Lactoferrin-rich active whey powder as well as preparation and application thereof

Abstract

The invention discloses active whey powder rich in lactoferrin as well as preparation and application of the active whey powder, and belongs to the technical field of preparation of dairy products. The preparation method comprises the following steps: adding sodium chloride into sterilized skimmed milk to release lactoferrin, performing membrane filtration to obtain membrane filtration permeate (lactoferrin-rich whey protein phase), and finally performing ultrafiltration, desalination and spray drying to obtain the lactoferrin-rich whey powder. The lactoferrin-rich whey powder prepared by the preparation method disclosed by the invention is higher in lactoferrin content and higher in activity. In addition, lactoferrin and lactoferrin-free whey protein can be separated from membrane filtration permeate through an ion exchange column, and then high-purity lactoferrin powder and lactoferrin-free whey protein are prepared.

Description

Technical Field

[0001] The invention relates to active whey powder rich in lactoferrin and preparation and application thereof, belonging to the technical field of dairy product preparation. Background Art

[0002] As people's health awareness increases and their living standards improve, the demand for high-quality nutritious food is growing. As an important bioactive protein, lactoferrin has attracted widespread attention due to its wide range of biological functions. Lactoferrin can not only regulate iron metabolism and promote iron absorption, but also has multiple biological activities such as antibacterial, antioxidant, anti-inflammatory and immunomodulatory. These characteristics make lactoferrin have broad application prospects in infant formula, health foods and clinical nutrition.

[0003] However, commercial whey protein products such as WPC and WPI are mainly prepared from whey, a byproduct of cheese processing. However, during the cheese production process, milk is subjected to heat and fermentation processes, and the resulting whey fraction needs to be further heated to inactivate the remaining bacteria. These thermal processing processes will cause the natural structure of whey protein to change and the active ingredients to be destroyed.

[0004] It can be seen that although the whey powder products on the market are rich in protein, the lactoferrin content is generally not high and its activity is difficult to maintain.

[0005] Moreover, casein in milk exists in the form of micelles with a particle size between 50-600nm, while the particle size of whey protein is generally around 2-5nm. Membrane filtration may be a better choice than traditional whey protein to separate whey protein directly from skim milk. However, the biggest problem in membrane filtration of whey protein ingredients is the low content of lactoferrin in whey. Because lactoferrin is a positively charged alkaline protein, it mainly binds to negatively charged casein in milk, resulting in most of the lactoferrin and other alkaline proteins being retained in the casein phase when separating whey protein.

[0006] Therefore, it is of great significance to develop an active whey powder that is rich in lactoferrin and maintains its biological activity to meet the market demand for high-quality nutritional foods.

[0007] Lactoferrin is a type of iron-bound active glycoprotein with immunological activity and broad-spectrum antibacterial properties. It can be used to promote and balance the immune system and adjust intestinal function in functional foods or beverages. It also has the potential to be used in special medical foods to assist in the treatment of diseases caused by pathogenic microorganisms. With the increasing demand of consumers for nutritional health, lactoferrin has attracted much attention around the world, and the market demand has also shown a significant increase. However, it is currently limited to the online enrichment and application of infant formula milk powder, and there is no commercially available lactoferrin ingredient. Therefore, preparing a high-purity and high-activity lactoferrin powder can solve the problem of lactoferrin ingredients on the one hand, and on the other hand, it can provide a feasibility basis for in-depth research and large-scale production of lactoferrin, and promote its application in the fields of food, medicine and health products.

[0008] In addition, whey protein is rich in amino acids, especially branched-chain amino acids. It is not only a high-quality protein source with high nutritional value, but also plays an important role in promoting muscle growth, regulating blood sugar, improving lipid metabolism, anti-aging, controlling weight, promoting intestinal health, and clinical nutritional support. At the same time, whey protein has emulsifying and thickening effects, and its use in baking and confectionery products is increasing. Whey protein can replace egg white and plays an important role in the production of frozen foods, cakes, biscuits, etc. Therefore, the preparation of highly active whey powder can promote its wide application in the food industry and increase the nutritional value and functionality of food. Summary of the invention

[0009] [Technical issues]

[0010] The existing processing technology of whey powder can hardly retain the activity of functional proteins in whey;

[0011] The active protein in conventional whey powder products is partially denatured and its biological activity is reduced; and the lactoferrin content is low, resulting in a large gap between the prepared whey powder and whey.

[0012] [Technical solution]

[0013] In order to solve the above problems, the present invention provides an active whey powder rich in lactoferrin and its preparation and application; specifically, the present invention releases lactoferrin by adding sodium chloride to sterilized skim milk, then obtains membrane filtration permeate (lactoferrin-rich whey protein) by membrane filtration, and finally performs ultrafiltration, desalination, and spray drying to obtain lactoferrin-rich whey powder. The lactoferrin-rich whey powder prepared by the present invention has a higher lactoferrin content and higher activity.

[0014] Moreover, the present invention further separates lactoferrin and lactoferrin-free whey from lactoferrin-rich active whey through a cation exchange column, and then prepares high-purity lactoferrin powder and lactoferrin-free whey protein after drying, thereby making the application range of the ingredients wider.

