A method for increasing the content of osteopontin in whey and a whey obtained by the method

Abstract

The application provides a method for increasing the content of osteopontin in whey and whey prepared by the method, and the method comprises the following steps: (1) adding sodium citrate to milk-containing raw materials, and stirring to obtain a mixture; (2) placing the mixture at a temperature of 2-10 DEG C, and performing low-temperature treatment for 0.5-3 hours, and performing microfiltration separation to obtain whey containing osteopontin. Through the synergistic regulation of sodium citrate and low temperature, the yield and release rate of osteopontin in the whey phase can be significantly improved under the premise of maintaining the natural micellar structure of casein and avoiding acid denaturation, and the high activity of osteopontin is ensured; and the process steps are simple, no complex equipment is needed, the production cycle can be effectively shortened, and the comprehensive cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of dairy biotechnology, specifically relating to a method for increasing the osteopontin content in whey and the whey obtained therefrom. Background Technology

[0002] Osteopontin (OPN) is a whey protein with various biological activities, widely used in infant formula, functional foods, and other fields. However, in natural cow's milk, osteopontin is tightly bound to casein through calcium bridges, hydrophobic interactions, and electrostatic interactions. This results in extremely low osteopontin content in the whey phase when using conventional microfiltration techniques to separate skim milk, making it difficult to meet the needs of large-scale extraction and the development of high-value-added products.

[0003] In existing technologies, there are two main types of methods to increase the content of osteopontin in whey phase: one is acidification, which involves lowering the pH of skim milk to below 4.6 and controlling the calcium ion concentration to disrupt the binding between osteopontin and casein, thus achieving osteopontin dissociation. However, this method causes irreversible acid denaturation of casein, forming a dense curd structure, making the denatured casein unusable for the production of high-value-added dairy products such as processed cheese and infant formula, thus reducing the overall utilization rate of dairy raw materials. The second method uses ethylenediaminetetraacetic acid (EDTA) as a calcium ion chelating agent to disrupt calcium bridging and release osteopontin. However, EDTA residues are difficult to completely remove, posing a food safety risk. It also increases the difficulty and cost of subsequent osteopontin purification, which is not conducive to industrial-scale application.

[0004] Therefore, there is an urgent need for an extraction method that can safely and efficiently increase the osteopontin content in the whey phase while maintaining the natural structure of casein. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a method for increasing the osteopontin content in whey and the whey prepared therefrom, which can effectively avoid casein denaturation, increase the yield of osteopontin in the whey phase, and achieve efficient and safe extraction of osteopontin.

[0006] To achieve the above objectives, the present invention provides a method for increasing the osteopontin content in whey, wherein the method includes the following steps: (1) Add sodium citrate to the milk-containing raw material and stir to obtain a mixture; (2) The mixture is placed at 2-10°C for 0.5-3 hours and microfiltered to obtain a whey containing osteopontin.

[0007] According to a specific embodiment of the present invention, preferably, in step (1), the sodium citrate is a 10-20 w / v% sodium citrate solution.

[0008] In this invention, "w / v%" is the mass-volume percentage concentration, which refers to the number of grams of solute (g / 100mL) contained in 100mL of solution.

[0009] According to a specific embodiment of the present invention, preferably, in step (1), the final concentration of sodium citrate in the mixture is 1-5 mM.

[0010] According to a specific embodiment of the present invention, preferably, in step (1), the final concentration of sodium citrate in the mixture is 1-2.5 mM, more preferably 2.5 mM.

[0011] According to a specific embodiment of the present invention, preferably, in step (1), the stirring speed is 100-300 rpm and the time is 15-45 minutes. The stirring step can uniformly disperse sodium citrate in the mixture system without local excessive concentration; sodium citrate efficiently chelates calcium ions in the system, destroying the calcium bridge between osteopontin and casein, laying the foundation for osteopontin release.

[0012] According to a specific embodiment of the present invention, preferably, in step (2), the temperature of the low-temperature treatment is 4-6°C, and the treatment time is 2 hours. The present invention has found that after low-temperature treatment for more than 2 hours, osteopontin undergoes complexation, leading to a sharp drop in release rate and affecting the yield of osteopontin in the whey.

