A method for separating and preparing milk fat globule membrane

Abstract

The present invention discloses a method for separating and preparing milk fat globule membrane, belonging to the technical field of dairy product processing. The method for separating and preparing milk fat globule membrane according to the present invention uses fresh animal milk after centrifugation and sterilization as raw material, and realizes the preparation of buttermilk by controlling the ripening temperature and time of the cream; further, the casein in the buttermilk is removed by using the rennet method, and after regulating the rennet coagulation step, the extraction rates of MFGM protein and phospholipid reach 56.42% and 72.57%; further, after regulating the whey drainage step of the rennet, the flocculation structure of the casein becomes loose, which is beneficial to the recovery of milk fat globule membrane components in the buttermilk whey, and the extraction rates of MFGM protein and phospholipid increase to 68.90% and 80.57% respectively; further, with the assistance of ultrasonic physical extraction, the extraction rates of MFGM protein and phospholipid are further increased to 78.26% and 83.12% respectively.

Description

Technical Field

[0001] The present invention relates to a method for separating and preparing milk fat globule membrane, belonging to the technical field of dairy product processing. Background Art

[0002] Milk fat is one of the main components of animal milk, mainly existing in the form of fat globules. The surface of the fat globules is coated with three layers of membranes, and this membrane is the milk fat globule membrane (MFGM). Its existence keeps the fat globules in a stable emulsion state. MFGM is a protein-lipid complex, composed of components such as milk fat globule membrane protein, phospholipids, and gangliosides. Among them, phospholipids have the effects of promoting the development of the infant brain, nervous system, and digestive system, as well as improving cognitive ability, etc.; milk fat globule membrane protein has the functions of inhibiting the growth of breast cancer cells, inhibiting the growth of harmful bacteria such as Staphylococcus aureus, Escherichia coli, and Salmonella in the infant intestine, and resisting rotavirus infection, etc. Therefore, MFGM can be used as an excellent functional ingredient in infant formula or other functional foods. In addition, due to the amphiphilic properties of phospholipids, MFGM can also be used as an emulsifier or stabilizer, and as an embedding wall material for various functional active ingredients to keep them stable in different liquid environments.

[0003] Currently, commercially available MFGM products include Lacprodan MFGM-10, SureStart Lipid 70, and Hilmar7500, etc., which are mainly prepared by using whey, a processing by-product of cheese whey, to prepare whey buttermilk and further separating it. However, due to the low consumption of raw cheese in China, it is difficult to obtain cheese whey; and during the process of removing casein by enzyme coagulation in cheese whey, a large number of fat globules will be embedded in the casein gel network, and these fat globules are difficult to enter the whey phase when discharging whey, resulting in a very low content of MFGM in whey. In addition, during the processing of cheese and whey, due to multiple heat treatments, the activity of heat-sensitive MFGM protein is easily affected. Therefore, it is necessary to find alternative raw materials. Cream is the upper phase part collected by centrifuging whole milk for defatting. Its processing conditions are relatively mild, and it retains more original globule membrane components, which is a potential alternative raw material.

[0004] The composition of cream is complex, including milk fat globules and the fat globule membrane on their surface, casein, whey protein, lactose, salts and other substances. The difficulties in extracting MFGM from cream include: the content of MFGM components in the raw material is low, and the extraction process is complex; the particle size and isoelectric point of MFGM are relatively close to those of casein micelles, and it is easy to be removed together with casein during separation, resulting in a decrease in yield; the structure of MFGM is unstable, and it is easy to be damaged and lost during purification processes such as washing, and it is easy to break into small fragments and enter the permeate during membrane separation, resulting in loss; the protein activity of MFGM is easily affected by processing conditions and lost, etc. These factors limit the production and application of MFGM. According to the current reports on the separation and preparation methods of MFGM, it is found that: the current extraction methods have problems such as low extraction rate, low production efficiency, poor protein activity, etc., and are mainly used in the field of scientific research and are not suitable for large-scale industrial preparation.

[0005] For example: Patent CN111227039A uses cream as the raw material, first stirs and freezes it, then heats it up to break the fat globules, then adds a sucrose-salt eluent to elute and centrifuge to obtain a fat globule membrane suspension, and then enriches the MFGM phospholipid and protein components by microfiltration through a 0.1-0.2 μm membrane; however, the demulsification method of freezing combined with heating is not sufficient to completely break all fat globules, resulting in a low yield of MFGM, and there is no special process to remove casein, so the purity of MFGM is low. Patent CN113455551A uses cream as the raw material, adds a PBS eluent to wash, removes the casein and whey protein around the fat globules, then adds ethanol and ultrasonics to break the fat globule structure and release the fat globule membrane, and then adds cold acetone to remove non-polar lipids, and the solution is centrifuged to obtain the MFGM phospholipid component; however, this technology uses organic reagents, which will bring food safety problems, and only collects the phospholipid component, and does not collect the physiologically active MFGM protein component; in addition, due to the unstable structure of the fat globule membrane, part of the MFGM protein and phospholipid will be washed away during the washing process, resulting in a decrease in yield. Patent CN105455109A uses the by-product buttermilk of cream processing as the raw material, adds a chelating salt to dissociate casein, and then uses a 0.1-1.5 μm membrane to retain the MFGM protein and phospholipid components, but the use of chelating salt will dissociate MFGM fragments, and the dissociated MFGM will enter the permeate during membrane separation, resulting in a large loss of MFGM protein and phospholipid. Patent CN114478739A uses buttermilk as the raw material, adjusts the pH to 4.5-4.7 to precipitate casein, then uses heat calcium treatment and dialysis treatment to remove polar lipids and whey protein, and finally obtains the MFGM protein ingredient; this technology uses the acid precipitation method to remove casein, but since the isoelectric point of bovine milk MFGM is between 4.2-4.8, which is close to the isoelectric point of casein, the acid precipitation method will cause the MFGM protein and casein to precipitate together and cause loss, and the beneficial components such as MFGM phospholipid will be removed after heat calcium treatment. Summary of the Invention

[0006] [Technical Problem]

[0007] There are problems in the separation of MFGM using cheese whey as raw material, such as difficult access to raw materials, low content of milk fat globule membrane components, and low production efficiency.

[0008] There are food safety problems in the separation of MFGM using cream as raw material, which limits the application of the obtained isolate in foods with high requirements for raw materials such as infant foods. At the same time, there are also problems such as low yield or purity of MFGM components, low protein activity, only collecting phospholipids or MFGM proteins, and further reduction of the yield due to the unstable structure of MFGM leading to entering the permeate during membrane separation.

[0009] [Technical Solution]

[0010] To solve the above problems, the present invention provides a method for separating and preparing MFGM without food safety problems, high yield, high retention of protein activity, and simultaneous enrichment of MFGM protein and phospholipid components. The present invention first obtains buttermilk by regulating the ripening and demulsification conditions of cream; then uses the rennet method to remove casein in the buttermilk. By regulating the steps of curdling and draining whey, the flocculent structure of casein becomes loose, which is beneficial to the release of milk fat globule membrane components into the buttermilk whey to improve its extraction rate; further, ultrasonic-assisted extraction can further improve the extraction rate of MFGM; by utilizing the fact that temperature and pH affect the aggregation characteristics of MFGM, by regulating conditions such as membrane pore size, temperature, and pH during membrane filtration, the retention effect of MFGM in the concentration and purification steps is improved; combined with key operations such as drying, finally, an MFGM ingredient with a high extraction rate and high activity retention of MFGM is obtained.