[0015] The first object of the present invention is to provide an active whey powder rich in lactoferrin, and the preparation method thereof is as follows:

[0016] (1) Release of lactoferrin:

[0017] Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer;

[0018] Wherein, the concentration of sodium chloride added is 200-400mmol / L;

[0019] (2) Enrichment of lactoferrin:

[0020] The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin);

[0021] Among them, the filtrate used in membrane filtration is sodium chloride solution;

[0022] (3) Sterile concentration:

[0023] The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin;

[0024] (4) Spray drying:

[0025] The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

[0026] Furthermore, in step (1), the concentration of sodium chloride added is 300 mmol / L, the temperature is 37° C., and the pH is 6.6.

[0027] Furthermore, the stirring speed in step (1) is 300-800 rpm.

[0028] Furthermore, in step (1), lactoferrin is transferred from the casein phase to the whey phase.

[0029] Furthermore, in step (2), the membrane pore size used for membrane filtration is 90-110 nm, the temperature is 35-55° C., the concentration multiple is 2-4, and the number of diafiltrations is 3-5; further preferably, the membrane pore size used for membrane filtration is 100 nm, the temperature is 35-55° C., the concentration multiple is 3, and the number of diafiltrations is 4.

[0030] Furthermore, the operational endpoint of the membrane filtration in step (2) is determined by "the liquid volume in the membrane filtration retentate becomes one third of the initial volume".

[0031] Furthermore, the sodium chloride solution in step (2) is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

[0032] Furthermore, after each filtration in the membrane filtration of step (2) reaches the end point of the operation, the filtrate is backfilled to the initial volume and filtered again.

[0033] Furthermore, in step (3), the ultrafiltration membrane used for ultrafiltration has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C; further preferably, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; and the ultrafiltration temperature is 40°C.

[0034] Furthermore, in step (3), the desalination is carried out by using ultrapure water as the second ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After the ultrafiltration is completed, electrodialysis is performed for further desalination; the electrodialysis adopts a working current of 45A, a feed flow rate of 110mL / min, and a feed temperature of 35°C; the end point of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the permeate.

[0035] Furthermore, the spray drying parameters in step (4) are: material inlet temperature is 60-80°C, and material outlet temperature is 52-55°C.

[0036] Furthermore, the active whey powder rich in lactoferrin is stored in a sealed state at room temperature after processing.

[0037] The second object of the present invention is to use the active whey powder rich in lactoferrin in the field of infant formula milk powder, health food and clinical nutrition products.

[0038] The third object of the present invention is to provide a method for preparing active whey powder rich in lactoferrin, comprising the following steps:

[0039] (1) Release of lactoferrin:

[0040] Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer;

[0041] Wherein, the concentration of sodium chloride added is 200-400mmol / L;

[0042] (2) Enrichment of lactoferrin:

[0043] The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin);

[0044] Among them, the filtrate used in membrane filtration is sodium chloride solution;

[0045] (3) Sterile concentration:

[0046] The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin;

[0047] (4) Spray drying:

[0048] The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

[0049] The fourth object of the present invention is to provide a method for increasing the lactoferrin content in whey powder, comprising the following steps:

[0050] (1) Release of lactoferrin:

[0051] Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer;

[0052] Wherein, the concentration of sodium chloride added is 200-400mmol / L;

[0053] (2) Enrichment of lactoferrin:

[0054] The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (whey protein rich in lactoferrin);

[0055] Among them, the filtrate used in membrane filtration is sodium chloride solution;

[0056] (3) Sterile concentration:

[0057] The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin;

[0058] (4) Spray drying:

[0059] The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

[0060] A fifth object of the present invention is to provide a method for simultaneously preparing lactoferrin powder and whey powder without lactoferrin, comprising the following steps:

[0061] (1) Release of lactoferrin:

[0062] Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer;

[0063] Wherein, the concentration of sodium chloride added is 200-400mmol / L;

[0064] (2) Enrichment of lactoferrin:

[0065] The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin);

[0066] Among them, the filtrate used in membrane filtration is sodium chloride solution;

[0067] (3) Sterile concentration:

[0068] The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin;

[0069] (4) Further enriching lactoferrin and collecting whey without lactoferrin:

[0070] Passing the aseptically concentrated active whey rich in lactoferrin through a cation exchange column to transfer lactoferrin from the whey to the cation exchange column to obtain whey without lactoferrin; then eluting the lactoferrin on the cation exchange column with an aqueous solution of sodium chloride to remove salt and obtain lactoferrin;

[0071] (5) Drying:

[0072] The lactoferrin is freeze-dried to obtain lactoferrin powder; the whey without lactoferrin is spray-dried to obtain whey powder without lactoferrin.

[0073] Furthermore, in step (1), the concentration of sodium chloride added is 300 mmol / L, the temperature is 37° C., and the pH is 6.6.

[0074] Furthermore, the stirring speed in step (1) is 300-800 rpm.

[0075] Furthermore, in step (1), lactoferrin is transferred from the casein phase to the whey phase.

[0076] Furthermore, in step (2), the membrane pore size used for membrane filtration is 90-110 nm, the temperature is 35-55° C., the concentration multiple is 2-4, and the number of diafiltrations is 3-5; further preferably, the membrane pore size used for membrane filtration is 100 nm, the temperature is 35-55° C., the concentration multiple is 3, and the number of diafiltrations is 4.