[0013] In this invention, the temperature of the low-temperature treatment step is controlled at 2-10℃, which can effectively avoid protein precipitation, maintain hydrophobic interactions, and increase the dissociation effect; at the same time, the low-temperature treatment time is controlled to not exceed 3 hours to avoid excessive dissociation of casein to form insoluble complexes and reduce the osteopontin content in the whey.

[0014] According to a specific embodiment of the present invention, preferably, before step (1), the method further includes a step of pre-cooling the milk-containing raw material at 4-6°C for 10-60 minutes. Pre-cooling can keep the temperature of the milk-containing raw material stable and avoid temperature fluctuations affecting the subsequent chelation and dissociation effects.

[0015] According to a specific embodiment of the present invention, preferably, the milk-containing raw material is skim milk, and the fat content of the skim milk is ≤0.5%.

[0016] According to a specific embodiment of the present invention, preferably, the skimmed milk is obtained by skimming fresh cow's milk.

[0017] According to a specific embodiment of the present invention, preferably, the sodium citrate is food-grade sodium citrate.

[0018] This invention utilizes the synergistic regulation of sodium citrate chelating calcium ions and low temperature to disrupt the calcium bridging between osteopontin and casein, thereby achieving selective release and whey phase enrichment of osteopontin. Specifically, sodium citrate preferentially chelates calcium ions in milk, disrupting the calcium bridging between osteopontin and casein micelles; simultaneously, low temperature conditions inhibit casein thermal denaturation, weakening the hydrophobic interaction between osteopontin and casein. The synergistic effect of these two factors maintains the natural micelle structure of casein, achieving selective release and whey phase enrichment of osteopontin.

[0019] According to a specific embodiment of the present invention, preferably, in step (2), the microfiltration separation step includes: using a microfiltration membrane with a pore size of 0.05μm-0.2μm to perform cross-flow filtration under a pressure of 0.1MPa-0.3MPa.

[0020] According to a specific embodiment of the present invention, preferably, the microfiltration membrane includes a ceramic membrane, an organic polymer microfiltration membrane, or a hollow fiber membrane; more preferably, the microfiltration membrane is a hollow fiber membrane.

[0021] According to a specific embodiment of the present invention, preferably, the pore size of the microfiltration membrane is 0.1 μm, the pressure is 0.2 MPa, and the crossflow velocity is 3 m / s.

[0022] This invention achieves the enrichment of osteopontin in the whey phase through an optimized microfiltration process, efficiently increasing the content of osteopontin in the microfiltered whey phase while ensuring the bioactivity of osteopontin, thus obtaining a whey with high content and high activity.

[0023] On the other hand, the present invention also provides a whey solution, wherein the whey solution is prepared by the above method.

[0024] According to a specific embodiment of the present invention, preferably, the osteopontin content in the whey is 6.00-9.00 mg / L, more preferably 8.0-9.0 mg / L, and even more preferably 8.75-9.0 mg / L.

[0025] According to a specific embodiment of the present invention, preferably, based on the total mass of the whey liquid as 100%, the whey liquid contains: protein content of 0.1-0.3%, fat content of 0.05-0.3%, and solids content of 2-4%.

[0026] The method of this invention produces a whey solution containing osteopontin, which has a high osteopontin content and avoids the irreversible denaturation of casein caused by acidification treatment in existing technologies. Its natural micelle structure remains intact, allowing it to be directly used in the development of infant formula, immune-modulating foods, functional beverages, and other products, enhancing their bioactivity and market competitiveness. Furthermore, this whey solution can serve as a high-quality raw material for further purification of osteopontin, reducing purification costs and promoting the widespread application of osteopontin in pharmaceuticals, food, cosmetics, and other fields, demonstrating significant economic and social benefits.