[0011] The first object of the present invention is to provide a method for separating and preparing milk fat globule membrane, comprising the following steps:

[0012] (1) Separation and preparation of buttermilk:

[0013] Keep the pasteurized cream standing at 4 - 20 °C for ripening for 2 - 24 h; then stir to demulsify until buttermilk is obtained;

[0014] (2) Curdling in the preparation of buttermilk whey:

[0015] Keep the temperature of the buttermilk in step (1) at 30 - 55 °C, add 0.05 - 2 g / 100 kg of rennet, and react for 20 - 80 min to obtain the curdled buttermilk;

[0016] (3) Draining whey in the preparation of buttermilk whey:

[0017] Cut and stir the curdled buttermilk in step (2), add 0 - 9 mM of sodium citrate, then adjust the pH value to 6.5 - 8.5 and stir slowly for 30 - 50 min. At the same time, raise the whey drainage temperature to 35 - 55 °C, centrifuge at 500 - 10,000 g for 4 - 6 min, and collect the upper layer of buttermilk whey containing milk fat globule membrane.

[0018] (4) Preparation of milk fat globule membrane:

[0019] Filter the buttermilk whey through an ultrafiltration membrane and dry it to obtain milk fat globule membrane whey powder; filter the buttermilk whey through a microfiltration membrane and dry it to obtain milk fat globule membrane powder.

[0020] In one embodiment of the present invention, the preparation method of pasteurized cream in step (1) is as follows:

[0021] Take fresh animal milk, heat it to 35 - 50 °C, and defat it by a disc centrifuge to obtain cream with a fat content of 30 - 45%; then heat the cream to 70 - 75 °C and sterilize it for 10 - 30 s to obtain pasteurized cream;

[0022] Among them, fresh animal milk includes cow milk, goat milk, sheep milk, buffalo milk, yak milk, dromedary camel milk, bactrian camel milk, horse milk, donkey milk, etc.; the heating temperature of fresh animal milk is further preferably 40 - 45 °C; the fat content of the cream is further preferably 35 - 40%; the pasteurization conditions are further preferably sterilization at 72 °C for 15 s.

[0023] In one embodiment of the present invention, the ripening conditions in step (1) are further preferably ripening at 12 °C for 12 - 24 h, and most preferably ripening for 12 h.

[0024] In one embodiment of the present invention, the emulsion breaking in step (1) is carried out by using a small stirrer to break the emulsion or a butter churn to break the emulsion.

[0025] In one embodiment of the present invention, the temperature of the buttermilk in step (2) is further preferably 30 - 35 °C, and most preferably 35 °C.

[0026] In one embodiment of the present invention, the concentration of rennet in step (2) is further preferably 0.5 - 2 g / 100 kg, and most preferably 0.5 g / 100 kg.

[0027] In one embodiment of the present invention, the addition method of rennet in step (2) is a solution prepared from rennet powder; the solvent of the solution is water, and the concentration of the solution is 4 - 6 g / kg, and further preferably the solvent is deionized water and the concentration is 5 g / kg.

[0028] In one embodiment of the present invention, the average enzyme activity of the rennet in step (2) is 2200 IMCU / g, and it is purchased from Chr. Hansen (Beijing) Trading Co., Ltd.

[0029] In one embodiment of the present invention, the reaction time of the rennet in step (2) is further preferably 60 - 80 min, and most preferably 60 min.

[0030] In one embodiment of the present invention, the concentration of sodium citrate in step (3) is further preferably 1.5 - 4.5 mM, and most preferably 3 mM.

[0031] In one embodiment of the present invention, the sodium citrate can be added in the curdling step or the whey draining step, and preferably in the whey draining step.

[0032] In one embodiment of the present invention, the pH value in step (3) is further preferably 7.5 - 8.0, and most preferably 7.5.

[0033] In one embodiment of the present invention, the pH value in step (3) is adjusted using a 2M sodium hydroxide solution.

[0034] In one embodiment of the present invention, the time of slow stirring in step (3) is further preferably 50 min.

[0035] In one embodiment of the present invention, the temperature of whey draining in step (3) is further preferably 45 - 50 °C, and most preferably 50 °C.

[0036] In one embodiment of the present invention, after collecting the buttermilk whey containing milk fat globule membrane in the upper layer in step (3), the casein precipitate can also be repeatedly washed and centrifuged with deionized water, and the supernatant is collected and combined with the buttermilk whey. The preferred number of washing times is 1 time.

[0037] In one embodiment of the present invention, after the stirring time in the whey draining in step (3) is 50 min and the temperature is raised simultaneously, ultrasonic treatment can be carried out; the conditions of the ultrasonic treatment are: the ultrasonic power is 100 - 500 W, further preferably 200 - 300 W, and most preferably 300 W; the ultrasonic time is 10 - 30 min, and further preferably 15 min.

[0038] In one embodiment of the present invention, the conditions of centrifugation in step (3) are further preferably centrifugation at 1500 g for 5 min.

[0039] In one embodiment of the present invention, in the step (4), ultrafiltration membrane filtration is carried out by using a membrane with a membrane pore size of 5 - 100 kDa for filtration and concentration, and microfiltration membrane filtration is carried out by using a membrane with a membrane pore size of 10 - 200 nm for filtration and concentration; further preferably, ultrafiltration membrane filtration is carried out by using a membrane with a membrane pore size of 10 - 50 kDa for filtration and concentration, optimally 50 kDa, and microfiltration membrane filtration is carried out by using a membrane with a membrane pore size of 50 - 100 nm for filtration and concentration; the temperature of the material for ultrafiltration membrane filtration is 40 - 50 °C, and the pH of the material is 6.5 - 7.5; further preferably, the temperature of the material is 45 °C, and the pH of the material is 7.5; the temperature of the material for microfiltration membrane filtration is 25 - 55 °C, and the pH of the material is 5.5 - 7.5; further preferably, the temperature of the material is 35 °C, and the pH of the material is 6.0 - 6.5, optimally 6.5; the concentration multiple of membrane filtration is 2 - 5 times, and the number of washing and filtering times is 0 - 3 times; further preferably, the concentration multiple is 3 - 4 times, optimally 4 times, and the number of washing and filtering times is 1 - 2 times.

[0040] In one embodiment of the present invention, the drying in the step (4) includes one of spray drying, vacuum freeze drying, and low-temperature and low-pressure spray drying; among them, the conditions for spray drying are: inlet air temperature 185 - 190 °C, outlet air temperature 85 - 95 °C; the conditions for vacuum freeze drying are: freezing temperature -80 °C, vacuum degree 15 - 20 Pa; the conditions for low-temperature and low-pressure spray drying are: inlet air temperature 70 - 80 °C, outlet air temperature 50 - 60 °C, vacuum degree 0.02 - 0.04 Mpa.

[0041] The second object of the present invention is the milk fat globule membrane prepared by the method described in the present invention.

[0042] The third object of the present invention is the application of the milk fat globule membrane described in the present invention in the food field.