[0077] Furthermore, the operational endpoint of the membrane filtration in step (2) is determined by "the liquid volume in the membrane filtration retentate becomes one third of the initial volume".

[0078] Furthermore, the sodium chloride solution in step (2) is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

[0079] Furthermore, after each filtration in the membrane filtration of step (2) reaches the end point of the operation, the filtrate is backfilled to the initial volume and filtered again.

[0080] Furthermore, in step (3), the ultrafiltration membrane used for ultrafiltration has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C; further preferably, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; and the ultrafiltration temperature is 40°C.

[0081] Furthermore, in step (3), the desalination is carried out by using ultrapure water as the second ultrafiltration solution, and the amount of ultrafiltration solution used is 5-10 times that of the concentrated permeate. After the ultrafiltration is completed, electrodialysis is performed for further desalination; the electrodialysis adopts a working current of 45A, a feed flow rate of 110mL / min, and a feed temperature of 35°C; the end point of the desalination operation is determined by "the conductivity value is reduced to the normal permeate conductivity range", and the protein concentration is determined by the Kjeldahl method to determine the total protein concentration in the permeate.

[0082] Furthermore, the filler of the cation exchange column in step (4) is CM-Sepharose Fast Flow.

[0083] Furthermore, the concentration of the aqueous solution of sodium chloride in step (4) is 0.9-1.1M.

[0084] Furthermore, in step (4), desalination is carried out by dialysis, and the specification of the dialysis bag is 10Kda.

[0085] Furthermore, the parameters of spray drying in step (5) are: material inlet temperature is 60-80°C, and material outlet temperature is 52-55°C; the parameters of freeze drying are: drying at -80°C for 24h, and the vacuum degree is 30Pa.

[0086] The sixth object of the present invention is the lactoferrin powder and the whey powder without lactoferrin prepared by the method of the present invention.

[0087] The seventh objective of the present invention is the application of the lactoferrin powder and the whey powder without lactoferrin in the field of infant formula milk powder, health food and clinical nutrition products.

[0088] [Beneficial Effects]

[0089] (1) The whey powder rich in lactoferrin in the present invention is prepared by adding sodium chloride to skim milk to increase the ion concentration, promoting the dissociation of casein micelles and lactoferrin complexes, and releasing lactoferrin encapsulated in casein micelles from the lactoferrin complexes, and more lactoferrin is present in the whey phase in a free state; and then separating the whey protein rich in lactoferrin using a microfiltration membrane. The whey powder prepared by the method of the present invention has a significantly improved lactoferrin enrichment rate compared to whey protein that has not been treated for lactoferrin release.

[0090] (2) Compared with whey obtained as a byproduct of cheese processing, the whey powder prepared by the present invention avoids the heating and fermentation process in the cheese production process and well retains proteins such as lactoferrin in the whey.

[0091] (3) The lactoferrin powder prepared by the present invention has a purity of lactoferrin of more than 98% and a recovery rate of more than 70%. The lactoferrin activity is well retained by using membrane filtration sterilization in the preparation process. Based on the effects of lactoferrin on enhancing immunity, promoting iron absorption and regulating intestinal health, the lactoferrin powder prepared by the present invention has important application value in nutritional supplementation, disease prevention and treatment, etc.

[0092] (4) The lactoferrin-free whey powder prepared by the present invention retains the vast majority of whey proteins and the purity of the protein is as high as more than 90%, and can be used as a nutritional ingredient and scientific research; in addition, the whey powder is almost not affected by heat treatment, and the protein activity is well retained, thereby improving the nutritional and functional activity in food applications.

[0093] In summary, more lactoferrin is retained in the whey powder prepared by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 The present invention is a process flow chart of the method for preparing active whey powder rich in lactoferrin.

[0095] Figure 2 This is a graph showing the retention rate of raw milk undenatured whey in Example 1 and Comparative Example 1.

[0096] Figure 3 The test graphs are for the contents of lactoferrin (a) and immunoglobulin IgG (b) in the whey powder of Example 1 and Comparative Example 1.

[0097] Figure 4 The test results of lactoferrin content in whey powder of Example 1 and Comparative Examples 2-6 are shown.

[0098] Figure 5 This is the HPLC chart of the lactoferrin sample obtained after the lactoferrin powder prepared in Example 2 was re-dissolved.

[0099] Figure 6 This is an SDS-PAGE picture of a whey sample containing no lactoferrin obtained after the whey powder containing no lactoferrin prepared in Example 2 was reconstituted.

[0100] Figure 7 The SDS-PAGE diagram of the whey protein prepared in Example 2 and Comparative Example 7 is shown.

[0101] Figure 8 The recovery rate of lactoferrin in whey prepared in Example 2 and Comparative Example 7.