[0027] This invention, through the synergistic regulation of sodium citrate and low temperature, significantly improves the yield and release rate of osteopontin in the whey phase while maintaining the natural micelle structure of casein and avoiding its acid denaturation. Simultaneously, the method uses food-grade sodium citrate, ensuring high safety and no residue risk. Furthermore, the process is simple, requiring no complex equipment, effectively shortening the production cycle and reducing overall costs. The consistently mild low-temperature environment also ensures the bioactivity of osteopontin, allowing the resulting whey to be directly used in the development of high-value-added dairy products and providing a high-quality raw material for subsequent osteopontin purification, demonstrating promising industrial application prospects and comprehensive economic benefits. Attached Figure Description

[0028] Figure 1 The effect of different sodium citrate concentrations on osteopontin release rate in skim milk; Figure 2 To investigate the effect of different low-temperature reaction times on the release rate of osteopontin in skim milk; Figure 3 The effect of different sodium citrate concentrations on the yield of osteopontin in natural whey; Figure 4 The particle size test results of casein micelles at different sodium citrate concentrations are shown. Figure 5 The results of zeta potential tests for casein dispersions at different sodium citrate concentrations; Figure 6 To investigate the effect of different low-temperature reaction times on the yield of osteopontin in natural whey; Figure 7 The particle size test results of casein micelles at different low-temperature reaction times; Figure 8 The results of zeta potential tests for casein dispersion systems at different low-temperature reaction times; Figure 9 The effect of different EDTA concentrations on the release rate of osteopontin in skim milk. Detailed Implementation

[0029] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0030] It should be noted that, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0032] It should be understood that the terms “comprising,” “including,” and / or “containing” as used herein specify the presence of the stated features, integers, steps, components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0033] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0034] In the following examples and comparative examples, the osteopontin content was detected using the method described in "T / TDSTIA 028-2022 Determination of osteopontin in infant food and dairy products by high performance liquid chromatography".

[0035] Example 1: Experiment on Optimization of Sodium Citrate Concentration

[0036] Fresh milk is taken and defatted by disc centrifugation to control the fat content of the defatted milk to <0.1%. The defatted milk is collected and pre-cooled at 6℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0037] A 15% (w / v) sodium citrate solution was added to the pretreated skim milk, and the final concentrations of the system were adjusted to 0, 1, 2.5, 5, 10, 15, 20, 25, 30, and 35 mmol / L. The mixture was stirred at 180 rpm for 30 min to ensure uniform dispersion of the sodium citrate. The 0 mmol / L sodium citrate group served as the control group, and the other concentration groups served as the experimental groups.

[0038] As the concentration of sodium citrate increases, it gradually chelates the calcium phosphate in the skim milk micelles, disrupting the calcium bridges on the surface of casein micelles. This results in a looser micelle structure, altered particle size distribution, and a significant change in the colloidal dispersion state of the system. The appearance of the skim milk gradually changes from milky white to yellowish-green, and the viscosity of the mixture decreases. Referring to Table 1, with the 0 mmol / L group as a reference, the color difference value ΔE of the mixture continuously increases, indicating a change in the colloidal dispersion state and optical properties of the skim milk. This demonstrates that sodium citrate can effectively act on casein micelles and promote the separation of osteopontin.

[0039] The osteopontin (OPN) content in each mixture was measured, and the OPN release rate was calculated; OPN release rate = (OPN content in experimental group - OPN content in control group) / OPN content in control group × 100%. See also Figure 1 The analysis results are as follows: With the 0 mmol / L group as the control, the osteopontin release rate after treatment with 2.5 mmol / L sodium citrate was 14.52%; when the concentration increased to 10 mmol / L, the release rate decreased; in the range of 10-20 mmol / L, the osteopontin release rate gradually increased with the increase of concentration, reaching a peak of 37.07% at 20 mmol / L; after the concentration exceeded 20 mmol / L, the release rate showed a decreasing trend.

[0040] The effect of different concentrations of sodium citrate on osteopontin release in skim milk showed a phased pattern; at 0-0.5 mM, sodium citrate only chelated free Ca²⁺ in trace amounts. + The casein micelle structure was not damaged, and the OPN content in whey was basically the same as that in the control group. Sodium citrate at 1-2.5 mM could completely chelate free Ca²⁺ in whey. + It disrupts the loose casein calcium phosphate (CCP) on the surface of the micelles and breaks weak calcium bridges, prompting a small amount of OPN to be released at a rate much greater than the rebinding rate. The whey OPN content increases significantly. At 5-10 mM, the dense CCP inside the micelles migrates to the surface to form new calcium bridges. At the same time, the negative charge on the micelle surface increases, prompting OPN to be adsorbed and rebinded a second time. The whey OPN content decreases slightly. At 15-35 mM, the chelating ability of sodium citrate far exceeds the migration and replenishment rate of CCP. The micelle structure may be significantly damaged, and a large number of calcium bridges break. OPN is released rapidly and reaches a peak at 20 mM. The release amount tends to saturate at 25-35 mM.

