Establishment method and application of breast milk phospholipid evaluation model
By establishing a breast milk phospholipid evaluation model, infant formula formulas that are closer to breast milk phospholipid are screened out, which solves the problem of the difference between phospholipid supplements and breast milk phospholipids in existing infant formula milk powder and improves the nutritional quality of formula milk powder.
Patent Information
- Application Number
- CN202411683119.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-05-06
AI Technical Summary
There are significant differences between plant-derived phospholipid supplements used in existing infant formula and breast milk phospholipids, which fail to effectively simulate the diversity and structure of breast milk phospholipids.
A breast milk phospholipid evaluation model was established. By detecting the total amount of phospholipids, fatty acid content, different types of phospholipids and molecular structure of different natural phospholipids, the offset rate was calculated and the evaluation model was established based on the Topsis method, and formulas that were closer to breast milk phospholipids were screened out.
The breast-emulsified phospholipid formula screened through this evaluation model is closer to human breast-emulsified phospholipids in terms of total phospholipids, fatty acids, types and molecular structure, and improves the nutritional quality of infant formula.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of dairy product analysis and detection, and in particular to a method for establishing a breast milk phospholipid evaluation model and application thereof. Background Art
[0002] Breast milk phospholipids account for 0.2-2.0% of the total breast milk fat. It is a polar lipid with a hydrophobic tail and a hydrophilic head group. It is mainly composed of glycerophospholipids and sphingomyelin. Glycerophospholipids mainly contain phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). Breast milk phospholipids are rich in polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which play an indispensable role in the development of infants and young children. They have the effects of improving cognitive ability, promoting neurodevelopment, and anti-inflammatory. However, due to insufficient breast milk or the mother's ongoing drug treatment, some infants cannot accept breastfeeding. At this time, infant formula milk powder has become an ideal substitute for breast milk. However, at present, infant formula milk powder uses plant-derived phospholipids as supplements to simulate breast milk phospholipids, such as soybean phospholipids or sunflower phospholipids, and the addition form is single, focusing only on the total amount of phospholipids added in infant formula milk powder, and no more detailed simulation of breast milk phospholipids has been reported.
[0003] Buttermilk (BM) is a byproduct of fresh cream during the butter production process, which is mainly divided into sweet buttermilk and whey buttermilk. The composition of buttermilk is similar to that of skim milk, such as protein, lactose, ash, etc., but buttermilk is rich in polar lipids, because a large amount of milk fat globule membrane (MFGM) enters the buttermilk during the production process. The main lipids in buttermilk are triglycerides, accounting for about 65% of the total lipids. The remaining substances are mainly polar lipids, such as glycerophospholipids, sphingomyelin, gangliosides, etc., and their concentration is about 6 times that of skim milk. Studies have shown that these polar lipids are rich in polyunsaturated fatty acids such as linoleic acid and linolenic acid, and the phosphatidylcholine and sphingomyelin in buttermilk can provide choline, which can be used as an important source of choline for infant formula. In industrial production, buttermilk is often used as waste or used for animal feed processing due to its huge output. In recent years, with the in-depth study of milk fat globule membrane by researchers, buttermilk is often used as an important source of milk fat globule membrane separation, and then used in infant formula.
[0004] Currently, soybean lecithin is mostly used as a lecithin supplement in infant formula, and the addition form is single, resulting in significant differences between the lecithin in infant formula and the lecithin in breast milk. Summary of the invention
[0005] In order to better study the differences between different breast-milked phospholipid formulas or different types of natural phospholipids and breast milk phospholipids, the present invention first provides a method for establishing a breast milk phospholipid evaluation model, including: respectively detecting four indicators of total phospholipid content, phospholipid fatty acid content, content of different types of phospholipids and phospholipid molecular structure of different natural phospholipids, respectively comparing the similarities between natural phospholipids and human breast milk phospholipids in different indicators, calculating the deviation rates of natural phospholipids and human breast milk phospholipids in different indicators, assigning points to different indicators of natural phospholipids according to the deviation rates, and establishing a breast milk phospholipid evaluation model based on the Topsis method using the measured data of different indicators and the assigned scores.
[0006] Preferably, the total score of the four categories of indicators is 100 points, and the score of each category of indicators is 25 points.
[0007] Preferably, the higher the similarity between the different indicators of natural phospholipids and human breast milk phospholipids, the greater the assigned score.
[0008] Preferably, the natural lecithin is buttermilk lecithin, sunflower lecithin, egg lecithin and soybean lecithin.