[0102] Fig. 9 These are the test results of separating and purifying lactoferrin using two ion exchange resins in Example 3. DETAILED DESCRIPTION

[0103] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0104] Test method:

[0105] 1. Active whey extraction:

[0106] The whey powder and water were dissolved in a mass ratio of 1:8 under magnetic stirring at 45°C for 1 h to obtain a whey sample;

[0107] Then pour the whey sample into a centrifuge tube, centrifuge at 8000g for 15 minutes at 4°C, scrape off the upper layer of milk fat with a small spoon, adjust the pH of the obtained whey to 4.6, and then place it at 4°C for 1 hour to allow the casein and denatured whey protein to completely precipitate. During this period, pay attention to monitoring and adjusting the pH;

[0108] The supernatant sample was transferred to an ultra-high speed centrifuge tube and centrifuged at 31200 rpm and room temperature for 1.5 h. After centrifugation, the sample was divided into three layers (the top layer was residual milk fat, the middle layer was whey, and the bottom layer was aggregated casein micelles);

[0109] Use a pipette to carefully draw the whey sample from the middle layer into a clean centrifuge tube for later use;

[0110] The extracted whey was diluted for subsequent index determination.

[0111] 2. Determination of undenatured whey:

[0112] According to the method provided by the BCA kit:

[0113] Dilute 2 mg / mL bovine serum albumin standard with PBS in a gradient manner (0.025 mg / mL-2.0 mg / mL). Mix the provided A and B working solutions at a mass ratio of 1:50 and set aside;

[0114] The obtained whey was diluted 10 times with PBS, 25 μL of whey and a series of standards were taken into a 96-well plate, 200 μL of mixed working solution was added to each well using a dispenser, and after mixing, it was sealed with plastic wrap to prevent liquid evaporation. After incubation in the dark at 37°C for 30 minutes, the plate was taken out, and the absorbance at a wavelength of 562 nm was read using an ELISA reader. Three replicate wells were set for each sample, and distilled water was used as a blank control.

[0115] 3. Determination of lactoferrin content:

[0116] Take the bovine lactoferrin standard and perform gradient dilution to make its concentration between 500-7.8 ng / mL, then dilute the sample 500-2000 times according to the concentration range of the standard curve (determined by different standard curve ranges);

[0117] Add 100 μL of gradient concentration standard and previously extracted active whey (active whey diluted 10,000 times), set up 3 replicate wells, react at room temperature for 1 hour, then wash the wells repeatedly and pat dry;

[0118] After adding 100 μL of horseradish peroxidase-conjugated goat anti-bovine lactoferrin detection antibody, incubate at room temperature for 1 h, and wash the plate 5 times;

[0119] After adding 100 μL of TMB (enzyme substrate), the 96-well plate was placed in the dark and incubated at room temperature for 15 min. After adding 100 μL of stop solution (0.18 mol H2SO4), the plate was shaken in a microplate reader for 5 s and the absorbance at a wavelength of 450 nm was read.

[0120] Finally, Soft-Max Pro was used to perform 4-parameter curve fitting of the standard curve and calculate the lactoferrin concentration in the sample, with the sample dilution as the blank control.

[0121] 4. Determination of immunoglobulin G content:

[0122] Same as the method for determining lactoferrin content, the active whey sample was diluted 10,000 times.

[0123] 5. Determination of lactoferrin purity:

[0124] The purity of LFs was determined by RP-HPLC.

[0125] The sample was prepared by mixing equal volumes of LFs solution and buffer (0.1 M Bis-Tris propane, 8 M urea, 20 mM DTT, 1.3% sodium citrate, adjusted to pH 7) and then filtering with a 0.22 μm organic membrane.

[0126] RP-HPLC analysis was performed on a C430 nm 3.5 μm particle size column equilibrated in 95% mobile phase A (0.1% trifluoroacetic acid in acetonitrile) and 5% mobile phase B (0.1% trifluoroacetic acid in acetonitrile).

[0127] The chromatographic analysis was monitored by absorbance at a wavelength of 280 nm.

[0128] The column temperature was set at 60°C, the injection volume was 30 μL, and the bound protein was eluted with a linear gradient of 38-60% mobile phase B at a flow rate of 1 mL / min and an elution time of 20 min.

[0129] The raw materials used in the embodiments and comparative examples are:

[0130] The preparation method of sterilized skim milk is as follows:

[0131] After preheating the raw milk to 42°C, it is centrifuged in a disc centrifuge to obtain cream and skimmed milk; the skimmed milk is then filtered using a 1.4μm microfiltration membrane with the temperature controlled at 40°C to remove most of the microorganisms and impurities in the milk to obtain sterilized skimmed milk.

[0132] BCA kit: Thermo Fisher Scientific;

[0133] Lactoferrin and immunoglobulin reagent test kit (ElisaKit): Bethyl Company, USA;

[0134] The remaining reagents were of analytical grade.

[0135] The normal permeate conductivity range is 4-7mS / m.

