, lactoglobulin, lactoperoxidase, etc. through pasteurization. ② High-temperature sterilized milk retains some active proteins such as lactalbumin, lactoglobulin, lactoperoxidase, etc. through direct sterilization. ③ Milk powder containing active proteins such as lactoferrin is added to liquid milk products after aseptic treatment.

[0007] However, the above research and solutions have the following problems: ① Pasteurization or direct sterilization will still cause partial inactivation of active proteins, and only a small amount of active proteins can be retained. The product needs to be stored at low temperature, and the shelf life is short, which is not conducive to long-distance transportation and long-term sales of the product. ② Direct thermal sterilization will cause most of the native active proteins in the raw milk to be inactivated, and the retention content is low. In addition, the incomplete inactivation of plasmin in the raw milk may cause the problem of bad packages during the shelf life. ③ The method of supplementing by adding additional milk powder will cause a waste of raw materials and increase production costs. ④ If added by raw powder or after aseptic addition, the product can only meet the standards of modulated milk, which does not meet the concept of no additives and natural originality pursued by consumers.

[0008] Specifically, the current preparation processes for active protein milk are as follows: pasteurized milk and stabilizers are mixed and then sterilized at ultra-high temperature, and heat-sensitive substances such as active proteins are sterilized and then aseptically added online in the ultra-high temperature sterilization rear section to prepare milk containing active proteins (CN 101569328 B); raw milk is separated from fat, the cream part is sterilized at high temperature, skim milk is physically sterilized and then combined and backfilled, and the mixed liquid is homogenized and sterilized to prepare high-nutrition active milk (CN 113826695 A); raw milk is pasteurized, RO concentrated, and directly sterilized to prepare room temperature active protein milk (CN 113693130A). However, none of the above methods can achieve room temperature storage of milk that retains a variety of other native multi-active proteins while retaining high retention of native lactoferrin.

[0009] Therefore, it is necessary to further study the preparation method of liquid milk. Summary of the invention

[0010] One of the purposes of the present invention is to provide a room temperature liquid milk with low fibrinolytic enzyme activity obtained by using animal milk as raw material and a preparation method thereof. Preferably, the liquid milk of the present invention also takes into account the activity and content of native proteins in various animal milks.

[0011] In order to achieve this object, the technical solution of the present invention is as follows:

[0012] A method for preparing liquid milk comprises mixing lysine with a casein micelle solution to be treated separated from animal milk, incubating and separating the mixture to obtain an incubated casein micelle solution, and mixing the incubated casein micelle solution with other components of liquid milk; the other components of the liquid milk include components in animal milk except the casein micelle solution to be treated.

[0013] In the method of the present invention, the molar ratio of plasmin to lysine in the casein micelle solution to be treated is 1:(1-10), preferably 1:(6-9), so as to take both cost and treatment efficiency into consideration; the incubation temperature is 40°C-50°C;

[0014] And / or, the incubation time is 30 minutes to 60 minutes;

[0015] And / or, separation after mixed incubation is performed using a MF membrane, and the pore size of the MF membrane is 100-120 kDa.

[0016] The present invention preferably uses MF membrane to separate casein micelles and plasmin, and the retentate is the casein micelle solution after incubation. The pore size of the MF membrane of the present invention is preferably 100 kDa, so as to take into account the filtration of the retention of effective substances and plasmin. It is understandable that those skilled in the art can also use MF membranes with larger pore sizes according to common sense in the art, as long as the effective separation of the casein micelles and plasmin of the present invention can be guaranteed.

[0017] It is well known in the art that when adjusting the nutrients in raw milk (animal milk), a commonly used method is to separate each target component, and then adjust the proportioning backfilling, this method can improve the content of the target nutritional component, and provide a liquid milk with better effect. However, raw milk contains both fibrinolytic enzyme and target nutritional component casein, because fibrinolytic enzyme will be combined with casein micelles, and then when separating casein, fibrinolytic enzyme will be separated together with casein, becoming a component of liquid milk, and under the subsequent storage (especially room temperature storage) of the product, fibrinolytic enzyme will decompose casein, so that the nutrient casein content is reduced, and precipitation and bitterness are produced, thereby greatly affecting the mouthfeel and shelf life of the product. For this reason, the present invention has found that when the casein micelle solution (with fibrinolytic enzyme combined thereon) separated from animal milk is treated in a specific manner, the purpose of separating fibrinolytic enzyme from casein micelles can be achieved, so as to reduce the fibrinolytic enzyme content in the product, and avoid the quality problems such as precipitation and bitter bag in the product shelf life.

[0018] In the present invention, compared with the plasmin in the casein micelle solution to be treated, lysine is added in excess, which can ensure a better plasmin removal effect.

[0019] Animal milk in the present invention may be milk obtained from animals such as cows, sheep, goats, camels, mares or any other animals that produce milk suitable for human consumption, or any liquid substance derived therefrom.