[0009] Preferably, the method for separating and purifying buttermilk phospholipids comprises: washing the buttermilk with a SMUF solution and then centrifuging.
[0010] The present invention finds that the effect of removing protein when washing buttermilk with SMUF solution is better than that of removing protein by water washing method and acid precipitation method.
[0011] Preferably, the number of washings is 1 or more (eg, 2, 3, or 4 times), preferably 4 or more.
[0012] Preferably, the method for preparing buttermilk comprises: centrifuging raw milk using a disc centrifuge to obtain cream, and then whipping the cream to obtain buttermilk.
[0013] Furthermore, the present invention provides the application of the breast milk phospholipid evaluation model constructed by the establishment method in screening breast milk phospholipid formulas.
[0014] Furthermore, the present invention provides a breast milk phospholipid formula obtained by screening the breast milk phospholipid evaluation model, comprising: buttermilk phospholipids, egg phospholipids and soybean lecithin.
[0015] Preferably, the weight ratio of the buttermilk lecithin, egg lecithin and soybean lecithin is (4-5):(0.2-0.4):1.
[0016] Compared with the single soybean lecithin supplement in the current infant formula, the breast milk lecithin formula obtained by the above screening is closer to human breast milk lecithin in terms of total phospholipid amount, phospholipid fatty acids, phospholipid types, and phospholipid molecular structure, and can further improve the nutritional quality of infant formula.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a method for establishing a breast milk phospholipid evaluation model, which can better study the differences between different breast milk phospholipid formulas or different types of natural phospholipids and breast milk phospholipids, and a breast milk phospholipid formula that is closer to human breast milk phospholipids in terms of total phospholipid content, phospholipid fatty acids, phospholipid types and phospholipid molecular structure is obtained through screening by the breast milk phospholipid evaluation model. The breast milk phospholipid formula is applied to infant formula milk powder to improve the nutritional quality of the infant formula milk powder, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the electrophoresis diagram of buttermilk protein after washing buttermilk in water and SMUF solution, where M represents Marker, N represents buttermilk before washing, water 1 represents washing with water once, water 2 represents washing with water twice, water 3 represents washing with water three times, water 4 represents washing with water four times, S1 represents washing with SMUF solution once, S2 represents washing with SMUF solution twice, S3 represents washing with SMUF solution three times, and S4 represents washing with SMUF solution four times; 1 represents milk fat globule membrane protein, 2 represents casein, and 3 represents whey protein.
[0019] Figure 2 The figure is the electrophoresis diagram of casemilk protein obtained by acid precipitation and SMUF solution washing method, wherein M represents Marker, N represents buttermilk before washing, pH represents casemilk protein obtained by acid precipitation, S1 represents washing with SMUF solution once, S2 represents washing with SMUF solution twice, S3 represents washing with SMUF solution three times, S4 represents washing with SMUF solution four times, powder represents commercially available buttermilk powder, powder pH represents casemilk protein obtained by acid precipitation with commercially available buttermilk powder; 1 represents milk fat globule membrane protein, 2 represents casein, and 3 represents whey protein. DETAILED DESCRIPTION
[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0021] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.
[0022] Example 1 Isolation and purification of buttermilk This example uses different separation and purification methods to prepare buttermilk, and compares the effects of different separation and purification methods on removing protein from buttermilk. The steps are as follows: Raw milk was centrifuged using a disc centrifuge to obtain cream, which was then whipped to obtain buttermilk. (1) Purification of buttermilk by washing: The buttermilk was washed 1, 2, 3, and 4 times in deionized water and SMUF solution (homemade, containing Tris (0.02 mol / L), NaCl (0.05 mol / L), CaCl2 (0.003 mol / L), pH 6.7), respectively, and centrifuged again. (2) Purification of buttermilk by acid precipitation: Food-grade citric acid was added to the buttermilk to precipitate the buttermilk at 40°C, and then centrifuged at 1500g for 10 minutes to obtain buttermilk without casein, and then adjusted to the original pH.
[0023] The protein concentration was determined using a BCA kit, and the casein protein was qualitatively analyzed using SDS-PAGE. The protein concentration test results are shown in Table 1, and the SDS-PAGE results are shown in Table 2. Figure 1~Figure 2 shown.
[0024] Table 1 Protein concentration in emulsion obtained by different separation and purification methods
[0025] As shown in Table 1, the best effect of removing protein was achieved by washing buttermilk with SMUF solution for 4 times.
