A method for extracting and separating milk fat globule membranes from butter wastewater

Abstract

This invention belongs to the field of milk fat globule membrane separation and extraction technology, specifically relating to a method for extracting and separating milk fat globule membranes from cream wastewater. This method utilizes the gas-liquid interface of micro-nano bubbles introduced into the cream wastewater to simply and efficiently adsorb, separate, and enrich the milk fat globule membrane, followed by ultrasonic and filtration concentration. Compared with MFGM separation and enrichment technologies based on enzymatic methods, thermal treatment, or organic solvent extraction, this method has the advantages of being more environmentally friendly, energy-saving, and simple, making it suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of milk fat globule membrane separation and extraction technology, specifically relating to a method for extracting and separating milk fat globule membranes from cream wastewater. Background Technology

[0002] Milk fat globules are secreted by mammary epithelial cells and consist of triglycerides containing different fatty acids. They are enclosed by a unique three-layer membrane structure (an outer double layer and an inner single layer), which is composed of polar lipids, cholesterol, and proteins. This membrane is called the milk fat globule membrane (MFGM) and accounts for more than 90% of the dry weight of milk fat globules. MFGM has broad application prospects due to its various benefits, including improving gut health, promoting brain development, anti-cancer properties, antioxidant effects, and improving frailty in the elderly.

[0003] Cheese byproducts or buttermilk wastewater containing whey and buttermilk are the main sources for separating MFGM. Whey, the liquid portion discharged during cheese production, contains whey protein, as well as small amounts of casein, fat, lactose, and trace amounts of MFGM and minerals. Buttermilk, the liquid discharged after churning butter during butter processing, contains whey protein, casein, lactose, and a small amount of fat, but also is rich in MFGM, with an MFGM content approximately 2-5 times higher than that of whey. Currently, there are various methods for extracting MFGM from milk, byproducts, and wastewater, including centrifugation, enzymatic methods, heat treatment, ultrasound, ultrafiltration, pulsed electric field extraction, and organic solvent extraction.

[0004] The current difficulties in extracting MFGM include: (1) the content of MFGM in the raw material is low, and the extraction process is complex; (2) because the particle size of MFGM (100-4000 nm) overlaps with that of casein micelles (50-600 nm), and the isoelectric point of MFGM components is close to that of casein micelles, it is easy to be extracted together with casein during separation, which affects the purity of the product; (3) the structure of MFGM is unstable, and it is easy to break and form smaller membrane fragments during purification, resulting in loss due to lack of collection; (4) methods such as organic solvent extraction are prone to introducing other additional harmful components or unnecessary impurities, affecting the safety of the product in the food field.

[0005] To address the aforementioned issues, CN202011263223.9 (A method for separating and preparing milk fat globule membrane protein from buttermilk, a byproduct of butter) provides a method for preparing MFGM, which requires complex steps such as heating, acidification, centrifugation, hot calcium treatment, ultrasonic treatment, dialysis, and ultrafiltration to increase the product purity to 80%~95%. CN202310585228.0 (A method for separating and preparing milk fat globule membrane) provides a method for preparing MFGM, which has a relatively high content of milk fat globule membrane protein, but requires fresh milk as raw material and treatment with rennet, and the process is complex with an extraction rate of less than 85% for milk fat globule membrane protein. Summary of the Invention

[0006] To address the above problems, the purpose of this invention is to provide a method for extracting and separating milk fat globule membranes from cream wastewater. This invention includes the following technical solution:

[0007] A method for extracting and separating milk fat globule membranes from cream wastewater, the method comprising the following steps:

[0008] S1 Butter Wastewater Pretreatment: The butter wastewater is filtered through a screen to remove insoluble matter, resulting in pretreated butter wastewater.

[0009] S2 Primary enrichment of milk fat globule membrane: The pretreated cream wastewater obtained in step S1 was treated using a micro-nano bubble generator, and the upper foam layer was collected.

[0010] S5 Drying: The obtained foam layer product is dried to obtain milk fat globule film dry powder.

[0011] Preferably, in step S2, the flow rate of the micro-nano bubble generator is 20 L / h to 200 L / h, and the processing time for each continuous introduction of micro-nano bubbles is 10 min to 30 min.

[0012] Preferably, in step S2, the micro-nano bubble generator processes the material 1 to 5 times.