[0020] In the present invention, when preparing liquid milk, the step of separating the casein micelle solution to be treated from animal milk can be combined with the step of separating other substances. Preferably, the method of separating the casein micelle solution to be treated from animal milk comprises:

[0021] (1) Separating fat from animal milk to obtain skim milk and cream;

[0022] (2) treating the skim milk with a cationic chromatographic column to separate a whey protein-containing solution and a casein micelle solution to be treated from the flow-through liquid of the chromatographic column; the filler in the cationic chromatographic column is agarose gel;

[0023] Further preferably, the method for obtaining the whey protein-containing solution and the casein micelle solution to be treated by separation from the chromatographic column flow-through liquid is:

[0024] The chromatographic column flow-through liquid is treated with an MF membrane with a pore size of 100-120 kDa, the membrane inlet temperature is 45-55° C., and the concentration multiple is 2-4 times to obtain MF permeate and MF retentate; the MF permeate is the whey protein-containing solution, and the MF retentate is the casein micelle solution to be treated.

[0025] The flow-through liquid of the ion chromatography column in the present invention can be separated from whey and casein micelles through a microfiltration membrane (MF) (the pore size of the MF membrane of the present invention is preferably 100 kDa, but it is understandable that those skilled in the art can use a MF membrane with a larger pore size according to common sense in the art, as long as the effective separation of whey and casein micelles can be guaranteed), the MF retentate is a casein micelle solution to be treated, and after incubation, MF is used to separate casein micelles and fibrinolytic enzyme, the MF secondary retentate is the casein micelle solution after incubation, which is used for fat standardization with cream, and the MF permeate is a whey phase, containing a variety of active proteins, which is used for mixing with a cation chromatography eluent and then performing low-temperature absolute sterilization. The final product can be stored at room temperature for 30 days and has a good taste.

[0026] In the method of the present invention, in step (2), after the skim milk is treated with a cation chromatography column, the method further comprises the step of eluting to obtain an eluate containing lactoferrin;

[0027] Preferably, when the skim milk is treated with a cation chromatography column, the feed temperature is 45-55° C., the feed linear flow rate is 4-8 m / h, and after feeding, 3-6 times the column volume of 0.3-0.5 M NaCl is first used to elute impurities, and then 3-6 times the column volume of 0.7-0.9 M NaCl is used to elute lactoferrin to obtain an eluate containing lactoferrin; the elution flow rates for impurity elution and lactoferrin elution are both 4-8 m / h.

[0028] More preferably, in the method of the present invention, when eluting to obtain an eluate containing lactoferrin, the method further comprises: after eluting impurities, first using 3-6 times the column 0.55-0.65M NaCl to elute lactoperoxidase to obtain an eluate containing lactoperoxidase, and then eluting lactoferrin; the elution flow rate of the lactoperoxidase elution is 4-8m / h.

[0029] Further preferably, the method for preparing liquid milk further comprises: respectively concentrating and desalting the lactoferrin-containing eluate and the lactoperoxidase-containing eluate to obtain a lactoferrin concentrate and a lactoperoxidase concentrate;

[0030] Alternatively, the step of mixing the lactoferrin-containing eluate and the lactoperoxidase-containing eluate and then concentrating and desalting to obtain a mixed concentrate of lactoferrin and lactoperoxidase is performed.

[0031] The preferred concentration and desalination method is: the lactoferrin-containing eluate and / or the lactoperoxidase-containing eluate is concentrated 10-15 times by UF membrane, and then desalinated by hollow fiber membrane; the pore size of the UF membrane is 10-30 kDa, and those skilled in the art can select the pore size of the UF membrane according to common sense, as long as it is conducive to desalination and subsequent operations.

[0032] In the present invention, the method for concentrating and desalting the lactoferrin-containing eluate and the lactoperoxidase-containing eluate separately or concentrating and desalting them after mixing is the same.

[0033] The desalination operation is a well-known method in the art. After desalination in the present invention, the solution conductivity is less than 1 mS / cm.

[0034] The skimmed milk of the present invention is subjected to cation exchange chromatography to achieve retention of lactoferrin and lactoperoxidase, because the charged characteristics of the two proteins under the pH conditions of raw milk can be retained and eluted on the cation exchange chromatography, so the lactoferrin yield can be increased and the lactoferrin activity can be retained by cation chromatography.

[0035] In the method of the present invention, skim milk after fat separation can be directly subjected to lactoferrin extraction without heating or cooling, thereby improving production efficiency and reducing the time for transfer. According to the cation chromatography column extraction method of the present invention, the natural activity of lactoferrin can be retained under the coordination of a specific temperature and elution conditions, with high yield and high purity. In the lactoferrin concentrate, the lactoferrin yield can be 75-95% (preferably 86.5-95%), and the purity is preferably 80-99.5%.

[0036] The method of the present invention further comprises:

[0037] The step of concentrating the whey protein-containing solution to obtain a whey egg concentrate; preferably, the concentration is performed using a UF membrane with a pore size of 10-30 kDa, preferably concentrating to a protein concentration (i.e., total protein concentration) of 25-35 g / 100 g;

[0038] And / or, the method for separating the fat in the animal milk in step (1) is: preheating the animal milk to 45-55° C. and then centrifuging it at a speed of 6300-6700 r / min; preferably, the fat content of the skim milk obtained after separation is 0.06-0.3%, and the fat content of the cream is 35%-40% (temporarily stored at 1-7° C. for standby use);

[0039] And / or, before step (1), the method further comprises sterilizing the animal milk; preferably, the sterilization method comprises preheating the animal milk to 45-55° C. and then centrifuging the animal milk at a speed of 4800-5300 r / min and a flow rate of 5-10 T / h.