[0026] Example 2 Establishment of a breast milk phospholipid evaluation model 1. Separation and purification of buttermilk lecithin by gradient elution The total lipid sample (300 mg) of the buttermilk powder obtained after removing the protein using the SMUF solution in Example 1 was dissolved in 1 mL of chloroform / methanol (2:1, v / v). The total lipids were purified using a silica gel bonded cartridge. The bonded cartridge was first treated with hexane (3 mL), and then the sample was loaded into the bonded cartridge. Then, the non-polar lipids were eluted with 2 mL of n-hexane / ether (8:2, v / v) and 1 mL of n-hexane / ether (1:1, v / v). Next, the phospholipids were recovered by adding 6 mL of methanol and 2 mL of a mixture of chloroform / methanol / water (3:5:2, v / v). Finally, the purified chloroform was dried with nitrogen to obtain buttermilk phospholipids.
[0027] 2. Detection of phospholipid fatty acids by gas chromatography The buttermilk lecithin, purchased sunflower lecithin, egg lecithin, and soybean lecithin in step 1 were methylated with 300 μL of BF3 methanol (14%) at 100°C for 90 minutes. Then, hexane (600 μL) and saturated Na2CO3 solution (500 μL) were added, and the organic phase was collected. After removing trace water and passing through a 0.22 μm filter, the fatty acid composition of the lecithin was analyzed using an Agilent 8890A chromatograph equipped with a flame ionization detector.
[0028] The contents of phospholipid fatty acids in different ingredients are shown in Table 2.
[0029] Table 2 Phospholipid fatty acid content in different ingredients
[0030] 3. Determination of phospholipid types and molecules by HPLC-MS Buttermilk lecithin, sunflower lecithin, egg lecithin and soybean lecithin were dissolved in a mixture of chloroform and methanol (1 / 2, v / v). Then, 20 μL of the sample solution was pipetted into a liquid chromatography vial, and then methanol (containing 10 mmol / L ammonium formate and 0.1% formic acid) and internal standard solution were added to dilute the sample 50 times. The liquid chromatography setup process was as follows: mobile phase A was isopropanol / acetonitrile / formic acid (90 / 10 / 0.1, v / v / v), mobile phase B was acetonitrile / water / formic acid (70 / 30 / 0.1, v / v / v), and mobile phase C was methanol / water / ammonium hydroxide (90 / 10 / 0.1, v / v / v). Both mobile phases A and B contained 10 mmol / L ammonium formate. A BEH C18 column (1.7 μm, 2.1 mm ID × 100 mm) and a short C18 column (5 μm, 2.1 mm ID × 20 mm, rd-0121-c185, Higgins Analytical, Southborough, MA) were used in positive and negative ionization modes, respectively.
[0031] Mass spectrometry was performed by an API 4500 Q-TRAP mass spectrometer equipped with a data analysis and acquisition system. Nitrogen was used for the nebulizer and desolvator. The curtain gas was 25, the ion source gas (1) was 45, and the ion source gas (2) was 20. Positive ion multiple reaction monitoring (MRM) and negative ion multiple reaction monitoring modes were set at electrospray voltages of 5500 and 4500 at 500°C and 25°C, respectively. When running MRM-IDA-EPI, the information dependent acquisition (IDA) threshold was set to 200 counts per second (CPS) and the collision energy spread (CES) was set to 15. Finally, dynamic filling time (DFT) was used to prevent space charge.
[0032] The types and total amounts of phospholipids in different ingredients are shown in Table 3.
[0033] Table 3 Types and total amount of phospholipids in different ingredients
[0034] The contents of phospholipid molecules in different ingredients are shown in Table 4.
[0035] Table 4 Phospholipid content in different ingredients 4. Establishment of a breast milk phospholipid evaluation model A breast milk phospholipid evaluation model was established based on the Topsis method. The evaluation conditions included four categories, namely the total phospholipid content, phospholipid fatty acid content, the content of different types of phospholipids and phospholipid molecular structure.
[0036] Among them, G, G1, G2, G3, and G4 are the total scores of the similarity between the sample (natural phospholipid mixture) and breast milk phospholipids in the scoring model, the total score of the similarity between the total amount of phospholipids in the sample and breast milk phospholipids, the total score of the similarity between the relative content of phospholipid fatty acids and breast milk phospholipids, the total score of the similarity between the content of different phospholipids and breast milk phospholipids, and the total score of the similarity between the relative content of phospholipid molecules and breast milk phospholipids. The maximum scores are 100, 25, 25, 25, and 25, respectively.