[0041] Considering the osteopontin release effect and the casein system stability, a final sodium citrate concentration of 1-5 mmol / L is suitable for addition in the mixture system, and 2.5 mmol / L is more preferred. This condition is more environmentally friendly and economical, and has the potential for industrial-scale application.

[0042] Table 1. Effects of different sodium citrate concentrations on color difference parameters of skim milk

[0043] Example 2: Low-Temperature Reaction Time Optimization Experiment

[0044] Fresh milk is taken and defatted by disc centrifugation to control the fat content of the defatted milk to <0.1%. The defatted milk is collected and pre-cooled at 6℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0045] Add 15% (w / v) sodium citrate solution to the pretreated skim milk to adjust the final concentration of sodium citrate in the system to 2.5 mmol / L. Stir at 180 rpm for 30 minutes to uniformly disperse the sodium citrate and obtain a mixture.

[0046] The stirred mixture samples were subjected to low-temperature reactions at 4°C for 0, 0.5, 1, 2, 6, 24, 48, and 72 hours to promote the dissociation of casein micelles. During the low-temperature reaction, the container holding the mixture was inverted once every 0.5 hours. The mixture sample that was not refrigerated after stirring (0 hours) served as the control group, and the other treatment groups served as the experimental groups.

[0047] Referring to Table 2, as the low-temperature reaction time increases, the dispersion state of the defatted emulsion and the protein aggregation behavior change, resulting in a decrease in the brightness of the mixture system and a shift of the yellow-blue hue towards blue. With the 0-hour group as a reference, the color difference value ΔE of the sample first increases to 2.6 (2h) and then gradually decreases to 1.6 (72h), indicating the dynamic changes in the dispersion state and optical properties of the defatted emulsion.

[0048] After the low-temperature reaction of each group was completed, the osteopontin content in the mixture samples of each group was detected. Detection was performed immediately after the treatment time for each group. After the low-temperature reaction time of all experimental groups was completed and the detection results of each group were obtained, the release rate data was calculated uniformly using the same formula as shown in Example 1. The detection results are shown below. Figure 2The analysis is as follows: After adding 2.5 mM sodium citrate to the skim milk system, the OPN release rate showed a trend of first increasing and then decreasing with the extension of low temperature reaction time. Within 0-2 h, the OPN release rate rapidly increased from the initial 9.02% to a peak of 26.13%, mainly because sodium citrate can chelate calcium ions in casein micelles, destroy the calcium-phosphorus stable structure of the micelles, and promote the gradual dissociation of bound OPN into free OPN. At the same time, low temperature can inhibit protease activity, delay OPN degradation, and allow free OPN to continue to accumulate. After 2 h, the release rate continued to decrease, dropping to 6.13% at 6 h, 2.18% at 48 h, and even showing a negative release rate of -3.13% at 72 h. The main reason is that under prolonged low-temperature conditions, the released free OPN is prone to self-aggregation or recombination with casein micelles, resulting in a decrease in the detectable free OPN content. At the same time, the calcium-phosphorus structure of casein micelles is partially reconstructed over time, re-encapsulating OPN. Combined with the slow degradation effect of low-temperature resistant proteases, this leads to a decrease in the release rate. The negative release rate observed at 72 h indicates the presence of apparent effects such as protein precipitation, adsorption, or detection bias.

[0049] Considering both the osteopontin release effect and the stability of the mixture system, a low-temperature reaction of 0.5-2 hours is the suitable process condition, with 2 hours being more preferable, as it balances the simplicity of operation with the efficiency of additive utilization.

[0050] Table 2. Effects of different low-temperature reaction times on color difference parameters of skim milk

[0051] Example 3

[0052] Fresh milk is taken and defatted by disc centrifugation to control the fat content of the defatted milk to <0.1%. The defatted milk is collected and pre-cooled at 6℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0053] Add 15% (w / v) sodium citrate solution to the pretreated skim milk to adjust the final concentration of sodium citrate in the system to 0, 1, 2.5, 10, and 20 mmol / L, respectively. Stir at 180 rpm for 30 min to ensure uniform dispersion of sodium citrate and obtain the mixtures of each group.