[0040] The invention can separate the cream by a fat separator, and the cream is used for standardization of mixing with subsequent feed liquid, so that the concentration of protein and fat reaches a certain ratio, which neither causes fat floating of the final product during the shelf life nor avoids the poor taste of the product.

[0041] The present invention adopts a double-effect centrifugal sterilization method to perform preliminary sterilization on animal milk first, which can better ensure product quality. The double-effect centrifugal sterilization has the advantages of high sterilization efficiency, easy equipment cleaning, simple operation, etc. After sterilization and separation, the animal milk can be directly separated from fat without heating or cooling, thereby improving production efficiency and reducing the transfer time.

[0042] In the method of the present invention, the method of mixing the incubated casein micelle solution with other components of liquid milk comprises:

[0043] The step of mixing the incubated casein micelle solution with the cream and sterilizing the mixture to obtain a first mixed solution;

[0044] The step of mixing the lactoferrin concentrate or the mixed concentrate of lactoferrin and lactoperoxidase with the whey egg concentrate and sterilizing the mixture to obtain a second mixed solution;

[0045] And, a step of mixing the first mixed solution and the second mixed solution.

[0046] Preferably, after the incubated casein micelle solution is mixed with the cream, it is first concentrated (according to product requirements) (e.g., concentrated using an RO membrane with a pore size of 1 nm) to adjust to the target fat and protein content of the liquid milk and then sterilized (and then temporarily stored in a sterile tank);

[0047] According to product requirements, the protein concentration of the present invention after fat standardization and RO concentration can be 3.3-10g / 100mL, preferably 3.8-6g / 100mL, and the fat concentration can be 0.1-6g / 100mL, preferably 4.6-5.5g / 100mL.

[0048] When preparing the second mixed solution, the addition flow rate can be set according to product index requirements to perform aseptic online addition.

[0049] More preferably, the sterilization is high temperature sterilization, which can be tube-type sterilization (the temperature and time of tube-type sterilization are 137°C and 4-6s respectively) or steam direct sterilization; preferably steam direct sterilization, the sterilization temperature is 151-154°C and the time is 0.25-0.5s. Steam direct sterilization can reduce the degree of heat exposure of the feed liquid and reduce the Maillard reaction.

[0050] The sterilization is carried out by membrane filtration, preferably by three-stage series sterilization membrane filtration, more preferably by three-stage series sterilization membrane filtration of 0.45um-0.22um-0.22um. The three-stage series sterilization membrane filtration can make the sterilization efficiency greater than log7, thus achieving the purpose of sterilization.

[0051] When the first mixed solution and the second mixed solution are mixed, the addition flow rate can be set according to the product index requirements, and aseptic online addition is performed to achieve the preparation of liquid milks with different formula contents.

[0052] After the liquid milk of the invention is aseptically filled, the lactoferrin of the product is ≥60 mg / 100 mL, the α-lactalbumin is ≥1000 mg / 100 mL, and the β-lactoglobulin is ≥2000 mg / 100 mL. The product is stored at room temperature and has a shelf life of 30 days. The system is stable and the taste is good.

[0053] Taking cow's milk as a raw material, a specific embodiment of the present invention for preparing liquid milk comprises the following steps:

[0054] 1. Double-effect centrifugal sterilization of raw milk;

[0055] 2. The sterilized raw milk is put into a centrifuge for fat separation, and skim milk and cream are obtained and refrigerated for temporary storage;

[0056] 3. Let skim milk enter the cationic chromatographic column for separation, and lactoferrin and lactoperoxidase are subjected to gradient elution (or only lactoferrin can be eluted), concentration, and desalination to become lactoferrin concentrate and lactoperoxidase concentrate, and the remaining components become the chromatographic column flow-through liquid;

[0057] 4. The column flow-through liquid enters the MF membrane system for separation, and milk white, lactoglobulin, immunoglobulin and serum albumin become MF permeate. The MF permeate is concentrated by UF to become whey protein concentrate.

[0058] 5. The casein micelles become MF retentate, which is incubated with lysine solution and then separated from the casein micelles and plasmin by MF. The casein micelles after incubation become MF secondary retentate and are refrigerated at 1-7°C;

[0059] 6. The MF secondary retentate and the cream in step 2 are subjected to fat standardization and RO membrane concentration;

[0060] 7. The mixed liquid in step 6 is sterilized in a sterilizer and temporarily stored in a sterile tank;

[0061] 8. The lactoferrin concentrate and lactoperoxidase concentrate in step 3 are mixed with the whey protein concentrate in step 4, and subjected to low-temperature membrane sterilization and aseptic online addition.

[0062] 9. After standardized mixing of the liquids in step 7 and step 8, aseptic filling is performed and stored at room temperature.

[0063] The active protein content of the product of the invention meets certain requirements, the system is stable and the taste is good, and the shelf life can reach 30 days.