[0037] They refer to the scores deducted for each evaluation indicator in different samples, and the calculation formula is: Among them, Di and They refer to the weight of a certain indicator in the sample in the total corresponding indicator. The value of Di is set to the average value of a certain indicator of breast milk phospholipids. The actual values of the natural phospholipid investigation indicators are compared with the standard value range of the corresponding indicators in breast milk phospholipids, and the deviation rate of each investigation indicator in natural phospholipids is calculated. Ci is the deviation rate of the measured value of a certain indicator in the natural phospholipid mixture deviates from its standard value. The calculation formula is: Where Ai represents the upper or lower limit of the reference value (95% confidence interval) of a certain indicator in breast milk phospholipids. When the value of a certain indicator content (Bi) in the sample is higher than the upper limit of the indicator content (Ai) in the standard reference group (breast milk phospholipids), the value of Ai takes its upper limit; conversely, if the value of Bi is lower than the lower limit of Ai, Ai takes its lower limit. In these two cases, the farther the value of Bi deviates from the value of Ai, the greater the floating rate of the fatty acid content, the greater the corresponding deduction item Ei value, the lower the total score of the four simulation conditions calculated by formula (2), (3), (4), (5), and the lower the final calculated sample total score G value, indicating that the difference between the fatty acid composition and position distribution of the test sample group and human milk fat (standard reference group) is greater, that is, their similarity is lower. If the value of Bi is within the reference value of Ai, at this time, the value of C is 0, and the value of E calculated by formula (6) is 0, then the final G value is infinitely close to 100 points. Among them, M = Max (Min (Ai) - Min (Bi), Max (Bi) - Max (Ai)).
[0038] Based on the test data in Tables 2 to 4, the conditions for physical mixing of natural phospholipids to simulate breast milk phospholipids were set as shown in Table 5, and a better breast milk phospholipid formula was obtained using the breast milk phospholipid evaluation model.
[0039] Table 5 Conditions for physical mixing of natural phospholipids to simulate breast milk phospholipids
[0040] After screening by the breast milk phospholipid evaluation model, several breast milk phospholipid formulas with higher scores were finally obtained, as shown in Table 6.
[0041] Table 6 Optimal breast milk lecithin formula
[0042] It can be seen that the optimal breast milk lecithin formula is 78.4% buttermilk lecithin, 4.4% egg lecithin, and 17.2% soybean lecithin.
[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for establishing a breast milk phospholipid evaluation model, characterized in that: include: Four indicators, including the total phospholipids content, phospholipid fatty acid content, content of different types of phospholipids and phospholipid molecular structure, of different natural phospholipids were tested respectively. The similarities between natural phospholipids and human breast milk phospholipids in different indicators were compared, and the deviation rates of natural phospholipids and human breast milk phospholipids in different indicators were calculated. Points were assigned to different indicators of natural phospholipids according to the deviation rates. An evaluation model for breast milk phospholipids was established based on the Topsis method using the measured data of different indicators and the assigned scores.
2. The establishment method according to claim 1, characterized in that: The total score of the four categories of indicators is 100 points, and the score of each indicator is 25 points.
3. The establishment method according to claim 1, characterized in that: The higher the similarity between the different indicators of natural phospholipids and human breast milk phospholipids, the greater the score assigned.
4. The establishment method according to claim 1, characterized in that: The natural lecithin is buttermilk lecithin, sunflower lecithin, egg lecithin and soybean lecithin.
5. The establishment method according to claim 4, characterized in that: The separation and purification method of buttermilk phospholipids comprises: washing buttermilk with SMUF solution and then centrifuging.
6. The establishment method according to claim 5, characterized in that: The number of washings is 1 or more, preferably 4 or more.
7. The establishment method according to claim 5, characterized in that: The method for preparing buttermilk comprises: centrifuging raw milk with a disc centrifuge to obtain cream, and then whipping the cream to obtain buttermilk.
8. Application of the breast milk phospholipid evaluation model constructed by the establishment method described in any one of claims 1 to 7 in screening breast milk phospholipid formulas.
9. A breast milk phospholipid formula, characterized in that: include: Buttermilk lecithin, egg lecithin and soy lecithin.
10. The breast milk phospholipid formula according to claim 9, characterized in that The weight ratio of the buttermilk lecithin, egg lecithin and soybean lecithin is (4-5):(0.2-0.4):1.