[0043] In one embodiment of the present invention, the application includes being used for preparing infant foods, and in particular, being used for preparing infant formula milk powder.

[0044] [Beneficial effects]

[0045] (1) The method for separating and preparing milk fat globule membrane (MFGM) according to the present invention uses fresh animal milk as raw material, specifically the skim milk obtained by centrifuging and pasteurizing fresh animal milk. By controlling the ripening temperature and time of the skim milk, efficient preparation of buttermilk is achieved. Further, by using the rennet method to remove casein from the buttermilk, after regulating the rennet coagulation step, and preferably under the condition of not adding Ca, the extraction rates of MFGM protein and phospholipid reach 56.42% and 72.57% respectively. Further, after regulating the whey drainage step, the flocculent structure of casein becomes loose, which is beneficial for the components of the milk fat globule membrane to enter the buttermilk whey and be recovered, and the extraction rates of MFGM protein and phospholipid increase to 68.90% and 80.57% respectively. Further, with the assistance of ultrasonic physical extraction, the extraction rates of MFGM protein and phospholipid are further increased to 78.26% and 83.12% respectively. In addition, washing the casein precipitate after separating the buttermilk whey can further improve the extraction rates of MFGM protein and phospholipid.

[0046] (2) The method for separating and preparing milk fat globule membrane (MFGM) according to the present invention can enrich MFGM protein and phospholipid simultaneously and preferably retains the active components of the MFGM component. Compared with the buttermilk whey prepared by the acid precipitation method and the whey buttermilk prepared by cheese whey, the activity of the typical active protein xanthine oxidase in the milk fat globule membrane prepared by the present invention is 11.42 U / L, higher than 4.41 U / L and 9.24 U / L of the former two. In terms of the extraction rate and separation efficiency of the MFGM component, it is also superior to the raw materials obtained by the other two separation and preparation methods.

[0047] (3) The present invention utilizes the dependence of the aggregation state of MFGM itself on conditions such as temperature and pH, that is, moderate low temperature and moderate low pH can better maintain the stability of the MFGM structure. Thus, by regulating the pore size, temperature and pH during membrane filtration, the retention of MFGM in the concentration and purification step is improved.

[0048] (4) The method of the present invention does not have food safety problems, can improve the utilization value of butter processing by-products (buttermilk), and broaden its application in foods with high requirements for raw materials such as infant foods, especially infant formula milk powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the process flow chart of the method for separating and preparing milk fat globule membrane according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0050] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0051] Test method:

[0052] 1. MFGM protein analysis:

[0053] Take buttermilk and buttermilk whey samples, dilute them 5-fold with deionized water and mix them 1:1 with the buffer, then water bath in boiling water for 5 min. Inject 10 μL of the sample into a gel plate with a 4% stacking gel and a 12% separating gel. Use a Bio-Rad ChemiDoc XRS+ imager to measure the protein band intensity on each gel lane.

[0054] The casein residue rate is the ratio of the casein gel band intensity in buttermilk whey to that of buttermilk.

[0055] 2. Determination of xanthine oxidase (XO) activity:

[0056] Take buttermilk and buttermilk whey samples, dilute them 50-fold with phosphate buffer, then take 100 μL of the diluted sample into a 96-well plate and add an equal volume of reaction solution. Place the plate in a SpectraMax M5 multi-functional microplate reader to continuously record the fluorescence change within 10 min, and calculate the XO activity using formula (1).

[0057]

[0058] Note: F1 and F2 are the differences in fluorescence intensities at two endpoints within time T, and K is the slope of the standard curve.

[0059] 3. Determination of immunoglobulin (IgG) concentration:

[0060] Use an Elisa kit (Cat.NO.7520; Alpha Diagnostic, USA) to determine the IgG concentration in goat buttermilk whey. Dilute goat buttermilk whey 1000 - 5000 times for IgG determination, and fit and quantify it using the parametric equation of the standard curve.

[0061] 4. Determination of lactoferrin concentration:

[0062] Use liquid chromatography - mass spectrometry to determine the lactoferrin concentration in goat buttermilk whey. After enzymolysis of goat buttermilk whey protein, add internal standard peptide segments for on-machine detection, and calculate the lactoferrin concentration using the internal standard peptide concentration.

[0063] 5. MFGM lipid analysis:

[0064] The determination of fat content refers to the alkaline hydrolysis method in the national standard GB5009.6-2016 "Determination of Fat in Foods". The determination of phospholipid content uses high performance liquid chromatography combined with evaporative light scattering detector (HPLC-ELSD). The chromatographic column used is ZorbaxRx-SIL silica gel column. Single standards and mixed standards with different concentration gradients of PE, PI, PS, PC and SM are injected respectively to confirm the retention time of each type of phospholipid and complete further quantification. The determination of ganglioside content uses ultra-high performance liquid chromatography combined with time-of-flight mass spectrometry detector (UPLC-Q-TOF MS). The chromatographic column used is Aquity BEH C18. Single standards and mixed standards with different concentration gradients of GM3 and GD3 are injected respectively to confirm the retention time of each type of ganglioside and complete further quantification. The determination of cholesterol content uses gas chromatography-mass spectrometry (GC-MS). The chromatographic column used is DB-5. Cholesterol standard is injected to confirm its retention time and complete further quantification.

[0065] 6. The definitions of MFGM protein and phospholipid extraction rates are as follows:

[0066] The MFGM protein extraction rate is the ratio of the MFGM protein band intensity in the buttermilk whey rich in MFGM protein to that in the buttermilk.

[0067] The phospholipid extraction rate is the ratio of the phospholipid content in the buttermilk whey rich in phospholipids to that in the buttermilk.

[0068] The average enzyme activity of the rennet used in the examples is 2200 IMCU / g, purchased from Chr. Hansen (Beijing) Trading Co., Ltd.; the addition method of rennet is the solution prepared from rennet powder; the solvent of the solution is deionized water, and the concentration of the solution is 5 g / kg.

[0069] Unless otherwise specified, "%" involved in the examples all refers to mass percentage.

[0070] Separation and preparation of buttermilk in Example 1: Influence of different ripening temperatures and ripening times of cream

[0071] (1) Regulation of cream ripening temperature:

[0072] Take fresh goat milk, heat it to 40 °C, and degrease it by a disc centrifuge to obtain cream with a fat content of 40%; heat the cream to 72 °C and sterilize it for 15 s to obtain pasteurized cream; let the pasteurized cream stand at 4, 8, 12, 16, 20 °C respectively and ripen for 12 h; then use a cream separator to break the emulsion until buttermilk is obtained, and collect it after filtering with double-layer filter cloth.

[0073] Analyze the relative contents of MFGM protein and phospholipids in the buttermilk (relative to the content at the ripening temperature of 4 °C), and the results are shown in Table 1.

[0074] Analysis results of the ripening temperature of cream in Example 1 in Table 1

[0075]

[0076] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content corresponding to the ripening temperature of 4°C.