[0064] The present invention achieves the purpose of separating plasmin through special treatment and specific binding to plasmin, thereby reducing plasmin in the product. Combined with further optimization method steps, the present invention also solves the problem of few types and low content of active proteins in room temperature white milk, and provides a preparation method for retaining native active protein milk, which can greatly retain the content and natural activity of lactoferrin, and can retain multiple active proteins at the same time, which is not only conducive to the coordinated active functions of various proteins and meets the needs of consumers for native nutrition, but also effectively solves the problem of waste of raw milk active proteins due to heat processing inactivation.

[0065] The present invention can accurately fill the extracted active protein components back into the product through aseptic online addition according to product requirements, adjust the content of each active protein, and realize large-scale production of liquid milk.

[0066] The present invention also provides a liquid milk, which is prepared by the above method.

[0067] The present invention further provides an application of the method for preparing liquid milk in reducing the activity of plasmin in liquid milk.

[0068] In the application of the present invention, the liquid milk is stored at room temperature.

[0069] The beneficial effects of the present invention are at least:

[0070] The present invention adopts a combination of incubation + separation process to simply and effectively separate plasmin and casein micelles, reduce plasmin components that affect product stability, solve the problems of bitter bags and precipitation during the shelf life of the product caused by incomplete inactivation of plasmin due to direct heat sterilization, realize the room temperature storage and sales of the product, and extend the product sales cycle.

[0071] In addition, after further combining the steps of the preferred method, the present invention also uses a method of combining membrane separation with cation exchange chromatography, and sets specific conditions of process series connection, which not only retains lactoferrin, but also retains lactalbumin and lactoglobulin at the same time, and better restores the nutritional components in the raw milk. The specific elution conditions improve the yield and purity of lactoferrin, and also retain a variety of active proteins in the raw milk, which is more conducive to the coordinated development of active functions such as immunity and antibacterial, and the product tastes smooth and fresh, retaining the natural and additive-free characteristics of the product, achieving the purpose of retaining a variety of active proteins in the product and providing high-quality nutrition. DETAILED DESCRIPTION

[0072] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0073] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0074] The cation chromatography column used in the specific implementation of the present invention was purchased from GE Healthcare, model SP Sepharose Big Beads. The pore size of the UF membrane is 30 kDa. The pore size of the MF membrane is 100 kDa. The pore size of the RO membrane is 1 nm. The hollow fiber membrane is made of polysulfone membrane, purchased from Cytiva, model UFP-30-C-35 (pore size is 30 kDa).

[0075] The raw material used in the specific implementation of the present invention is the same as raw cow milk, wherein the content of plasmin is 3.5 mg / L.

[0076] The characterization of the detection indexes and the detection means of the present invention are shown in Table 1. For the index detection, three parallel tests were performed on each sample and the results were presented as the average value or the average value and the variance.

[0077] Table 1

[0078]

[0079]

[0080] Example 1

[0081] This embodiment provides a method for preparing high-activity protein milk, which specifically comprises the following steps:

[0082] (1) Raw milk is collected after passing the inspection according to GB19302 and temporarily stored in the milk silo at a temperature of 4°C.

[0083] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology with a sterilization temperature of 55°C, a rotation speed of 5300 r / min, and a flow rate of 5 T / h.

[0084] (3) The sterilized raw milk was centrifuged at a temperature of 55° C. and a speed of 6700 r / min. The skim milk obtained had a fat content of 0.06% and the cream had a fat content of 40%, which were temporarily stored at 1° C.

[0085] (4) All skim milk was transferred to a cation chromatography column, the feed temperature was 55°C, the feed linear flow rate was 8 m / h, 6 column volumes of 0.3 M NaCl were used for impurity elution, 6 column volumes of 0.9 M NaCl were used for lactoferrin elution, the elution linear flow rate was 8 m / h, and lactoferrin eluate was obtained. The lactoferrin eluate was concentrated 10 times by UF membrane, and then desalted by hollow fiber membrane (pressure 1.5 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of lactoferrin concentrate were detected to characterize its biological activity.

[0086] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, with a membrane inlet temperature of 55° C. and a concentration factor of 2. The MF permeate is concentrated to a protein content of 25 g / 100 g by a UF membrane to obtain a whey protein concentrate.

[0087] (6) The MF retentate obtained in step (5) was mixed with 1% volume of 0.063 M lysine solution, incubated at 40° C. for 60 minutes, and then separated by MF membrane to obtain MF secondary retentate - casein micelles.

[0088] (7) The MF secondary retentate obtained in step (6) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 3.8 g / 100 mL of protein and 4.6 g / 100 mL of fat.

[0089] (8) Steam direct sterilization, 154℃ / 0.25s, temporarily stored in a sterile tank.

[0090] (9) The lactoferrin concentrate obtained in step (4) is mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0091] (10) The product was stored at room temperature for 30 days, and the product stability during the offline and shelf life was tracked. The test indicators and results are shown in Table 2.

[0092] Table 2

[0093]

[0094] Example 2

[0095] This embodiment provides a method for preparing high-activity protein milk, which specifically comprises the following steps:

[0096] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0097] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology, with a sterilization temperature of 45°C, a rotation speed of 4800r / min, and a flow rate of 10T / h.

[0098] (3) The sterilized raw milk was centrifuged at a temperature of 45° C. and a speed of 6300 r / min. The skim milk obtained had a fat content of 0.3% and the cream had a fat content of 35%, which were temporarily stored at 7° C.