[0013] Preferably, the extraction and separation method further includes the following steps:

[0014] S3 Ultrasonic treatment: The foam layer obtained in step S2 is ultrasonically treated to obtain ultrasonic products;

[0015] S4 Secondary enrichment of milk fat globule membrane: The ultrasonic product obtained in step S3 is filtered through an ultrafiltration membrane, and the concentrate is collected as the secondary enrichment product.

[0016] The ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 3kDa to 110kDa.

[0017] Preferably, in step S3, the ultrasonic treatment power is 150W~450W, and the ultrasonic treatment time is 5min~30min.

[0018] Preferably, in step S4, the liquid temperature during ultrafiltration is 10℃~25℃.

[0019] Preferably, in step S4, the concentration factor of the membrane filtration treatment is 5 to 10 times.

[0020] Preferably, the extraction and separation method further includes the following steps:

[0021] S5 Drying: The secondary enrichment product from step S4 is dried to obtain milk fat globule membrane powder.

[0022] Preferably, the drying method is one of freeze drying and spray drying.

[0023] Beneficial effects:

[0024] The method for preparing milk fat globule membrane of the present invention uses cream wastewater as raw material, without the need to add additional enzymes, organic solvents, etc. The process steps are simple and the product has a high content of milk fat globule membrane protein and phospholipid, making it extremely suitable for large-scale industrial production.

[0025] Micro- and nanobubbles (typically referring to groups of microbubbles with a diameter of 10-50 micrometers and nanobubbles smaller than 200 nanometers) possess a large specific surface area and persistent interfacial properties, and they rise slowly in liquids. This invention has discovered that the gas-liquid interface of micro- and nanobubbles can be used to simply and efficiently adsorb and separate milk fat globule membranes, achieving highly efficient separation and enrichment. Compared to centrifugation-based MFGM separation and enrichment techniques, micro- and nanobubble technology offers advantages in terms of energy efficiency and high separation efficiency. Compared to MFGM separation and enrichment techniques based on enzymatic methods, thermal treatment, or organic solvent extraction, it is more environmentally friendly, energy-efficient, and simple, making it highly suitable for industrial application. Attached Figure Description

[0026] Figure 1 The images show actual samples of cream wastewater from different steps in Experiment 1 of Example 1. (A) is pretreated cream wastewater after sieving, (B) is untreated cream wastewater, and (C) is cream wastewater after removing the foam layer.

[0027] Figure 2The images show the sample processing of different experimental examples in Example 1. (A) is the cream wastewater at the beginning of the micro-nano bubble treatment in Example 2 of Example 1; (B) is the cream wastewater after 10 min of micro-nano bubble treatment in Example 2 of Example 1; (C) is the cream wastewater after 20 min of micro-nano bubble treatment in Example 2 of Example 1; (D) is the cream wastewater after 20 min of the third micro-nano bubble treatment in Example 5 of Example 1; (E) is the sample obtained by combining the three foam layers in Example 5 of Example 1; and (F) is the cream wastewater sample after the foam layer is removed in Example 5 of Example 1.

[0028] Figure 3 The images shown are of the products prepared in Examples 6, 8, and 9 of Example 2. (A) is the product of the ultrasonic step in Example 6 of Example 2; (B) is the sample of the 30kDa ultrafiltration membrane that did not permeate after treatment in Example 8 of Example 2; (C) is the sample of the 30kDa ultrafiltration membrane that permeated after treatment in Example 8 of Example 2; (D) is the sample of the 10kDa ultrafiltration membrane that did not permeate after treatment in Example 9 of Example 2; and (E) is the sample of the 10kDa ultrafiltration membrane that permeated after treatment in Example 9 of Example 2. Detailed Implementation

[0029] The technical solutions and effects of the present invention will be shown and described below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection.

[0030] In the following embodiments, the micro-nano bubble generator used is a commercially available micro-nano bubble generator (micro-nano bubbles are a group of bubbles composed of micron bubbles with a particle size of 10-50 micrometers and nano bubbles with a particle size of less than 200 nanometers).

[0031] In the following examples, the cream wastewater is from the same enterprise and the same preparation process. This refers to cream wastewater generated during the centrifugal process for preparing cream (milk). The main byproducts in this wastewater are buttermilk and whey, and it contains milk fat globule membranes. For example, in patent document CN116636562A, the product obtained after centrifugation and whey removal from the preparation of light cream contains buttermilk, whey, and milk fat globule membranes; the byproducts of butter preparation in patent document CN114478739A also contain buttermilk and milk fat globule membranes. In the following examples, the cream wastewater used in the experimental examples and comparative examples of the same embodiment is from the same batch; the cream wastewater used in different embodiments is from different batches.