[0136] Example 1

[0137] A method for preparing active whey powder rich in lactoferrin (such as Figure 1 ), comprising the following steps:

[0138] (1) Release of lactoferrin:

[0139] Add 300 mmol / L of sodium chloride to the sterilized skim milk while stirring (500 rpm), control the temperature at 37° C. and the pH at 6.6, until the sodium chloride is completely dissolved, to obtain skim milk after lactoferrin phase transfer;

[0140] (2) Enrichment of lactoferrin:

[0141] The skim milk after the lactoferrin phase transfer is filtered using a 100 nm microfiltration membrane to separate the whey protein phase from the casein phase to obtain a membrane filtration permeate (lactoferrin-rich whey protein);

[0142] The filtration temperature is controlled at 37±2℃, and the end point of the filtration operation is determined by "the volume of the liquid in the membrane filtration retentate becomes one third of the initial volume", that is, the concentration multiple is 3 times;

[0143] The filtration was performed using a 300 mmol / L sodium chloride aqueous solution as the filtrate. After each filtration reached the end point of the operation, the filtrate was backfilled to the initial volume and re-filtered, and this was repeated 4 times.

[0144] (3) Sterile concentration:

[0145] The membrane filtered permeate (lactoferrin-rich whey protein) is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, with a concentration multiple of 8 times and an ultrafiltration temperature of 40°C; ultrapure water is then used as a second ultrafiltration solution, and the ultrafiltration solution usage is 8 times that of the concentrated permeate; after the ultrafiltration is completed, electrodialysis (operating current is 45A, feed flow rate is 110 mL / min, and feed temperature is 35°C) is further desalted, and the desalination operation endpoint is judged by "the conductivity value is reduced to the normal permeate conductivity range"; active whey rich in lactoferrin after aseptic concentration is obtained (this step skips the sterilization unit, and the microorganisms therein are removed through membrane concentration to obtain active whey rich in lactoferrin); the total protein concentration in the permeate is determined by the Kjeldahl method;

[0146] (4) Spray drying:

[0147] The aseptically concentrated active whey rich in lactoferrin is spray-dried (material inlet temperature is 75° C., material outlet temperature is 53° C.) to obtain active whey powder rich in lactoferrin; and the powder is sealed and light-proofed for subpackaging.

[0148] Comparative Example 1 (control group)

[0149] Several WPC concentrated whey protein products were purchased from the market, which were marked as WPC1, WPC2, WPC3 and WPC4, and their main components were protein (≥80%), fat (≤8%), ash (≤6%), and moisture (≤5%); the WPC concentrated whey protein product was mixed with warm water at a mass ratio of 1:8, the temperature of the warm water was 45°C, and the mixture was stirred and mixed at a speed of 500 rpm while maintaining the temperature. After re-dissolution, the indicators were compared with those of the whey powder in Example 1.

[0150] The test results are as follows:

[0151] Figure 2 is the retention rate diagram of undenatured whey in Example 1 and Comparative Example 1, from Figure 2 It can be seen that the retention rates of undenatured whey protein in Example 1, WPC1, WPC2, WPC3 and WPC4 are 94.06%, 72.09%, 94.64%, 99.84% and 96.44%, respectively. Except for WPC1, the retention rates of undenatured whey in the remaining whey powders are all higher than 94%, and most of the whey powders are not denatured.

[0152] Figure 3 The following is a test chart of the content of lactoferrin (A) and immunoglobulin IgG (B) in the whey powder of Example 1 and Comparative Example 1. Figure 3 It can be seen from (A) that the active lactoferrin content of the concentrated whey powder of Example 1 is significantly higher than that of each group of samples in Comparative Example 1, reaching 1997.23 mg / 100 g protein. Among the samples in Comparative Example 1, the highest lactoferrin content is WPC1, which is 844.99 mg / 100 g protein, and the other samples are 221.05, 94.32 and 193.40 mg / 100 g protein (WPC2, WPC3 and WPC4), respectively. Figure 3 It can be seen from (B) that the active IgG content in the WPC of WPC1 in the concentrated whey powder of Example 1 and the comparative example is the highest, which is 3669.80 and 3253.35 mg / 100g protein respectively, significantly exceeding 573.27, 2315.19 and 2678.69 mg / 100g protein of WPC2, WPC3 and WPC4 in content. As can be seen from the above results, the active lactoferrin content of the concentrated whey powder of Example 1 significantly exceeds each group of WPC products in the comparative example 1, indicating that the process adopted by Example 1 can effectively enrich and improve the lactoferrin content in whey. In addition, the content of immunoglobulin IgG of the whey powder product of Example 1 is also the highest in each group of products, proving that while enriching lactoferrin, the immunoglobulin in the enriched whey can also be well retained.

[0153] Comparative Example 2 Effect of Selection of Different Microfiltration Washing Filtrates on Lactoferrin Enrichment Rate in Whey Powder

[0154] The washing filtrate sodium chloride solution in step (2) of Example 1 was adjusted to deionized water, and the rest was kept consistent with Example 1 to obtain whey powder.

[0155] Comparative Example 3 Effect of lactoferrin phase separation step on lactoferrin enrichment rate in whey powder

[0156] The lactoferrin phase separation step in step (1) of Comparative Example 2, i.e., the operation of adding sodium chloride, was omitted, and the rest was kept the same as in Comparative Example 2 to obtain whey powder.