[0054] The mixtures were then placed at 4°C for 2 hours for a low-temperature reaction. During the low-temperature reaction, the containers holding the mixtures were inverted once every 0.5 hours.

[0055] After the low-temperature reaction, the mixtures from each group were microfiltered using a 0.1 μm pore size microfiltration membrane at a pressure of 0.25 MPa and a cross-flow rate of 3 m / s, and natural whey containing osteopontin was collected. The microfiltration group with 0 mmol / L sodium citrate was used as the control group, and the other concentration groups were used as experimental groups.

[0056] The osteopontin content in the whey obtained from microfiltration of each mixture was measured, and the OPN yield was calculated as follows: OPN yield = (OPN content in whey × whey volume) / (OPN content in skim milk × skim milk volume). The relative yield was calculated as: OPN yield in the experimental group / OPN yield in the control group × 100%. (See results below.) Figure 3 Compared with the control group, the osteopontin yield of sodium citrate increased in the range of 0-2.5 mmol / L, reaching 24.73% at 2.5 mmol / L, with a relative yield of 133.10%. The osteopontin yield decreased when the concentration increased to 10 mmol / L, and reached 24.82% at 20 mmol / L, with a relative yield of 133.58%. Sodium citrate concentrations exceeding 10 mM increased the microfiltration membrane pressure differential, reducing microfiltration efficiency and causing membrane blockage, resulting in the OPN protein content in whey not reaching the expected level.

[0057] Particle size and zeta potential were characterized for casein dispersions in microfiltration-treated whey solutions subjected to different sodium citrate concentrations. Particle size results are shown below. Figure 4 Compared with the blank CN group (control group), the hydrodynamic particle size of casein increased to 177.1 nm after isothermal treatment with 1 mM sodium citrate for 2 h; when the sodium citrate concentration was adjusted to 2.5 mM and treated for 2 h, the particle size of the system decreased to the lowest among all groups (144.9 nm); when the concentration continued to increase to 5 mM and 10 mM, the particle size showed a slight upward trend; when the sodium citrate concentration reached 20 mM, the particles agglomerated significantly, and the particle size increased sharply to 309.6 nm.

[0058] See the results of the zeta potential test. Figure 5 The blank group had a potential of only -14.7 mV. After the introduction of sodium citrate, the negative charge density on the particle surface was significantly increased. The potential of the 2.5 mM, 2 h treatment group reached -25.37 mV, with the best electrostatic repulsion effect. The absolute values ​​of the potentials of the other concentration groups were all lower than this condition, indicating weaker dispersion stability of the system.

[0059] Considering both osteopontin release yield and microfiltration continuous treatment efficiency, the preferred concentration of sodium citrate is 1~2.5 mmol / L, with 2.5 mmol / L being the optimal process point. This parameter condition allows casein micelles to reach a suitable particle size and the electrostatic repulsion of the system to reach its peak, achieving optimal casein colloidal dispersion stability while efficiently dissociating osteopontin.

[0060] Example 4

[0061] Fresh milk is taken and defatted by disc centrifugation to control the fat content of the defatted milk to <0.1%. The defatted milk is collected and pre-cooled at 6℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0062] Add 15% (w / v) sodium citrate solution to the pretreated skim milk to adjust the final concentration of sodium citrate in the system to 2.5 mmol / L. Stir at 180 rpm for 30 min to uniformly disperse the sodium citrate and obtain a mixture.

[0063] The mixture was then subjected to low-temperature reactions at 4°C for 0 hours, 1 hour, 2 hours, 24 hours, and 72 hours, respectively. During the low-temperature reaction, the container holding the mixture was inverted once every 0.5 hours.

[0064] After the low-temperature reaction, the mixtures from each group were microfiltered using a 0.1 μm pore size microfiltration membrane at a pressure of 0.25 MPa and a cross-flow rate of 3 m / s, and natural whey containing osteopontin was collected. The 0-hour low-temperature reaction time group served as the control group, and the other low-temperature reaction time groups served as the experimental groups.