[0099] (4) All the skim milk was transferred to a cation chromatography column with a feed temperature of 45° C. and a feed linear flow rate of 4 m / h. Impurities were eluted using 3 times the column volume of 0.5 M NaCl, lactoperoxidase was eluted using 3 times the column volume of 0.55 M NaCl, and lactoferrin was eluted using 3 times the column volume of 0.7 M NaCl. The elution linear flow rate was 4 m / h to obtain a lactoperoxidase eluate and a lactoferrin eluate.

[0100] The lactoperoxidase eluate was concentrated 15 times by UF membrane and desalted by hollow fiber membrane (pressure 2 bar) to obtain lactoperoxidase concentrate (conductivity <1 mS / cm).

[0101] The lactoferrin eluate was concentrated 15 times by UF membrane and desalted by hollow fiber membrane (pressure 2 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of lactoferrin concentrate were tested to characterize its biological activity.

[0102] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, with a membrane inlet temperature of 45° C. and a concentration factor of 4. The MF permeate is concentrated by a UF membrane to a protein content of 25 g / 100 g to obtain a whey protein concentrate.

[0103] (6) The MF retentate obtained in step (5) was mixed with 1% volume of 0.042M lysine solution, incubated at 50°C for 30 minutes, and then separated by MF membrane to obtain MF secondary retentate - casein micelles.

[0104] (7) The MF secondary retentate obtained in step (6) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 6.0 g / 100 mL of protein and 5.5 g / 100 mL of fat.

[0105] (8) Tubular sterilization, 137℃ / 5s, temporarily stored in a sterile tank.

[0106] (9) The lactoferrin concentrate obtained in step (4) is mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0107] (10) The product was stored at room temperature for 30 days, and the product stability during the offline and shelf life was tracked. The test indicators and results are shown in Table 3.

[0108] Table 3

[0109]

[0110]

[0111] In this embodiment, since the protein is concentrated to 6.0 g / 100 mL and the fat is concentrated to 5.5 g / 100 mL by RO membrane, the protein and fat contents after concentration are higher than those in Embodiments 1 and 3, that is, the concentration multiple is greater, so the corresponding final protein concentrations are also higher.

[0112] Example 3

[0113] This embodiment provides a method for preparing high-activity protein milk, which specifically comprises the following steps:

[0114] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0115] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology with a sterilization temperature of 50°C, a rotation speed of 5200 r / min, and a flow rate of 8 T / h.

[0116] (3) The sterilized raw milk was centrifuged at a temperature of 50° C. and a speed of 6700 r / min. The skim milk obtained had a fat content of 0.1% and the cream had a fat content of 38%, which were temporarily stored at 4° C.

[0117] (4) All skim milk was transferred to a cation chromatography column, the feed temperature was 50°C, the feed linear flow rate was 6 m / h, 5 column volumes of 0.4 M NaCl were used for impurity elution, 5 column volumes of 0.65 M NaCl were used for lactoperoxidase elution, 5 column volumes of 0.8 M NaCl were used for lactoferrin elution, and the elution linear flow rate was 6 m / h. Finally, lactoperoxidase eluate and lactoferrin eluate were obtained.

[0118] The lactoperoxidase eluate was concentrated 12 times by UF membrane and desalted by hollow fiber membrane (pressure 1.8 bar) to obtain lactoperoxidase concentrate (conductivity <1 mS / cm).

[0119] The lactoferrin eluate was concentrated 12 times by UF membrane and desalted by hollow fiber membrane (pressure 1.8 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of lactoferrin concentrate were tested to characterize its biological activity.

[0120] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, the membrane inlet temperature is 50° C., the concentration factor is 2.5, and the MF permeate is concentrated to a protein content of 35 g / 100 g through a UF membrane to obtain a whey protein concentrate.

[0121] (6) The MF retentate obtained in step (5) was mixed with 1% volume of 0.0525 M lysine solution, incubated at 45° C. for 45 minutes, and then separated by MF membrane to obtain MF secondary retentate - casein micelle solution.

[0122] (7) The MF secondary retentate obtained in step (6) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 3.8 g / 100 mL of protein and 4.6 g / 100 mL of fat.

[0123] (8) Steam direct sterilization, 154℃ / 0.25s, temporarily stored in a sterile tank.

[0124] (9) The lactoperoxidase concentrate and lactoferrin concentrate obtained in step (4) are mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0125] (10) The product was stored at room temperature for 30 days, and the product stability during the offline and shelf life was tracked. The test indicators and results are shown in Table 4.

[0126] Table 4

[0127]

[0128] Example 4

[0129] This embodiment provides a method for preparing milk, which specifically comprises the following steps (basically the same as that of Embodiment 1, the main difference being that the processing temperature is different):

[0130] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0131] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology with a sterilization temperature of 58°C, a rotation speed of 5300 r / min, and a flow rate of 5 T / h.

[0132] (3) The sterilized raw milk was centrifuged at a temperature of 58° C. and a speed of 6700 r / min. The skim milk obtained had a fat content of 0.06% and the cream had a fat content of 40%, which were temporarily stored at 1° C.