[0077] It can be seen from Table 1 that as the ripening temperature of the cream increases, the relative contents of MFGM protein and phospholipids in the buttermilk both show a trend of first increasing and then decreasing, reaching the maximum value at 12°C. The ripening temperature mainly affects the aggregation process of fat globules by influencing the solid fat content. A lower ripening temperature will result in a higher solid fat content of the fat globules, lacking sufficient liquid fat, which is not conducive to full demulsification and leads to a decrease in the MFGM content in the buttermilk; a higher ripening temperature will make most of the fat in a liquid state, and some fat globules do not crystallize sufficiently, which is not conducive to whipping and demulsification. Therefore, both lower and higher temperatures are not conducive to the ripening process of the cream. The contents of MFGM protein and phospholipids in the buttermilk obtained by ripening at 12°C are the highest, and 12°C can be preferably selected as the ripening temperature of the cream.

[0078] (2) Regulation of the ripening time of cream:

[0079] Take fresh goat milk, heat it to 40°C, and skim it through a disc centrifuge to obtain cream with a fat content of 40%; heat the cream to 72°C and sterilize it for 15 s to obtain pasteurized cream; let the pasteurized cream stand at 12°C and ripen for 2, 4, 8, 12, and 24 h respectively; then use a butter churn to whip and demulsify until buttermilk is obtained, and collect it after filtering with double-layer filter cloth.

[0080] Analyze the relative contents of MFGM protein and phospholipids in the buttermilk (relative to the content at a ripening time of 2 h), and the results are shown in Table 2.

[0081] Table 2 Analysis results of the ripening time of cream in Example 1

[0082]

[0083] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content at a ripening time of 2 h.

[0084] As can be seen from Table 2, as the ripening time of the cream prolonged, the relative contents of MFGM protein and phospholipids in the buttermilk both showed a gradually increasing trend and reached the maximum value within the range of 12 - 24 h. This indicates that the crystallization destabilization of fat globules is a dynamic process that requires a certain equilibration time. With the prolongation of the ripening time, the protein and phospholipid contents of MFGM gradually increased from 2 - 12 h and leveled off from 12 - 24 h. These results suggest that the cream ripening reaches equilibrium around 12 h, thereby fully destabilizing the fat globules to increase the enrichment rate of the MFGM component in the buttermilk. Therefore, a ripening time of 12 - 24 h can be preferably selected for the cream, and most preferably 12 h.

[0085] Therefore, the optimal conditions for buttermilk preparation obtained are as follows: the ripening temperature of the cream is 12°C, the ripening time is 12 h, and the optimal buttermilk preparation conditions are applied to the subsequent examples.

[0086] Regulation of the curdling step in the preparation of buttermilk whey in Example 2: Effects of rennet concentration, curdling temperature, and curdling time

[0087] (1) Regulation of rennet concentration:

[0088] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, and add 0.05, 0.2, 0.5, 1, 2 g / 100 kg of rennet respectively, and react for 60 min to obtain the curdled buttermilk;

[0089] Centrifuge at 1500 g for 5 min to collect the upper-layer buttermilk whey containing milk fat globule membranes.

[0090] Analyze the MFGM protein, phospholipid extraction rate, and casein residue rate of the buttermilk whey, and the results are shown in Table 3.

[0091] Table 3 Analysis results of rennet concentration regulation in Example 2

[0092]

[0093] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05).

[0094] It can be seen from Table 3 that as the concentration of chymosin increases, the extraction rates of MFGM protein and phospholipids in buttermilk whey both gradually decrease, and the residual rate of casein also gradually decreases. Chymosin can specifically hydrolyze κ-casein that is stably located on the surface of casein micelles. The higher the concentration of chymosin, the faster the hydrolysis rate of κ-casein, thereby destabilizing casein. In the whey obtained at chymosin concentrations of 0.05 g / 100 kg and 0.2 g / 100 kg, the extraction rates of MFGM protein and phospholipids are high, but a large amount of casein still remains in the whey. Compared with 0.2 g / 100 kg, in the whey obtained at 0.5 g / 100 kg, the extraction rates of MFGM protein and phospholipids decrease, but a large amount of casein is removed. As the enzyme concentration continues to increase, there is no obvious change in the extraction rate of MFGM protein and the residual rate of casein, and the extraction rate of phospholipids decreases significantly at 2 g / 100 kg. Therefore, a chymosin concentration condition of 0.5 - 2 g / 100 kg can be preferably used for obtaining buttermilk whey, and the most preferred is 0.5 g / 100 kg.

[0095] (2) Regulation of curdling temperature:

[0096] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 30, 35, 40, 45, 50, and 55 °C respectively, add 0.5 g / 100 kg of chymosin, and react for 60 min to complete chymosin curdling; centrifuge at 1500 g for 5 min to collect the upper-layer buttermilk whey containing milk fat globule membrane.

[0097] Analyze the extraction rates of MFGM protein, phospholipids and the residual rate of casein in the buttermilk whey, and the results are shown in Table 4.

[0098] Table 4 Analysis results of curdling temperature regulation in Example 2

[0099]

[0100] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05).

[0101] It can be seen from Table 4 that: as the curdling temperature increases, there is no significant difference in the extraction rate of MFGM protein and the residual rate of casein in buttermilk whey between 30 - 35 °C. The extraction rate of phospholipids increases slightly numerically and there is also no significant difference. Between 35 - 40 °C, the extraction rate of MFGM protein increases slightly, the extraction rate of phospholipids increases significantly, but the residual rate of casein also increases significantly. Between 40 - 50 °C, the extraction rate of MFGM protein and the residual rate of casein do not change significantly, and the extraction rate of phospholipids increases slightly. The increase in the extraction rate of phospholipids with the increase in temperature may be due to two reasons. On the one hand, some phospholipids bind to casein. After increasing the temperature, the residual rate of casein increases, so the extraction rate of phospholipids also increases. On the other hand, as the temperature increases, the molecular movement accelerates, making the diffusion and dissolution rate of phospholipids from the precipitate faster. In the whey obtained at 55 °C, the extraction rates of MFGM protein, phospholipids and the residual rate of casein all increase significantly. It may be that the temperature affects the activity of chymosin. The increase in temperature gradually reduces the enzyme activity and severely inactivates it at 55 °C, resulting in a weakened ability to hydrolyze κ-casein, and a large amount of casein remains in the whey. Therefore, 30 - 35 °C can be preferably selected as the curdling temperature condition for obtaining buttermilk whey, and 35 °C is most preferred.

[0102] (3) Regulation of curdling time:

[0103] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35 °C, add 0.5 g / 100 kg of chymosin, and react for 20, 40, 60, and 80 min respectively to complete chymosin curdling; centrifuge at 1500 g for 5 min to collect the upper-layer buttermilk whey containing milk fat globule membrane.

[0104] Analyze the extraction rates of MFGM protein, phospholipids and the residual rate of casein in the buttermilk whey, and the results are shown in Table 5.

[0105] Table 5 Analysis results of curdling time regulation in Example 2

[0106]

[0107] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05).

[0108] It can be seen from Table 5 that as the curdling time extends, the extraction rates of MFGM protein and phospholipids and the residual rate of casein in buttermilk whey gradually decrease. In the whey after 20 minutes of curdling, κ-casein is being hydrolyzed, and a large number of stable casein micelles are still contained in the whey; subsequently, when the time is extended to 40 minutes, there is still partially incompletely hydrolyzed κ-casein, resulting in too high a retention of casein in the whey; in the whey at 60 minutes of curdling, the residual rate of casein significantly decreases, and the MFGM protein and phospholipids in the whey can be well retained; there is no significant difference in the MFGM extraction rate and the residual rate of casein in the whey at 60 minutes and 80 minutes of curdling. Therefore, 60 - 80 minutes can be preferably selected as the curdling equilibrium time condition for obtaining buttermilk whey, and 60 minutes is most preferred.