[0032] The testing methods for different indicators in the following examples are as follows:

[0033] Milk fat globule membrane (MFGM) protein analysis: MFGM protein in the sample was analyzed by polyacrylamide gel electrophoresis. For details, please refer to patent document CN116636562A.

[0034] Detection of phospholipids: Phospholipids in buttermilk were detected using high-performance liquid chromatography combined with evaporative light scattering detector (HPLC-ELSD). The extracted total fat was redissolved in 1 g chloroform / methanol (2:1, v / v) solution, and separation was performed using a Zorbax Rx-SIL silica gel column. 10 µL of the sample was loaded. Mobile phase A was chloroform-methanol-ammonia (80:19.5:0.5, v / v / v), and mobile phase B was chloroform-methanol-ammonia-water (60:34:0.5:5.5, v / v / v / v). The elution gradient was 0 min, 100% A; 14 min, 0% A; 23 min, 0% A; 35 min, 100% A; 40 min, 100% A. The evaporative light scattering detector temperature was 55 °C, the nitrogen generator carrier gas flow rate was 1.5 L / min, and the gain was 1. Single and mixed standards of different concentration gradients of phosphatidylethanolamine (PE), lecithin (PC), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) were injected to confirm the retention time of each type of phospholipid and to complete further quantification.

[0035] The MFGM protein extraction rate is the ratio of the intensity of the MFGM protein band in the treated sample to the intensity of the MFGM protein band in the untreated sample.

[0036] Phospholipid extraction rate is the ratio of phospholipid content in the treated sample to that in the untreated sample.

[0037] Unless otherwise specified, the filtration, micro / nano bubble treatment, and ultrasonic treatment in the following examples do not require additional heating; that is, the treatment temperature is room temperature. If the temperature is too high during ultrafiltration, it should be controlled below 25°C (e.g., 10°C to 25°C, with 20°C as an example in the specific examples).

[0038] Example 1: Primary enrichment of MFGM

[0039] This embodiment examines and compares the enrichment effects of different MFGM single-enrichment methods, involving 5 embodiments and 1 comparative example.

[0040] The MFGM primary enrichment method in Experiment Example 1 is as follows:

[0041] (1) Pretreatment of butter wastewater: Collect the wastewater containing whey and buttermilk generated during butter processing, filter it through a sieve (200 mesh) to remove solid insoluble matter in the butter wastewater, and obtain pretreated butter wastewater.

[0042] (2) Enrichment of MFGM: A large number of micro-nano bubbles were continuously introduced into the pretreated cream wastewater through a micro-nano bubble generator (the micro-nano bubbles were introduced from the bottom of the cream wastewater at a flow rate of 50 L / h; air was introduced, not ozone). After 10 min of micro-nano bubble treatment, the foam layer on the surface of the wastewater was collected.

[0043] (3) Vacuum freeze drying: Collect the foam layer and freeze dry it: Pre-freeze at a pre-freezing temperature of -30℃ for 3h; sublime dry at a sublimation drying temperature of -20℃ for 24h, and sublimation drying pressure of 20Pa; desorption dry at a desorption drying temperature of 35℃ for 3h, and desorption drying pressure of 20Pa to obtain the sample of Experimental Example 1.

[0044] Figure 1 These are actual photos of cream wastewater samples taken at different steps in this experiment.

[0045] The only difference between the MFGM one-time enrichment method in Experiment 2 and Experiment 1 is that the micro-nano bubble treatment time is 20 min, and the obtained sample is the same as that in Experiment 2.

[0046] The only difference between the MFGM one-time enrichment method in Experiment 3 and Experiment 1 is that the micro-nano bubble treatment time is 30 min, and the obtained sample is the same as that in Experiment 3.

[0047] The only difference between the MFGM enrichment method in Experiment 4 and that in Experiment 2 is that: after micro-nano bubble treatment for 20 min, the foam layer on the surface of the wastewater is collected, and then deionized water is added to the remaining pretreated cream wastewater. After micro-nano bubble treatment for 20 min again using the same method, the foam layer on the surface of the wastewater is collected. The foam layers obtained from the two treatments are combined and then freeze-dried under vacuum. The sample obtained is the sample of Experiment 4.

[0048] The only difference between the MFGM enrichment method in Experiment 5 and that in Experiment 4 is that after two micro-nano bubble treatments, deionized water was added to the remaining pretreated cream wastewater according to the second micro-nano bubble treatment method, and the foam layer on the surface of the wastewater was collected after another 20 minutes of micro-nano bubble treatment using the same method. The foam layers obtained from the three treatments were combined and then freeze-dried under vacuum. The sample obtained was the sample of Experiment 5.