[0133] (4) All skim milk was transferred to a cation chromatography column, the feed temperature was 58°C, the feed linear flow rate was 8 m / h, 6 column volumes of 0.3 M NaCl were used for impurity elution, 6 column volumes of 0.9 M NaCl were used for lactoferrin elution, the elution linear flow rate was 8 m / h, and lactoferrin eluate was obtained. The lactoferrin eluate was concentrated 10 times by UF membrane, and the hollow fiber membrane was desalted (pressure 1.5 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of the lactoferrin concentrate were detected to characterize its biological activity.

[0134] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, with a membrane inlet temperature of 58° C. and a concentration factor of 2. The MF permeate is concentrated through a UF membrane to a protein content of 25 g / 100 g to obtain a whey protein concentrate.

[0135] (6) The MF retentate obtained in step (5) was mixed with 1% volume of 0.063 M lysine solution, incubated at 40° C. for 60 minutes, and then separated by MF membrane to obtain MF secondary retentate - casein micelle solution.

[0136] (7) The MF secondary retentate obtained in step (6) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 3.8 g / 100 mL of protein and 4.6 g / 100 mL of fat.

[0137] (8) Steam direct sterilization, 154℃ / 0.25s, temporarily stored in a sterile tank.

[0138] (9) The lactoferrin concentrate obtained in step (4) is mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0139] (10) The product was stored at room temperature for 30 days, and the product stability during the offline and shelf life was tracked. The test indicators and results are shown in Table 5.

[0140] Table 5

[0141]

[0142]

[0143] Compared with Example 1, the contents of lactoferrin, lactalbumin and lactoglobulin are reduced.

[0144] Example 5

[0145] This embodiment provides a method for preparing milk, which specifically comprises the following steps (basically the same as that of embodiment 2, the main difference being that the elution gradient is changed):

[0146] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0147] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology, with a sterilization temperature of 45°C, a rotation speed of 4800r / min, and a flow rate of 10T / h.

[0148] (3) The sterilized raw milk was centrifuged at a temperature of 45° C. and a speed of 6300 r / min. The skim milk obtained had a fat content of 0.3% and the cream had a fat content of 35%, which were temporarily stored at 7° C.

[0149] (4) All skim milk was transferred to a cation chromatography column, the feed temperature was 45°C, the feed linear flow rate was 4 m / h, 3 column volumes of 0.6 M NaCl were used for impurity elution, 3 column volumes of 0.8 M NaCl were used for lactoperoxidase elution, 3 column volumes of 1.0 M NaCl were used for lactoferrin elution, and the elution linear flow rate was 4 m / h. Finally, lactoperoxidase eluate and lactoferrin eluate were obtained.

[0150] The lactoperoxidase eluate was concentrated 15 times by UF membrane and desalted by hollow fiber membrane (pressure 2 bar) to obtain lactoperoxidase concentrate (conductivity <1 mS / cm).

[0151] The lactoferrin eluate was concentrated 15 times by UF membrane and desalted by hollow fiber membrane (pressure 2 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of lactoferrin concentrate were tested to characterize its biological activity.

[0152] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, with a membrane inlet temperature of 45° C. and a concentration factor of 4. The MF permeate is concentrated by a UF membrane to a protein content of 25 g / 100 g to obtain a whey protein concentrate.

[0153] (6) The MF retentate obtained in step (5) was mixed with 1% volume of 0.042M lysine solution, incubated at 50°C for 30 minutes, and then separated by MF membrane to obtain MF secondary retentate - casein micelle solution.

[0154] (7) The MF secondary retentate obtained in step (6) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 6.0 g / 100 mL of protein and 5.5 g / 100 mL of fat.

[0155] (8) Tubular sterilization, 137℃ / 5s, temporarily stored in a sterile tank.

[0156] (9) The lactoperoxidase concentrate and lactoferrin concentrate obtained in step (4) are mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0157] (10) The product was stored at room temperature for 30 days, and the product's stability during the off-line and shelf life, fibrinolytic enzyme activity, taste and other characteristics were tracked. The test indicators and results are shown in Table 6.

[0158] Table 6

[0159]

[0160] Compared with Example 2, the contents of lactoferrin, lactalbumin and lactoglobulin are reduced.

[0161] Comparative Example 1

[0162] This comparative example provides a method for preparing milk, which specifically comprises the following steps (basically the same as Example 3, except that there is no plasmin incubation treatment step):

[0163] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0164] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology with a sterilization temperature of 50°C, a rotation speed of 5200r / min, and a flow rate of 8T / h.

[0165] (3) The sterilized raw milk was centrifuged at a temperature of 50° C. and a speed of 6700 r / min. The skim milk obtained had a fat content of 0.1% and the cream had a fat content of 38%, which were temporarily stored at 4° C.

[0166] (4) All skim milk was transferred to a cation chromatography column, the feed temperature was 50°C, the feed linear flow rate was 6 m / h, 5 column volumes of 0.4 M NaCl were used for impurity elution, 5 column volumes of 0.65 M NaCl were used for lactoperoxidase elution, 5 column volumes of 0.8 M NaCl were used for lactoferrin elution, and the elution linear flow rate was 6 m / h. Finally, lactoperoxidase eluate and lactoferrin eluate were obtained.