[0157] Comparative Example 4 Effect of microfiltration temperature (30°C) on the enrichment rate of lactoferrin in whey powder

[0158] The filtration temperature in step (2) of Comparative Example 3 was adjusted to 30°C, and the other conditions were kept the same as those of Comparative Example 3 to obtain whey powder. Comparative Example 5 Effect of microfiltration temperature (45°C) on the enrichment rate of lactoferrin in whey powder

[0159] The filtration temperature in step (2) of comparative example 3 was adjusted to 45° C., and the other conditions were kept the same as those of comparative example 3 to obtain whey powder. Comparative example 6 Effect of the timing of sodium chloride addition on the enrichment rate of lactoferrin in whey powder

[0160] The timing of adding sodium chloride is changed to adding it to casein, as follows:

[0161] (1) Preheating raw milk to 42° C., centrifuging the milk with a disc centrifuge to obtain cream and skim milk; then filtering the skim milk with a 1.4 μm microfiltration membrane at a temperature controlled at 40° C. to remove most of the microorganisms and impurities in the milk, thereby obtaining sterilized skim milk;

[0162] (2) filtering skim milk with a 0.1 μm microfiltration membrane to separate the whey protein phase from the casein phase, and obtaining a membrane filtration permeate and a membrane filtration retentate (casein); the filtration temperature is controlled at 37±2° C., the pH is 6.6, and the end point of the filtration operation is determined by “the liquid volume in the membrane filtration retentate becomes one-third of the initial volume”; the filtration is performed using a deionized water solution as a filtration solution, and after each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and re-filtered, and the process is repeated 4 times; and the permeate obtained from each filtration is collected;

[0163] (3) The membrane filtration retentate (casein) is further filtered using a 0.1 μm microfiltration membrane, and a sodium chloride solution containing 450 mmol is backfilled to the volume before step (2) to make the final sodium chloride concentration 300 mmol, and the filtration is continued under this condition; subsequently, a 300 mmol sodium chloride solution is used as the second filtration solution; the end point of the filtration operation is determined by "the liquid volume in the membrane filtration retentate becomes one-third of the initial volume". After each filtration reaches the end point of the operation, the filtration solution is backfilled to the initial volume and filtered again, and the above filtration is repeated 4 times; the permeate obtained from each filtration is collected;

[0164] (4) The permeate is then subjected to ultrafiltration, concentration and desalination, and finally spray-dried to obtain whey powder.

[0165] The whey powder obtained in the examples and comparative examples was reconstituted, and the protein and active lactoferrin contents of the reconstituted whey were measured.

[0166] Figure 4 The lactoferrin content in whey powder of Example 1 and Comparative Examples 2-6 is as follows; Figure 4 It can be seen that the lactoferrin content in the whey powder of embodiment 1 and comparative examples 2-6 is specifically 1997.23, 1111.84, 493.93, 187.91, 286.75, 1214.03mg / 100g protein. For comparative example 2, after adopting deionized water to carry out filtration washing, the lactoferrin content in the whey powder is significantly reduced, this is because after the deionized water filtration washing, the ionic strength in the system is rapidly reduced, and the remaining lactoferrin and casein binding trend are strengthened again, thereby reducing the lactoferrin content in the finished product whey powder. For comparative examples 3-5, after omitting the lactoferrin phase separation step, respectively under 30 ℃, 37 ℃ and 45 ℃ conditions, and using deionized water filtration washing, in the final whey powder product obtained, the lactoferrin content in the whey powder corresponding to 37 ℃ is the highest, reaching 493.93mg / 100g protein, so 37 ℃ is the optimal condition. For example, in Comparative Example 6, after changing the timing of adding sodium chloride, the effect of filtering lactoferrin from the casein retentate was not as good as that in Example 1, and the lactoferrin content in the final whey powder was 1214.03 mg / 100 g protein. In summary, the processing conditions adopted in Example 1 can obtain better whey powder rich in lactoferrin.

[0167] Example 2

[0168] A method for preparing active whey powder rich in lactoferrin (such as Figure 1 ), comprising the following steps:

[0169] (1) Release of lactoferrin:

[0170] Add 300 mmol / L of sodium chloride to the sterilized skim milk while stirring (500 rpm), control the temperature at 37° C. and the pH at 6.6, until the sodium chloride is completely dissolved, to obtain skim milk after lactoferrin phase transfer;

[0171] (2) Enrichment of lactoferrin:

[0172] The skim milk after the lactoferrin phase transfer is filtered using a 100 nm microfiltration membrane to separate the whey protein phase from the casein phase to obtain a membrane filtration permeate (lactoferrin-rich whey protein);

[0173] The filtration temperature is controlled at 37±2℃, and the end point of the filtration operation is determined by "the volume of the liquid in the membrane filtration retentate becomes one third of the initial volume", that is, the concentration multiple is 3 times;

[0174] The filtration was performed using a 300 mmol / L sodium chloride aqueous solution as the filtrate. After each filtration reached the end point of the operation, the filtrate was backfilled to the initial volume and re-filtered, and this was repeated 4 times.