[0049] Figure 2 Images of the samples prepared in Experiments 2 and 5 are shown. It can be seen that the treatment time for micro / nano bubble water increases with the extension of the treatment time (within the range of 0-20 min). Figure 2 A, B, C); As the number of micro-nano bubble treatments increased, by the third micro-nano bubble treatment, the amount of foam was reduced, and the total amount of foam obtained from enrichment was greater than that obtained from a single micro-nano bubble treatment, and the color of the cream wastewater tended to be milky white. Figure 2 (D, E, F).

[0050] The MFGM enrichment method for Comparative Example 1: The pretreated cream wastewater was centrifuged at 3000g for 5 min, and the suspended solids containing milk fat globule membranes in the upper layer were collected. The solids were then freeze-dried under vacuum, and the resulting sample was the sample of Comparative Example 1.

[0051] The extraction rates (%) of MFGM protein and phospholipids of the above experimental and comparative samples were detected and calculated, and the results are shown in Table 1.

[0052] Table 1. MFGM extraction rates of different samples using a single-step MFGM enrichment method

[0053] As can be seen from Table 1, the extraction rates of MFGM protein and phospholipids gradually increased with the increase of micro-nano bubble treatment time. This indicates that micro-nano bubbles can separate the milk fat globule membrane (including MFGM protein and phospholipids) in the cream wastewater in the form of foam. Although the extraction rates of MFGM protein and phospholipids after 30 min of treatment (Experiment 3) were higher than those after 20 min of treatment (Experiment 2), the improvement in extraction rate was not significant. Therefore, 20 min can be selected as the optimal time for one treatment with micro-nano bubbles.

[0054] With increasing micro / nanobubble treatment cycles, both MFGM protein and phospholipid extraction rates significantly increased, indicating that multiple micro / nanobubble treatments can improve the separation of milk fat globule membrane proteins from cream wastewater. The effects of two and three micro / nanobubble treatments were significantly higher than one treatment. The MFGM protein extraction rate after three micro / nanobubble treatments was slightly higher than that after two treatments, but the difference was not significant. The phospholipid extraction rate after three micro / nanobubble treatments was significantly higher than that after two treatments. Since three micro / nanobubble treatments are time-consuming, two treatments are the recommended number of cycles.

[0055] In contrast, the extraction rates of MFGM protein and phospholipids using the method in Comparative Example 1 were less than 60%, which is far lower than the extraction and separation method of this invention.

[0056] Example 2: Subsequent Refining of MFGM

[0057] This embodiment further explores the subsequent purification method of MFGM based on Example 1, involving 5 experimental cases and 1 comparative example.

[0058] Sample preparation for Experiment Example 6:

[0059] (1) Pretreatment of butter wastewater: Collect the wastewater containing whey and buttermilk generated during butter processing, filter it through a sieve (200 mesh) to remove solid insoluble matter in the butter wastewater, and obtain pretreated butter wastewater.

[0060] (2) Enrichment of MFGM: A large number of micro-nano bubbles are generated in the pretreated cream wastewater by a micro-nano bubble generator. After micro-nano bubble treatment (bubbles are introduced from the bottom and the gas flow rate is 50L / h) for 20 minutes, the foam layer on the surface of the wastewater is collected. Then, deionized water is added to the remaining cream wastewater and micro-nano bubble treatment is carried out again in the same way. After micro-nano bubble treatment for 20 minutes, the foam layer on the surface of the wastewater is collected and the foam layers obtained from the two micro-nano bubble treatments are combined.

[0061] (3) Ultrasonic treatment: Mix the foam layers collected twice and treat them with ultrasound for 10 minutes at a power of 350W to obtain the ultrasonic product.

[0062] (4) Secondary enrichment of milk fat globule membrane: The ultrasonic product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, with the temperature controlled below 25°C, the transmembrane pressure of about 3 bar, and the feed pump running at 35 Hz. Ultrafiltration continued until a volume concentration factor of ten times was achieved, and the concentrate was collected as the secondary enrichment product.

[0063] (5) Vacuum freeze drying: The secondary enriched product was freeze dried at a pre-freezing temperature of -30℃ for 3 hours; sublimation drying at a sublimation drying temperature of -20℃ for 24 hours and a sublimation drying pressure of 20Pa; and desorption drying at a desorption drying temperature of 35℃ for 3 hours and a desorption drying pressure of 20Pa to obtain the sample of Experimental Example 6.