[0167] The lactoperoxidase eluate was concentrated 12 times by UF membrane and desalted by hollow fiber membrane (pressure 1.8 bar) to obtain lactoperoxidase concentrate (conductivity <1 mS / cm).

[0168] The lactoferrin eluate was concentrated 12 times by UF membrane and desalted by hollow fiber membrane (pressure 1.8 bar) to obtain lactoferrin concentrate (conductivity <1 mS / cm). The yield, purity and antibacterial rate of lactoferrin concentrate were tested to characterize its biological activity.

[0169] (5) The chromatographic column flow-through liquid obtained in step (4) is completely transported to the MF membrane system, the membrane inlet temperature is 50° C., the concentration factor is 2.5, and the MF permeate is concentrated to a protein content of 35 g / 100 g through a UF membrane to obtain a whey protein concentrate.

[0170] (6) The MF retentate obtained in step (5) was mixed with the cream obtained in step (3), and the fat was standardized and concentrated by RO membrane to 3.8 g / 100 mL of protein and 4.6 g / 100 mL of fat.

[0171] (7) Steam direct sterilization, 154℃ / 0.25s, temporarily stored in a sterile tank.

[0172] (8) The lactoperoxidase concentrate and lactoferrin concentrate obtained in step (4) are mixed with the whey protein concentrate obtained in step (5), filtered through a 0.45um-0.22um-0.22um three-stage series sterilization membrane, aseptically added online to a sterile tank, and aseptically filled. The separated active proteins are backfilled into the product in a 1:1 ratio.

[0173] (9) The product was stored at room temperature for 30 days, and the product's stability during the off-line and shelf life, fibrinolytic enzyme activity, taste and other characteristics were tracked. The test indicators and results are shown in Table 7.

[0174] Table 7

[0175]

[0176] The activity of plasmin increases significantly during the shelf life, affecting the sensory properties of the product.

[0177] Comparative Example 2

[0178] This comparative example provides a method for preparing milk, which specifically comprises the following steps:

[0179] (1) Raw milk is tested and qualified according to GB19302 and stored in milk silos at a temperature of 4°C.

[0180] (2) The selected raw milk was sterilized using double-effect centrifugal sterilization technology with a sterilization temperature of 50°C, a rotation speed of 5200 r / min, and a flow rate of 8 T / h.

[0181] (3) RO membrane concentration to 3.8 g / 100 mL protein and 4.6 g / 100 mL fat.

[0182] (4) Pasteurization at 75℃ / 15s and aseptic filling.

[0183] (5) Track product characteristics such as product end-of-line and shelf life stability, taste, etc. See Table 8 for various test indicators and results.

[0184] Table 8

[0185]

[0186] In this comparative example, the contents of lactoferrin, lactalbumin and lactoglobulin are low, and the activity of plasmin is high.

[0187] Comparative Example 3

[0188] This comparative example provides a method for preparing milk, and the specific steps are basically the same as those in Example 3, except that step (6) is changed to: incubating the MF retentate obtained in step (5) with a 1% volume of a 0.0525M arginine solution at 45°C for 45 minutes, and then separating through an MF membrane to obtain a MF secondary retentate, namely, casein micelles. The remaining steps are the same as those in Example 3.

[0189] The product was stored at room temperature for 30 days, and the characteristics of the product such as fibrinolytic activity and taste were tracked during the product's offline and shelf life. The test indicators and results are shown in Table 9.

[0190] Table 9

[0191]

[0192] Comparative Example 4

[0193] This comparative example provides a method for preparing milk, and the specific steps are basically the same as those in Example 3, except that step (6) is changed to: the MF retentate obtained in step (5) is heat-pretreated by indirect heating (90° C., 5 minutes) to inactivate plasmin, and then step (7) is performed. The remaining steps are the same as those in Example 3.

[0194] The product was stored at room temperature for 30 days, and the characteristics of the product such as fibrinolytic activity and taste were tracked during the product's offline and shelf life. The test indicators and results are shown in Table 10.

[0195] Table 10

[0196]

[0197] The test results of the lactoferrin concentrate in the examples and comparative examples are shown in Table 11.

[0198] Table 11

[0199] project Lactoferrin yield% Lactoferrin purity % Antibacterial rate% Example 1 86.5 80.0 82 Example 2 91.2 89.3 92 Example 3 95.0 99.5 98 Example 4 75.0 76.1 74 Example 5 81.9 78.4 78 Comparative Example 1 94.6 99.3 97 Comparative Example 3 94.8 99.4 96 Comparative Example 4 95.0 99.5 97

[0200] The contents of lactoferrin, milk white and milk globules in the liquid milk of the present invention during the shelf life all meet the product index requirements; the fibrinolytic enzyme activity is low, and there is no precipitation or bitter bag problem during the shelf life; the yield, purity and antibacterial rate of lactoferrin are improved, indicating that the present invention not only realizes the extraction of lactoferrin with high purity and high yield, and retains its original activity and has antibacterial effect, but also realizes the original retention and backfilling of milk white and milk globules, and finally realizes the retention of multiple active proteins in the product under room temperature storage, the system is stable, and the product has a good taste.