[0065] After the low-temperature reaction of each group was completed, the osteopontin content in the whey obtained by microfiltration of the mixtures from each group was detected. After all treatment groups were completed and the detection results of each group were obtained, the yield and relative yield were calculated using the same formula as shown in Example 3. The detection results are shown below. Figure 6 Compared with the 0 h control group, the osteopontin yield showed an increasing trend in the 0-2 h range, with an osteopontin yield of 24.82% at 2 h and a relative yield of 120.46%; after 2 h, the osteopontin yield gradually decreased.

[0066] Using 2.5 mM sodium citrate as a fixed concentration, the effects of different low-temperature treatment times on the particle size and zeta potential of the casein system in the microfiltration whey were investigated. See [link to particle size results]. Figure 7Compared to the blank CN group (skim milk without added sodium citrate), the particle size of the sodium citrate-treated group increased to 162.0 nm after 0 h; as the holding time was extended to 2 h, the particle size decreased to the lowest of all groups at 144.9 nm; the particle size rebounded to 163.5 nm after 24 h and slightly decreased to 148.2 nm after 72 h, both higher than the 2 h group.

[0067] See potential results Figure 8 Adding sodium citrate can significantly increase the negative charge on the particle surface. The zeta potential of the 2 h treatment group reached -25.37 mV, and the electrostatic repulsion was better than that of the 0 h, 1 h, 24 h and 72 h groups.

[0068] Considering both particle size and surface charge characteristics, low-temperature treatment with 2.5 mM sodium citrate for 2 h is the optimal process to ensure stable dispersion of the casein system.

[0069] Based on a comprehensive assessment of osteopontin release yield, casein micelle size, and surface charge characteristics, a low-temperature reaction time of 2 hours was determined to be the optimal reaction time.

[0070] Comparative Example 1

[0071] Compared with Example 1, this comparative example replaces sodium citrate with EDTA and adjusts its concentration to 0, 0.5, 1, 2.5, 5, 10, and 20 mmol / L, while keeping the other steps the same as in Example 1.

[0072] Referring to Table 3, as the concentration of EDTA increases, the total color difference value ΔE of skim milk continues to increase: in the low concentration range of 0.5~2.5 mmol / L, ΔE is only 0.4~0.6, and the colloidal structure of skim milk only undergoes slight changes; when the concentration of EDTA increases to 5 mmol / L and above, the value of ΔE increases sharply, reaching 23.1 at 20 mmol / L, and the optical properties and colloidal dispersion state of skim milk undergo drastic changes, which are macroscopically manifested as obvious protein precipitation in the system.

[0073] See results for verification. Figure 9The osteopontin release rate increased with increasing EDTA concentration, reaching 14.45% at 20 mmol / L; precipitation began to occur in the system when the EDTA concentration exceeded 5 mmol / L. Considering both the osteopontin release effect and the microfiltration efficiency, 2.5 mmol / L EDTA was the optimal addition concentration, but the osteopontin release rate was low under this condition. In comparison, the OPN release rate under optimal EDTA conditions was only 14.52%, far lower than the peak release rate achieved using the sodium citrate process in this invention; moreover, EDTA easily damages casein micelles and produces precipitation, making casein recovery impossible. EDTA residues also pose food compliance risks, and high-concentration systems cannot be continuously microfiltered, making them unsuitable for industrial food production.

[0074] Table 3. Effects of different EDTA concentrations on color difference parameters of skim milk

[0075] Comparative Example 2

[0076] Raw material pretreatment: Fresh milk is taken and degreased by disc centrifugation. The fat content of the degreased milk is controlled to be <0.1%. The degreased milk is collected and pre-cooled at 6℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0077] Use a magnetic stirrer to continuously stir the skim milk at 180 rpm for 30 minutes.

[0078] Low-temperature co-processing: Transfer the skim milk to a sterile sealed container and store it in a 6°C constant temperature refrigerator for 2 hours, inverting the container once every 0.5 hours during this period.

[0079] Sampling and testing: After 2 hours, the osteopontin content in the skim milk was 61.36 mg / L, and the pH of the system remained stable at 6.8.

[0080] Microfiltration separation: A 0.1 μm pore size microfiltration membrane was used to filter the solution at a cross-flow velocity of 3 m / s under a pressure of 0.25 MPa, and the permeate was collected.

[0081] The osteopontin content in the permeate was determined using HPLC.