[0175] (3) Sterile concentration:

[0176] The membrane filtered permeate (lactoferrin-rich whey protein) is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, with a concentration multiple of 8 times and an ultrafiltration temperature of 40°C; ultrapure water is then used as a second ultrafiltration solution, and the ultrafiltration solution usage is 8 times that of the concentrated permeate; after the ultrafiltration is completed, electrodialysis (operating current is 45A, feed flow rate is 110 mL / min, and feed temperature is 35°C) is further desalted, and the desalination operation endpoint is judged by "the conductivity value is reduced to the normal permeate conductivity range"; active whey rich in lactoferrin after aseptic concentration is obtained (this step skips the sterilization unit, and the microorganisms therein are removed through membrane concentration to obtain active whey rich in lactoferrin); the total protein concentration in the permeate is determined by the Kjeldahl method;

[0177] (4) Further enriching lactoferrin and collecting whey without lactoferrin:

[0178] The aseptically concentrated active whey rich in lactoferrin is pumped into a cation exchange column filled with resin filler (CM-Sepharose Fast Flow) through a peristaltic pump, and the whey flowing out at the end of the column is collected, and the whey is the active whey without lactoferrin; then the column is flushed with pure water until all the residual whey is discharged;

[0179] Then, a 0.3M sodium chloride aqueous solution is used to elute the cation exchange column, and the eluted part is non-lactoferrin impurities. The liquid flowing out of the column tail is received and backfilled into the column whey. Subsequently, a 1.0M sodium chloride aqueous solution is used to elute the cation exchange column, and the liquid flowing out of the column tail is received; finally, a dialysis bag with a molecular weight cutoff of 10KDa is used to dialyze the liquid phase flowing out of the column tail, and the dialysate is ultrapure water, and the dialyzate is dialyzed until the conductivity of the lactoferrin phase reaches a normal range to obtain lactoferrin;

[0180] (5) Drying:

[0181] The lactoferrin sample was freeze-dried (temperature -80°C, time 36h and vacuum degree 30pa), and the whey without lactoferrin was spray-dried (material inlet temperature 75°C, material outlet temperature 53°C) to obtain whey powder without lactoferrin; and the mixture was sealed and packaged in a dark place.

[0182] The lactoferrin powder and whey powder obtained above were re-dissolved, the obtained lactoferrin powder was tested for purity, and the whey powder without lactoferrin was tested by SDS-PAGE index. The test results were as follows:

[0183] Figure 5 This is the HPLC chart of the lactoferrin sample obtained after the lactoferrin powder prepared in Example 2 was reconstituted. Figure 5It can be seen that the lactoferrin in the reconstituted lactoferrin sample of Example 2 has a higher purity. According to calculation, the lactoferrin peak area accounts for more than 98% of the total area.

[0184] Figure 6 This is an SDS-PAGE image of a whey sample containing no lactoferrin obtained after the whey powder containing no lactoferrin prepared in Example 2 was reconstituted. Figure 6 It can be seen that the original lactoferrin band should be at around 80KDa. After column separation, the obtained whey powder no longer contains lactoferrin, but the main α-lactalbumin and β-lactoglobulin and a small amount of low-abundance whey protein are still retained.

[0185] The above results show that the lactoferrin powder and the whey powder without lactoferrin obtained as described in Example 2 have excellent performance.

[0186] Comparative Example 7 Preparation of whey powder by acid precipitation

[0187] A method for preparing whey powder comprises the following steps:

[0188] (1) fresh milk is preheated and then centrifuged to skim to obtain skimmed milk;

[0189] (2) Filter the skim milk through a 1.4 μm pore size membrane to obtain sterilized milk, and take a sample;

[0190] (3) taking part of the sterilized milk obtained in step (2), adjusting the pH to 4.6 with 6M HCl solution, centrifuging at 4°C and 10,000 g for 15 minutes, and taking the supernatant;

[0191] (4) adjusting the pH of the supernatant obtained in step (2) to 6.8 using a 6 M NaOH solution, and then centrifuging at 4° C. and 10,000 g for 15 minutes to obtain the supernatant (whey);

[0192] (5) Sodium chloride was slowly added to the remaining sterilized milk in step (2) to a final concentration of 0.3 M, and 100 nm membrane separation was performed. The whey obtained from the first filtration was sampled. The remaining operations were the same as step (2) of Example 2.

[0193] The whey obtained by the first filtration of step (4) and step (5) was subjected to SDS-PAGE results. The recovery rate of all lactoferrin obtained in step (4) and step (5) was determined by ELISA kit, and the test results were as follows:

[0194] Figure 7 The SDS-PAGE diagram of the whey protein prepared in Example 2 and Comparative Example 7 is shown in FIG. Figure 7It can be seen that compared with the whey obtained by acid precipitation in Comparative Example 7, the lactoferrin band of the whey obtained by membrane filtration in Example 2 is darker, which means that the lactoferrin content is higher.

[0195] Figure 8 is the recovery rate of lactoferrin in the whey prepared in Example 2 and Comparative Example 7. Figure 8 It can be seen that the whey obtained by the membrane filtration treatment of Example 2 has a higher lactoferrin recovery rate than the acid-precipitated whey of Comparative Example 7, which are 70.64% and 45.80%, respectively. During the acid precipitation process, the casein micelle precipitation will entrain a large amount of lactoferrin, resulting in a decrease in the lactoferrin content of the entire system, thereby reducing the recovery rate of lactoferrin. Therefore, it is better to select the membrane filtration treatment of Example 2.