[0109] Regulation of the curdling step in Comparative Example 1: Influence of calcium ion concentration

[0110] Adding calcium ions during curdling:

[0111] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add calcium ions (calcium chloride solution at 0.2 g / mL) to make its final mass concentration 0.02% of the buttermilk, and then add 0.5 g / 100 kg of rennet, and react for 60 minutes to complete rennet curdling; centrifuge at 1500 g for 5 minutes to collect the upper-layer buttermilk whey containing milk fat globule membrane.

[0112] Analyze the MFGM protein, phospholipid extraction rate and casein residual rate of the buttermilk whey in the above Example 2 (under the optimal conditions in part (3)) and Comparative Example 1, and the results are shown in Table 6.

[0113] Table 6 Analysis results of calcium ion concentration regulation in Example 2 and Comparative Example 1

[0114]

[0115] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05).

[0116] It can be seen from Table 6 that the concentration of calcium ions in the system has a great influence on the recovery rate of MFGM components in buttermilk whey. After adding 0.02% calcium ions in the rennet curdling step, the extraction rates of MFGM protein and phospholipids in buttermilk whey significantly decrease, but the residual rate of casein also significantly decreases. This indicates that calcium ions can make the curd structure formed by casein denser and intercept more MFGM components in the gel network, resulting in a decrease in the extraction rates of MFGM protein and phospholipids.

[0117] Example 3 Regulation of the Whey Draining Step in the Preparation of Buttermilk Whey: Effects of Centrifugal Force, Whey Draining Temperature, Whey Draining pH Value, Sodium Citrate Concentration during Whey Draining, and Number of Washings

[0118] (1) Regulation of centrifugal force:

[0119] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 0.5 g / 100 kg of rennet, and react for 60 min to obtain the coagulated buttermilk;

[0120] Then centrifuge at 500, 1500, 3000, 6000, and 10000 g for 5 min respectively to collect the buttermilk whey containing milk fat globule membrane in the upper layer.

[0121] Analyze the extraction rates of MFGM protein, phospholipids, and the residual rate of casein in the buttermilk whey, and the results are shown in Table 7.

[0122] Table 7 Analysis Results of the Regulation of Centrifugal Force in Example 3

[0123]

[0124] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P < 0.05).

[0125] It can be seen from Table 7 that as the centrifugal force increases, the extraction rates of both MFGM protein and phospholipids in the buttermilk whey gradually decrease, and the residual rate of casein also gradually decreases. On the one hand, it is due to the action of centrifugal force, and on the other hand, it may be that casein binds to some MFGM fragments during precipitation, resulting in their co-precipitation. In the whey centrifuged at 500 g, the extraction rate of MFGM protein accounts for 72.91% of the original buttermilk, and 36.77% of casein remains. In the whey centrifuged at 10000 g, casein can be well removed, but due to the large centrifugal force, the extraction rate of MFGM protein drops to 48.34%, and most of it is lost in the casein precipitate. Compared with 500 g, the whey centrifuged at 1500 g can further reduce the residual rate of casein (16.96%), and the extraction rate of MFGM protein is higher than that at 6000 g (56.28% > 51.41%). Therefore, 1500 g can be selected as the optimal condition for MFGM protein recovery. As the centrifugal force increases, the extraction rate of phospholipids in the whey decreases significantly between 500 g - 1500 g and 6000 g - 10000 g. Compared with 3000 g, the whey centrifuged at 1500 g can retain more phospholipid components. Therefore, 1500 g can be preferably selected as the centrifugation condition for obtaining buttermilk whey.

[0126] (2) Regulation of whey draining temperature:

[0127] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 0.5g / 100kg rennet, and react for 60min to complete rennet coagulation;

[0128] The buttermilk after the coagulation step was cut and stirred, and the temperature of the whey was adjusted to 35, 40, 45, 50, and 55°C, and centrifuged at 1500g for 5 minutes to collect the upper layer of buttermilk whey containing milk fat globule membranes.

[0129] The MFGM protein, phospholipid extraction rate and casein residual rate of buttermilk whey were analyzed. The results are shown in Table 8.

[0130] Table 8 Analysis results of whey temperature control in Example 3

[0131]

[0132] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P<0.05).

[0133] It can be seen from Table 8 that as the whey discharge temperature increases, the MFGM protein extraction rate decreases slightly, the phospholipid extraction rate gradually increases, and the casein residual rate hardly changes. This may be because the casein particles shrink at a higher temperature, causing part of the MFGM protein to be wrapped in the casein. As the temperature increases, the diffusion and dissolution rate of phospholipids from the precipitate accelerates, which increases the phospholipid content in the whey. Therefore, in order to increase the phospholipid extraction rate in buttermilk whey and avoid the adverse effects of excessively high temperatures on active proteins, 45-50°C can be preferably used as the whey discharge temperature condition for obtaining buttermilk whey, and 50°C is most preferred.

[0134] (3) Regulation of whey pH value:

[0135] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 0.5g / 100kg rennet, and react for 60min to complete rennet coagulation;

[0136] The buttermilk after the coagulation step was cut and stirred, and then the pH value was adjusted to 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, and slowly stirred for 30 minutes, while the temperature of the whey was raised to 50°C; centrifuged at 1500g for 5 minutes to collect the upper layer of buttermilk whey containing milk fat globule membrane.

[0137] The MFGM protein, phospholipid extraction rate and casein residual rate of buttermilk whey were analyzed. The results are shown in Table 9.

[0138] Table 9 Analysis results of whey pH control in Example 3

[0139]

[0140] Note: Different lowercase letters in the same column indicate significant differences between corresponding data (P<0.05).

[0141] As can be seen from Table 9: With the increase of the pH value of the drained whey, the extraction rates of MFGM protein and phospholipids gradually increase. The residual rate of casein shows no obvious change between 6.0 - 6.5, significantly increases between 7.0 - 7.5, then shows no obvious change between 7.5 - 8.0, and the casein band significantly increases at 8.5. This may be because the increase in pH value makes the system far from the isoelectric points of MFGM and casein. After chymosin hydrolyzes κ-casein, calcium ions in the whey will bind to phosphorylated casein (α s1 、α s2 and β-casein) inside the casein micelles through charge attraction, and the remaining free calcium ions will combine with negatively charged phospholipids to form precipitates. The increase in pH value will increase the negative charges in the system, thereby chelating the calcium ions bound to casein and free calcium ions, making the structure of the precipitated casein become loose and releasing MFGM protein and phospholipids. The increase in pH value not only moves away from the isoelectric points of MFGM protein and casein, but also reduces the calcium ions in the system, improving the extraction rate of MFGM in the whey. Compared with a drained whey pH value of 7.0, the casein content slightly increases at 7.5 (from 15.42% to 16.79%), but significantly improves the extraction rate of MFGM in the whey. Therefore, 7.5 - 8.0 can be preferably used as the drained whey pH condition for obtaining casein whey, and 7.5 is the most preferred to avoid the adverse effects caused by too high pH.