[0082] The results showed that the osteopontin content in the whey phase prepared in this comparative example was 6.56 mg / L, and the osteopontin yield was 18.58%. Based on the total mass of the whey liquid as 100%, the whey liquid contained: 0.23% protein, 0.076% fat, and 4.62% solids.

[0083] Example 5

[0084] This embodiment provides a method for increasing the osteopontin content in whey fluid, the specific steps of which are as follows: Raw material pretreatment: Fresh milk is taken and degreased by disc centrifugation. The fat content of the degreased milk is controlled to be <0.1%. The degreased milk is collected and pre-cooled at 4℃ for 30 minutes to stabilize the system temperature. All equipment and utensils that come into contact with the materials are sterilized.

[0085] Sodium citrate addition and mixing: Add 15 w / v% food-grade sodium citrate solution to the pre-cooled skim milk to adjust the final concentration of sodium citrate in the system to 2.5 mmol / L. Stir continuously for 30 minutes using a magnetic stirrer at 180 rpm to ensure uniform dispersion of sodium citrate and obtain a mixture.

[0086] Low-temperature co-processing: Transfer the above mixture to a sterile sealed container and store it in a 4°C constant temperature refrigerator for 2 hours, inverting the container once every 0.5 hours during this period.

[0087] Sampling and testing: Samples were taken and tested after 2 hours of low-temperature reaction. The osteopontin content in the mixture after the reaction was 75.09 mg / L, and the pH of the system was stable at 6.8.

[0088] Microfiltration separation: A 0.1 μm pore size microfiltration membrane was used to filter the solution at a cross-flow velocity of 3 m / s under a pressure of 0.25 MPa. The permeate was collected to obtain a natural whey containing osteopontin.

[0089] The osteopontin content of the natural whey obtained above was determined by HPLC.

[0090] The results showed that the osteopontin content in the whey phase prepared in this embodiment was 8.75 mg / L, which was 2.19 mg / L higher than that in Comparative Example 2, and the osteopontin yield was increased by 33.38%. Based on the total mass of the whey solution as 100%, the whey solution contained: 0.24% protein, 0.097% fat, and 3.61% solids.

[0091] Comparative Example 3

[0092] This comparative example provides a method for isolating osteopontin. Compared with Example 5, the low-temperature reaction time in Example 5 is adjusted to 0 hours, while other steps remain the same as in Example 5.

[0093] In this comparative example, the osteopontin content in the mixture was 67.56 mg / L; the osteopontin content in the whey obtained by microfiltration was 7.17 mg / L. Based on the total mass of the whey (100%), the whey contained: 0.14% protein, 0.099% fat, and 3.42% solids.

[0094] The above embodiments illustrate and describe the main features and advantages of the present invention, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for increasing the osteopontin content in whey, characterized in that, The method includes the following steps: (1) Add sodium citrate to the milk-containing raw material and stir to obtain a mixture; (2) The mixture is placed at 2-10°C for 0.5-2 hours and then microfiltered to obtain a whey solution containing osteopontin; The final concentration of sodium citrate in the mixture is 1-5 mM.

2. The method according to claim 1, characterized in that, In step (1), the stirring speed is 100-300 rpm and the time is 15-45 minutes.

3. The method according to claim 1, characterized in that, In step (2), the low-temperature treatment is performed at a temperature of 4-6°C for 2 hours.

4. The method according to claim 1, characterized in that, Before step (1), the process also includes a step of pre-cooling the milk-containing raw materials at 4-6°C for 10-60 minutes.

5. The method according to claim 1, characterized in that, The milk-containing raw material is skim milk, and the fat content of the skim milk is ≤0.5%.

6. The method according to claim 1, characterized in that, In step (2), the microfiltration separation step includes: using a microfiltration membrane with a pore size of 0.05μm-0.2μm to perform cross-flow filtration under a pressure of 0.1MPa-0.3MPa.

7. A whey liquid, characterized in that, The whey solution was prepared using the method described in any one of claims 1-6; The osteopontin content in the whey solution is 6.00-9.00 mg / L.

8. The whey liquid according to claim 7, characterized in that, Based on the total mass of whey liquid (100%), the protein content is 0.1-0.3%, the fat content is 0.05-0.3%, and the total solids content is 2-4%.

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