[0196] Example 3 Screening of cation exchange column fillers

[0197] Select two gels, CM-Sephadex C-50 and CM-Sepharose Fast Flow; prepare two groups of 5 mL bovine whey, and pass the two groups of whey through 1 mL pre-loaded columns of the two gels. After loading, use pure water to elute unbound proteins, and then use 0.3M NaCl aqueous solution and 0.9M NaCl aqueous solution to elute some impurities and lactoferrin. Use SDS-PAGE to compare the separation effect, and combine the repeatability of the experimental operation to determine the choice of gel.

[0198] Fig. 9 The test results of separating and purifying lactoferrin by two ion exchange resins in Example 3 are shown in FIG. Fig. 9 It can be seen that after flushing the column with pure water using CM-Sephadex C-50, the washed protein band is deeper, indicating that the total amount of protein adsorbed is less, and its repeatability under the elution condition of 0.3M NaCl aqueous solution is poor, which may be related to its unstable adsorption. The protein eluted from CM-Sephadex C-50 in 0.9M NaCl aqueous solution has impurities, and the purity of lactoferrin is lower than that of CM-Sepharose Fast Flow. In addition, the elution filler has the problem of soft material. The operation repeatability of CM-Sepharose Fast Flow is better, the operation is relatively simple, and the separation effect is better. Therefore, CM-Sepharose Fast Flow was selected as the ion exchange separation resin.

[0199] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An active whey powder rich in lactoferrin, characterized in that: The preparation method is as follows: (1) Release of lactoferrin: Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer; Wherein, the concentration of sodium chloride added is 200-400mmol / L; (2) Enrichment of lactoferrin: The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin); Among them, the filtrate used in membrane filtration is sodium chloride solution; (3) Sterile concentration: The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin; (4) Spray drying: The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

2. The active whey powder rich in lactoferrin according to claim 1, characterized in that In step (2), the membrane filtration uses a membrane pore size of 90-110 nm, a temperature of 35-55° C., a concentration multiple of 2-4, and a filtration wash number of 3-5; the sodium chloride solution is a sodium chloride aqueous solution with a concentration of 200-400 mmol / L.

3. The active whey powder rich in lactoferrin according to claim 1, characterized in that The ultrafiltration membrane used in step (3) has a molecular weight cutoff of 9-11 kDa, a concentration multiple of 5-10 times, and an ultrafiltration temperature not exceeding 45°C.

4. The active whey powder rich in lactoferrin according to claim 1, characterized in that: The spray drying parameters in step (4) are: material inlet temperature is 60-80°C, and material outlet temperature is 52-55°C.

5. Use of the active whey powder rich in lactoferrin according to any one of claims 1 to 4 in the fields of infant formula milk powder, health food and clinical nutrition products.

6. A method for preparing active whey powder rich in lactoferrin, characterized in that: The steps include: (1) Release of lactoferrin: Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer; Wherein, the concentration of sodium chloride added is 200-400mmol / L; (2) Enrichment of lactoferrin: The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin); Among them, the filtrate used in membrane filtration is sodium chloride solution; (3) Sterile concentration: The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin; (4) Spray drying: The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

7. A method for increasing the lactoferrin content in whey powder, characterized in that: The steps include: (1) Release of lactoferrin: Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer; Wherein, the concentration of sodium chloride added is 200-400mmol / L; (2) Enrichment of lactoferrin: The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (whey protein rich in lactoferrin); Among them, the filtrate used in membrane filtration is sodium chloride solution; (3) Sterile concentration: The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin; (4) Spray drying: The active whey rich in lactoferrin is spray-dried to obtain whey powder rich in lactoferrin.

8. A method for simultaneously preparing lactoferrin powder and whey powder without lactoferrin, characterized in that: The steps include: (1) Release of lactoferrin: Adding sodium chloride to the sterilized skim milk while stirring, controlling the temperature at 35-39° C. and the pH at 6-7, until the sodium chloride is completely dissolved, thereby obtaining skim milk after lactoferrin phase transfer; Wherein, the concentration of sodium chloride added is 200-400mmol / L; (2) Enrichment of lactoferrin: The skim milk after the lactoferrin phase transfer is subjected to membrane filtration to obtain a membrane filtration permeate (a whey protein phase rich in lactoferrin); Among them, the filtrate used in membrane filtration is sodium chloride solution; (3) Sterile concentration: The membrane filtration permeate is ultrafiltered and desalted to obtain aseptically concentrated active whey rich in lactoferrin; (4) Further enriching lactoferrin and collecting whey without lactoferrin: Passing the aseptically concentrated active whey rich in lactoferrin through a cation exchange column to transfer lactoferrin from the whey to the cation exchange column to obtain whey without lactoferrin; then eluting the lactoferrin on the cation exchange column with an aqueous solution of sodium chloride to remove salt and obtain lactoferrin; (5) Drying: The lactoferrin is freeze-dried to obtain lactoferrin powder; the whey without lactoferrin is spray-dried to obtain whey powder without lactoferrin.

9. Lactoferrin powder and whey powder free of lactoferrin prepared by the method according to claim 8.

10. Use of the lactoferrin powder and the whey powder without lactoferrin according to claim 9 in the fields of infant formula milk powder, health food and clinical nutrition products.

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