[0142] (4) Regulation of sodium citrate concentration during drained whey:

[0143] Take the casein milk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 0.5 g / 100 kg of chymosin, and react for 60 min to complete chymosin coagulation of milk;

[0144] Cut and stir the casein milk after the coagulation step, add 0, 1.5, 3, 4.5, 6, 7.5, 9.0 mM of sodium citrate respectively, then adjust the pH value to 7.5 and slowly stir for 50 min, and at the same time raise the drained whey temperature to 50°C; Centrifuge at 1500 g for 5 min to collect the upper layer of casein whey containing milk fat globule membrane.

[0145] Analyze the extraction rates of MFGM protein, phospholipids and the residual rate of casein in the casein whey, and the results are shown in Table 10.

[0146] Table 10 Analysis results of the regulation of sodium citrate concentration during drained whey in Example 3

[0147]

[0148] Note: Different lowercase letters in the same column indicate significant differences between corresponding data (P < 0.05).

[0149] It can be seen from Table 10 that as the concentration of sodium citrate increases during whey drainage, the extraction rates of MFGM protein and phospholipids gradually increase, and the residual rate of casein also gradually increases. There is no significant difference in the extraction rates of MFGM protein and phospholipids at 0 - 1.5 mM, and then they gradually increase. When it is 1.5 - 3 mM, the extraction rate of MFGM protein increases from 64.43% to 69.09%, and the extraction rate of phospholipids increases from 78.95% to 80.60%; there is no significant difference in the residual rate of casein at 0 - 3 mM, and then it also gradually increases. When it is 3 - 4.5 mM, the residual rate of casein increases from 17.49% to 19.61%. This indicates that adding sodium citrate after whey drainage can chelate calcium ions in the system, making the already flocculated casein particles looser to release more MFGM protein and phospholipids into the whey. Generally speaking, adding sodium citrate with a concentration of 3 mM during whey drainage can effectively improve the recovery rates of MFGM protein and phospholipids. Therefore, 1.5 - 4.5 mM can be preferably used as the concentration of sodium citrate during whey drainage, and the optimal is 3 mM.

[0150] (5) Regulation of the washing times of casein precipitate after whey drainage:

[0151] Take the casein milk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 0.5 g / 100 kg of rennet, and react for 60 min to complete rennet coagulation of casein milk.

[0152] Cut and stir the casein milk after the coagulation step, add 3 mM of sodium citrate, then adjust the pH value to 7.5 and slowly stir for 50 min, and at the same time raise the whey drainage temperature to 50°C; centrifuge at 1500 g for 5 min to collect the upper layer of casein milk whey containing milk fat globule membrane. After collecting the casein milk whey, add deionized water accounting for 15% of the weight of the raw casein milk to the casein precipitate, stir on a rotary stirrer for 30 min and then centrifuge to collect the supernatant. Repeat the above operation and combine the supernatant with the whey casein milk.

[0153] Analyze the extraction rates of MFGM protein, phospholipids and the residual rate of casein in the combined solution of casein milk whey and supernatant, and the results are shown in Table 11.

[0154] Table 11 Analysis results of the regulation of the washing times of casein precipitate after whey drainage in Example 3

[0155]

[0156] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P<0.05).

[0157] As can be seen from Table 11: In the whey obtained under the optimal conditions for chymosin precipitation of casein, the recovery rates of MFGM protein and phospholipids were 68.90% and 80.57% respectively, and the residual rate of casein was 17.45%. After the first washing liquid was combined, more MFGM protein and phospholipids could still be recovered, while the residual rate of casein did not increase significantly. After the second washing liquid was combined, the extraction rates of MFGM protein and phospholipids did not change significantly compared with the first time, while the residual rate of casein increased significantly. This indicates that the first washing liquid contains a large amount of MFGM components. Therefore, in actual production, washing the casein precipitate can be considered to recover MFGM protein and phospholipids, and the preferred number of washing times is 1 time.

[0158] Regulation of the whey drainage step in Comparative Example 2: Influence of the sodium citrate addition process point

[0159] Add sodium citrate to chelate calcium ions during curdling:

[0160] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 35°C, add 3 mM citrate, then add 0.5 g / 100 kg of chymosin, and react for 60 min to complete chymosin curdling; cut and stir the buttermilk after the curdling step, then adjust the pH value to 7.5 and stir slowly for 50 min, while raising the whey drainage temperature to 50°C; centrifuge at 1500 g for 5 min to collect the upper layer of buttermilk whey containing milk fat globule membrane.

[0161] Analyze the MFGM protein, phospholipid extraction rate and casein residual rate of the buttermilk whey in Example 3 above (adjust the sodium citrate concentration to 3 mM during whey drainage in part (4) of Example 3, and keep other steps the same as in part (4) of Example 3) and Comparative Example 2. The results are shown in Table 12.

[0162] Table 12 Analysis results of the regulation of the sodium citrate addition process point in Example 3 and Comparative Example 2

[0163]

[0164] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P<0.05).

[0165] As can be seen from Table 12: Compared with adding sodium citrate to chelate calcium ions during curdling, adding sodium citrate during whey drainage can better remove casein and further improve the extraction rates of MFGM protein and phospholipids in buttermilk whey. This is because a decrease in the initial calcium content makes it difficult to remove casein, while a decrease in calcium content during whey drainage can make the already flocculated casein particles more porous, thus increasing the recovery of MFGM protein and phospholipids in buttermilk whey under the condition of low casein residue. Therefore, the method of adding sodium citrate to chelate calcium ions during whey drainage can be preferably adopted.

[0166] Comparative Example 3 Preparation of buttermilk whey by acid precipitation method

[0167] Take the buttermilk prepared under the optimal conditions in Example 1, keep its temperature at 50 °C, and adjust the pH of the buttermilk to 5.0 using 3M hydrochloric acid solution; centrifuge at 1500 g for 5 min to collect the upper-layer buttermilk whey containing milk fat globule membrane.

[0168] Comparative Example 4 Preparation of whey buttermilk from cheese whey

[0169] Heat fresh milk to 72 °C and sterilize for 15 s; after cooling the temperature to 30 - 32 °C, add 0.02% calcium chloride and 3.5 g / 100 kg starter, and ferment for 60 min; then add 0.9 g / 100 kg rennet and curdle for 30 min;

[0170] After curdling is completed, cut and stir the cheese for 5 min, raise the whey drainage temperature to 39 °C, and drain whey for 30 min; squeeze the cheese to drain the excess whey and collect it using cheesecloth, heat the whey temperature to 72 °C, and sterilize for 15 s;

[0171] Immediately cool to 4 °C, centrifuge at 5000 g for 10 min, and collect the upper-layer whey cream and the lower-layer skimmed whey;

[0172] Compound the whey cream and skimmed whey to a fat content of 40% to obtain whey cream;

[0173] After stirring and mixing the whey cream evenly, let it stand and ripen at 12 °C for 12 h, break the emulsion using a stirrer, and collect the whey buttermilk containing milk fat globule membrane using double-layer filter cloth.

[0174] For the above two milk fat globule membrane separation schemes, analyze the milk fat globule membrane components in Example 3 (under the optimal conditions in part (4)) and Comparative Examples 3 and 4, and the results are shown in Table 13.