[0064] The only difference between the sample preparation method in Experiment 7 and that in Experiment 6 is that the molecular weight cutoff of the ultrafiltration membrane used for secondary enrichment is 50 kDa.

[0065] The sample preparation method in Experiment 8 differs from that in Experiment 6 only in that the molecular weight cutoff of the ultrafiltration membrane used for secondary enrichment is 30 kDa.

[0066] The only difference between the sample preparation method in Experiment 9 and that in Experiment 6 is that the molecular weight cutoff of the ultrafiltration membrane used for secondary enrichment is 10 kDa.

[0067] The sample preparation method in Experiment 10 differs from that in Experiment 6 only in that the molecular weight cutoff of the ultrafiltration membrane used for secondary enrichment is 5 kDa.

[0068] Figure 3 The images show the products prepared in Experiments 6, 8, and 9.

[0069] The sample preparation method of Comparative Example 2 differs from that of Experimental Example 6 only in that the primary enrichment method in steps (1)-(2) adopts the primary enrichment method of Comparative Example 1 in Example 1, while the other ultrasonic treatment, secondary enrichment, freeze-drying methods and process parameters are the same.

[0070] The samples from Experimental Examples 6-10 and Comparative Example 2 were analyzed to determine the effect of membrane pore size (molecular weight cutoff) on the extraction efficiency of MFGM protein and phospholipids during ultrafiltration. The relative contents of MFGM protein and phospholipids in the treated samples (relative to the relative contents of MFGM protein and phospholipids in untreated butter wastewater) were analyzed. The results are shown in Table 2.

[0071] Table 2. Analytical results of samples from Experimental Examples 6-10 and Comparative Example 2

[0072] As shown in Table 2, when the membrane molecular weight cutoff is 5 kDa and 10 kDa, MFGM protein and phospholipids can be almost completely retained. When the membrane molecular weight cutoff is 100 kDa, 50 kDa, and 30 kDa, a small portion of MFGM protein and phospholipids permeate and are lost. Furthermore, the permeation rate of MFGM protein and phospholipids increases with increasing pore size. While decreasing the membrane pore size significantly improves the retention of MFGM protein and phospholipids, it also significantly reduces the membrane flux. Therefore, considering all factors, a 30 kDa molecular weight cutoff is the preferred pore size (molecular weight cutoff) for ultrafiltration. A comparison of the results from Experiments 6-10 and Comparative Example 2 shows that when ultrasound, ultrafiltration, and the single-enrichment method of Comparative Example 1 are combined, the extraction effect of MFGM protein and phospholipids from butter wastewater is very limited.

[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for extracting and separating milk fat globule membranes from cream wastewater, characterized in that, The extraction and separation method includes the following steps: S1 Butter Wastewater Pretreatment: The butter wastewater is filtered through a screen to remove insoluble matter, resulting in pretreated butter wastewater. S2 Primary enrichment of milk fat globule membrane: The pretreated cream wastewater obtained in step S1 was treated using a micro-nano bubble generator, and the upper foam layer was collected. S5 Drying: The obtained foam layer product is dried to obtain milk fat globule film dry powder.

2. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 1, characterized in that, In step S2, the flow rate of the micro-nano bubble generated by the micro-nano bubble generator is 20L / h to 200L / h, and the processing time for each continuous introduction of micro-nano bubbles is 10min to 30min.

3. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 2, characterized in that, In step S2, the micro-nano bubble generator is used to process the material 1 to 5 times.

4. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 1, characterized in that, The extraction and separation method further includes the following steps: S3 Ultrasonic treatment: The foam layer obtained in step S2 is ultrasonically treated to obtain ultrasonic products; S4 Secondary enrichment of milk fat globule membrane: The ultrasonic product obtained in step S3 is filtered through an ultrafiltration membrane, and the concentrate is collected as the secondary enrichment product. The ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 3kDa to 110kDa.

5. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 4, characterized in that, In step S3, the ultrasonic treatment power is 150W~450W, and the ultrasonic treatment time is 5min~30min.

6. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 4, characterized in that, In step S4, the liquid temperature is 10℃~25℃ during ultrafiltration.

7. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 4, characterized in that, In step S4, the concentration factor of the ultrafiltration membrane filtration process is 5 to 10 times.

8. The method for extracting and separating milk fat globule membranes from cream wastewater according to claim 1, characterized in that, The drying method is either freeze drying or spray drying.

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