[0201] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing liquid milk, characterized in that: Mixing lysine with a casein micelle solution to be treated separated from animal milk, incubating and separating the mixture to obtain an incubated casein micelle solution, and mixing the incubated casein micelle solution with other components of liquid milk; The other components of the liquid milk include components in animal milk except the casein micelle solution to be treated.

2. The method for preparing liquid milk according to claim 1, characterized in that: The molar ratio of plasmin to lysine in the casein micelle solution to be treated is 1:(1-10), preferably 1:(6-9); the incubation temperature is 40°C-50°C; And / or, the incubation time is 30 minutes to 60 minutes; And / or, separation after mixed incubation is performed using a MF membrane, and the pore size of the MF membrane is 100 kDa-120 kDa.

3. The method for preparing liquid milk according to claim 1 or 2, characterized in that: The method for separating the casein micelle solution to be processed from animal milk comprises: (1) Separating fat from animal milk to obtain skim milk and cream; (2) treating the skim milk with a cationic chromatographic column to separate a whey protein-containing solution and a casein micelle solution to be treated from the flow-through liquid of the chromatographic column; the filler in the cationic chromatographic column is agarose gel; Preferably, the method for obtaining the whey protein-containing solution and the casein micelle solution to be treated by separation from the chromatographic column flow-through liquid is: The chromatographic column flow-through liquid is treated with an MF membrane with a pore size of 100kDa-120kDa, the membrane inlet temperature is 45-55°C, and the concentration multiple is 2-4 times to obtain MF permeate and MF retentate; the MF permeate is the whey protein-containing solution, and the MF retentate is the casein micelle solution to be treated.

4. The method for preparing liquid milk according to claim 3, characterized in that: In step (2), after the skim milk is treated with a cation chromatography column, the method further comprises the step of eluting to obtain an eluate containing lactoferrin; Preferably, when the skim milk is treated with a cation chromatography column, the feed temperature is 45-55° C., the feed linear flow rate is 4-8 m / h, and after feeding, 3-6 times the column volume of 0.3-0.5 M NaCl is first used to elute impurities, and then 3-6 times the column volume of 0.7-0.9 M NaCl is used to elute lactoferrin to obtain an eluate containing lactoferrin; the elution flow rates for impurity elution and lactoferrin elution are both 4-8 m / h.

5. The method for preparing liquid milk according to claim 4, characterized in that: When eluting to obtain an eluate containing lactoferrin, the method further comprises: after eluting impurities, firstly eluting lactoperoxidase using 3-6 times column 0.55-0.65M NaCl to obtain an eluate containing lactoperoxidase, and then eluting lactoferrin; the elution flow rate of the lactoperoxidase elution is 4-8m / h; Preferably, the method for preparing liquid milk further comprises: respectively concentrating and desalting the lactoferrin-containing eluate and the lactoperoxidase-containing eluate to obtain a lactoferrin concentrate and a lactoperoxidase concentrate; Alternatively, the step of mixing the lactoferrin-containing eluate and the lactoperoxidase-containing eluate, and then concentrating and desalting to obtain a mixed concentrate of lactoferrin and lactoperoxidase; More preferably, the concentration and desalting method is: concentrating the lactoferrin-containing eluate and / or the lactoperoxidase-containing eluate by 10-15 times with a UF membrane, and then desalting with a hollow fiber membrane; the pore size of the UF membrane is 10 kDa-30 kDa.

6. The method for preparing liquid milk according to any one of claims 3 to 5, characterized in that: Also includes: The step of concentrating the whey protein-containing solution to obtain a whey egg concentrate; preferably, the concentration is performed using a UF membrane with a pore size of 10 kDa-30 kDa; And / or, the method for separating the fat in the animal milk in step (1) is: preheating the animal milk to 45-55° C. and then centrifuging it at a speed of 6300-6700 r / min; preferably, the fat content of the skim milk obtained after separation is 0.06-0.3%, and the fat content of the cream is 35%-40%; And / or, before step (1), the method further comprises sterilizing the animal milk; preferably, the sterilization method comprises preheating the animal milk to 45-55° C. and then centrifuging the animal milk at a speed of 4800-5300 r / min and a flow rate of 5-10 T / h.

7. The method for preparing liquid milk according to claim 6, characterized in that: The method of mixing the incubated casein micelle solution with other components of liquid milk comprises: The step of mixing the incubated casein micelle solution with the cream and sterilizing the mixture to obtain a first mixed solution; The step of mixing the lactoferrin concentrate or the lactoferrin and lactoperoxidase mixed concentrate with the whey egg concentrate and sterilizing the mixture to obtain a second mixed solution; and, a step of mixing the first mixed solution and the second mixed solution; Preferably, after the incubated casein micelle solution is mixed with the cream, it is concentrated to adjust to the target fat and protein content of the liquid milk and then sterilized; More preferably, the sterilization is high temperature sterilization, and further preferably, tube-type sterilization or steam direct sterilization; The sterilization is performed by membrane filtration, and more preferably by three-stage series sterilization membrane filtration.

8. A liquid milk, characterized in that: The method is prepared by any one of claims 1 to 7.

9. Use of the method for preparing liquid milk according to any one of claims 1 to 7 in reducing the activity of plasmin in liquid milk.

10. The use according to claim 9, characterized in that: The liquid milk is stored at room temperature.

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