[0175] Table 13 Analysis results of milk fat globule membrane components in Example 3 and Comparative Examples 3 and 4

[0176]

[0177] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P<0.05); the relative content refers to the percentage relative to the content corresponding to Example 3.

[0178] It can be seen from Table 13 that the relative content of MFGM protein, XO activity, immunoglobulin concentration, lactoferrin concentration, phospholipid content, ganglioside content, and cholesterol content in Example 3 are all higher than those in Comparative Examples 3 and 4; the immunoglobulin concentration in Comparative Example 3 is higher than that in Comparative Example 4, while the other indicators are lower than those in Comparative Example 4; the MFGM obtained in Comparative Example 4 is derived from cheese whey, similar to Example 3, both removing casein by the method of chymosin coagulation of milk, different from the acid precipitation method of removing casein in Comparative Example 3. The chymosin precipitation method completely hydrolyzes κ-casein under the optimal conditions, and then makes the casein precipitate by affecting the calcium ion environment and hydrophobic interaction in the whey. In Example 3, sodium citrate was added during the whey drainage, reducing the coagulation strength of casein and promoting the release of MFGM in the whey. The acid precipitation method of Comparative Example 3 gradually approaches the isoelectric point of casein, reducing the ionic strength in the whey, and relying on hydrophobic interaction to precipitate casein. However, MFGM is relatively sensitive to the ionic environment, so the acid precipitation method will cause a large loss of MFGM protein.

[0179] Preparation of buttermilk whey in Example 4: Influence of ultrasonic power

[0180] Take the buttermilk separated and prepared in Example 1, keep its temperature at 35°C, add 0.5 g / 100 kg of chymosin, and react for 60 min to complete chymosin coagulation of milk.

[0181] Cut and stir the buttermilk after the coagulation step, add 3 mM of sodium citrate, then adjust the pH value to 7.5 and stir slowly for 50 min, while raising the whey drainage temperature to 50°C, and then ultrasonicate for 15 min under the conditions of power of 0, 100, 200, 300, 400, and 500 W respectively; centrifuge at 1500 g for 5 min to collect the upper-layer buttermilk whey containing milk fat globule membrane.

[0182] Analyze the extraction rates of MFGM protein and phospholipids and the residual rate of casein in the buttermilk whey, and the results are shown in Table 14.

[0183] Table 14 Analysis results of ultrasonic power regulation during whey drainage in Example 4

[0184]

[0185] Note: Different lowercase letters in the same column indicate significant differences between the corresponding data (P<0.05).

[0186] As can be seen from Table 14, with the increase of ultrasonic power, the extraction rates of MFGM protein and phospholipids in buttermilk whey showed a trend of first increasing and then decreasing. The MFGM protein and phospholipids reached the maximum values at 300 W, which were 78.26% and 83.12% respectively. The residual rate of casein in buttermilk whey gradually increased numerically with the increase of ultrasonic power, but there was no significant difference. This indicates that weak ultrasound (100 - 300 W) can affect the network structure of casein, enabling some MFGM protein and phospholipids trapped in casein precipitation to be recovered in whey. When the ultrasonic power increased from 300 W to 500 W, the extraction rates of MFGM protein and phospholipids decreased, which may be due to the damage of the functional groups of the MFGM component caused by excessive ultrasonic intensity. Therefore, 200 - 300 W can be preferably selected as the ultrasonic power for discharging whey, and 300 W is the most preferred.

[0187] Example 5 Separation and preparation of milk fat globule membrane whey powder: Effects of membrane pore size, concentration factor and washing and filtration times

[0188] (1) Regulation of membrane pore size:

[0189] Separate and prepare buttermilk whey in the manner of Example 3 (under the optimal conditions of part (4)). Concentrate the buttermilk whey in polysulfone membranes with membrane pore sizes of 5, 10, 20, 50, and 100 kDa. The concentration factor is 4 times, the temperature of the ultrafiltered material is 45 °C, and the pH is 7.5. After vacuum freeze-drying the concentrated buttermilk whey, milk fat globule membrane whey powder is obtained.

[0190] At each membrane pore size, collect the permeate, calculate the membrane flux, and collect the concentrate. Dilute it 4 times with deionized water and measure the protein and phospholipid composition of the solution. The results are shown in Table 15.

[0191] Table 15 Analysis results of membrane pore size regulation in Example 5

[0192]

[0193] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content in buttermilk whey.

[0194] As can be seen from Table 15, with the increase of membrane pore size, the membrane flux becomes larger and larger, indicating that the filtration efficiency has been improved, and the MFGM protein and phospholipids are completely retained. However, when the membrane pore size is 100 kDa, some whey proteins pass through the ultrafiltration membrane and enter the permeate, resulting in a certain material loss. Therefore, 10 - 50 kDa can be preferably selected as the condition for the ultrafiltration membrane pore size, and 50 kDa is the most preferred.

[0195] (2) Regulation of concentration factor:

[0196] The buttermilk whey was separated and prepared in the manner of Example 3 (under the optimal conditions of part (4)), and the buttermilk whey was concentrated in a polysulfone membrane with a pore size of 50 kDa to 2, 3, 4, and 5 times respectively. The temperature of the ultrafiltered material was 45 °C and the pH was 7.5. After the concentrated buttermilk whey was vacuum freeze-dried, milk fat globule membrane whey powder was obtained.

[0197] At each concentration multiple, the permeate was collected and the membrane flux was calculated. The results are shown in Table 16.

[0198] Table 16 Analysis results of concentration multiple regulation in Example 5

[0199]

[0200] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05).

[0201] It can be seen from Table 16 that as the concentration multiple increases, the membrane flux continuously decreases; when the concentration multiple increases to 3 and 4 times, the membrane flux slightly decreases; when the concentration multiple increases to 5 times, the membrane flux significantly decreases, indicating that the concentration multiple of 5 reaches the membrane separation limit concentration. Therefore, considering the separation efficiency, 3 - 4 times can be preferably used as the condition of the concentration multiple, and the most preferred is 4 times.

[0202] (3) Regulation of washing and filtration times:

[0203] The buttermilk whey was separated and prepared in the manner of Example 3 (under the optimal conditions of part (4)), and the buttermilk whey was concentrated in a polysulfone membrane with a pore size of 50 kDa by 4 times. The temperature of the ultrafiltered material was 45 °C and the pH was 7.5. It was washed and filtered 0, 1, 2, and 3 times respectively; then the concentrated buttermilk whey was vacuum freeze-dried to obtain milk fat globule membrane whey powder.

[0204] The concentrated solution after washing and filtration was collected, diluted 4 times with deionized water, and the lactose content in the solution was measured. The results are shown in Table 17.

[0205] Table 17 Analysis results of washing and filtration times regulation in Example 5

[0206]

[0207] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05).

[0208] It can be seen from Table 17 that as the washing and filtration times increase, the membrane flux gradually increases and the lactose content continuously decreases. The protein and lipid content in the buttermilk whey is about 3%, and for the MFGM ingredients prepared from the buttermilk whey, the lactose content often needs to be lower than 20%; considering both the washing and filtration cost and the lactose removal efficiency, 1 - 2 times is preferably used as the condition of the washing and filtration times.

[0209] Example 6 Separation and Preparation of Milk Fat Globule Membrane Powder: Effects of Membrane Pore Size, Material Temperature, and Material pH

[0210] (1) Regulation of membrane pore size:

[0211] Separate and prepare buttermilk whey in the manner of Example 3 (under the optimal conditions of part (4)). Concentrate the buttermilk whey in ceramic membranes with pore sizes of 10, 20, 50, 100, and 200 nm. The concentration factor is 4 times. The material temperature for microfiltration is 45 °C, and the pH is 7.5. Subsequently, vacuum freeze-dry the concentrated buttermilk whey to obtain milk fat globule membrane powder rich in milk fat globule membrane.

[0212] At each membrane pore size, collect the permeate, calculate the membrane flux, and collect the concentrate. Dilute it 4 times with deionized water and measure the protein and phospholipid composition of the solution. The results are shown in Table 18.

[0213] Table 18 Analysis Results of Different Membrane Pore Sizes in Example 6

[0214]

[0215] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content in buttermilk whey.

[0216] It can be seen from Table 18 that when the membrane pore size is 10 and 20 nm, MFGM protein and phospholipids are completely retained; when the membrane pore size is 50 and 100 nm, a small part of MFGM protein and phospholipids permeate; when the membrane pore size is 200 nm, the permeation amount of MFGM protein and phospholipids increases significantly. As the membrane pore size increases, the membrane flux gradually increases. Especially when the membrane pore size increases to 50 nm, the membrane flux increases significantly. Therefore, 50 - 100 nm can be preferably used as the condition for the microfiltration membrane pore size.

[0217] (2) Regulation of material temperature:

[0218] Separate and prepare buttermilk whey in the manner of Example 3 (under the optimal conditions of part (4)). Subsequently, use a ceramic membrane with a pore size of 100 nm and conduct microfiltration concentration at 25, 35, 45, and 55 °C respectively. The concentration factor is 4 times, and the pH is 7.5. Vacuum freeze-dry the concentrated buttermilk whey to obtain milk fat globule membrane powder rich in milk fat globule membrane.

[0219] At each material temperature, collect the permeate, calculate the membrane flux, and collect the concentrate. Dilute it 4 times with deionized water and measure the protein and phospholipid composition of the solution. The results are shown in Table 19.

[0220] Table 19 Analysis Results of Material Temperature Regulation in Example 6

[0221]

[0222] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content in buttermilk whey.

[0223] It can be seen from Table 19 that as the microfiltration temperature increases from 25 °C to 55 °C, the membrane flux gradually increases; however, the relative content of MFGM protein and the phospholipid content continuously decrease. When the material temperature is 25 - 35 °C, there is no significant difference in the relative content of MFGM protein and the phospholipid content in buttermilk whey, but the membrane flux is higher at 35 °C. Therefore, 35 °C can be preferably selected as the condition of the microfiltration temperature.

[0224] (3) Regulation of the material pH:

[0225] Separate and prepare buttermilk whey in the manner of Example 3 (under the optimal conditions of part (4)), and use 3M hydrochloric acid solution to adjust the material pH to 7.5, 7.0, 6.5, 6.0, and 5.5 respectively; then use a ceramic membrane with a pore size of 100 nm, and the microfiltration material temperature is 35 °C. Stop microfiltration when the concentration multiple is 4; after vacuum freeze-drying the concentrated buttermilk whey, obtain milk fat globule membrane powder rich in milk fat globule membrane.

[0226] At each material pH, collect the permeate, calculate the membrane flux, and collect the concentrate, dilute it 4 times with deionized water, and measure the protein and phospholipid composition of the solution. The results are shown in Table 20.

[0227] Table 20 Analysis results of the regulation of the material pH in Example 6

[0228]

[0229] Note: Different lowercase letters in the same row indicate significant differences between the corresponding data (P < 0.05); the relative content refers to the percentage relative to the content in buttermilk whey.

[0230] It can be seen from Table 20 that as the material pH decreases from 7.5 to 6.5, the relative content of MFGM protein and the phospholipid content in the solution continuously increase, and then remain almost unchanged as the pH further decreases. When the material pH drops to 6.0, the membrane flux slightly decreases; when the material pH further decreases to 5.5, the membrane flux significantly decreases. Therefore, 6.0 - 6.5 can be preferably selected as the material pH condition, and the optimal is pH 6.5.

[0231] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for separating and preparing milk fat globule membrane, characterized in that, it comprises the following steps: (1) Separation and preparation of buttermilk: Leave the pasteurized cream to ripen at 4 - 20°C for 2 - 24 h; then stir to break the emulsion until buttermilk is obtained; (2) Coagulation of buttermilk whey: Keep the temperature of the buttermilk in step (1) at 30 - 55°C, add 0.05 - 2 g / 100 kg of rennet, and react for 20 - 80 min to obtain coagulated buttermilk; (3) Whey drainage in the preparation of buttermilk whey: Cut and stir the coagulated buttermilk in step (2), add 1.5 - 4.5 mM of sodium citrate, then adjust the pH value to 6.5 - 8.5 and stir slowly for 30 - 50 min. At the same time, raise the whey drainage temperature to 35 - 55°C, and centrifuge at 500 - 10000 g for 4 - 6 min to collect the upper layer of buttermilk whey containing milk fat globule membrane; (4) Preparation of milk fat globule membrane: Filter the buttermilk whey through an ultrafiltration membrane and dry it to obtain milk fat globule membrane whey powder; filter the buttermilk whey through a microfiltration membrane and dry it to obtain milk fat globule membrane powder; The ultrafiltration membrane filtration is carried out by filtering and concentrating with a membrane with a pore size of 5 - 100 kDa, and the microfiltration membrane filtration is carried out by filtering and concentrating with a membrane of 10 - 200 nm; the temperature of the material for ultrafiltration membrane filtration is 40 - 50°C, the material pH is 6.5 - 7.5, the temperature of the material for microfiltration membrane filtration is 25 - 55°C, the material pH is 5.5 - 7.5; the concentration multiple of ultrafiltration membrane filtration is 2 - 5 times, and the number of washing and filtering times is 1 - 2 times.

2. The method according to claim 1, characterized in that, the ripening condition in step (1) is to ripen at 12°C for 12 - 24 h.

3. The method according to claim 1, characterized in that, the temperature of the buttermilk in step (2) is 30 - 35°C.

4. The method according to claim 1, characterized in that, the concentration of rennet in step (2) is 0.5 - 2 g / 100 kg.

5. The method according to claim 1, characterized in that, the concentration of sodium citrate in step (3) is 3 mM; the pH value is 7.5 - 8.0; the whey drainage temperature is 45 - 50°C.

6. The method according to claim 1, characterized in that, the stirring time in the whey drainage in step (3) is 50 min; and ultrasonic treatment is carried out after heating at the same time; the conditions of ultrasonic treatment are: the ultrasonic power is 100 - 500 W; the ultrasonic time is 10 - 30 min.

7. Milk fat globule membrane whey powder prepared by the method according to any one of claims 1 - 6.

8. Milk fat globule membrane powder prepared by the method according to any one of claims 1 - 6.

9. Application of the milk fat globule membrane whey powder according to claim 7 or the milk fat globule membrane powder according to claim 8 in the food field.

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