CN116990419B - Evaluation method and application of breast milk fat-soluble vitamins - Google Patents

Evaluation method and application of breast milk fat-soluble vitamins Download PDF

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CN116990419B
CN116990419B CN202311235817.2A CN202311235817A CN116990419B CN 116990419 B CN116990419 B CN 116990419B CN 202311235817 A CN202311235817 A CN 202311235817A CN 116990419 B CN116990419 B CN 116990419B
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陈历俊
刘妍
王亚玲
乔为仓
赵军英
张明辉
刘茜
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Beijing Sanyuan Foods Co Ltd
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Abstract

The invention relates to an evaluation method and application of breast milk fat-soluble vitamins, which are carried out according to the following steps: (1) Collecting a breast milk sample, and collecting breast milk information corresponding to the breast milk sample at the same time of collecting the breast milk sample, wherein the breast milk information comprises mother basic information, infant basic information, meal intake information and infant feeding information; (2) preprocessing a breast milk sample to obtain a sample to be detected; (3) Measuring the content of fat-soluble vitamins in a sample to be measured, combining the content with breast milk information, and establishing a model of the fat-soluble vitamins in the breast milk; (4) And calculating the content level of the corresponding fat-soluble vitamin in the breast milk according to the model evaluation. The method can effectively establish a dynamic regression model, and can effectively judge the content level of the fat-soluble vitamins in the breast milk according to the dietary condition of the mother so as to infer whether the fat-soluble vitamins in the breast milk meet the growth requirement of infants or not, and feed back the deficiency or excess types of the fat-soluble vitamins in the breast milk.

Description

Evaluation method and application of breast milk fat-soluble vitamins
Technical Field
The invention mainly relates to the field of breast milk evaluation, in particular to an evaluation method and application of breast milk fat-soluble vitamins.
Background
Vitamins are a number of different types of low molecular weight organic compounds with different structures and functions. The human beings continuously perfect the demands for the nutrient substances, and the vitamin content becomes an important index for evaluating the nutrient components. Breast milk is the most perfect source of nutrition for infants from 0 to 6 months. The vitamin component in breast milk also has high bioavailability at low concentration, and can maintain life activity (metabolism, development and oxidation resistance) of infant. Vitamins in breast milk are classified into water-soluble vitamins and fat-soluble vitamins in terms of nutrition according to the dissolution mode. The water-soluble vitamins mainly comprise vitamin C and B vitamins. The fat-soluble vitamins mainly include vitamin a (vitamin a and carotenoid), vitamin D, vitamin E, and vitamin K.
Beta-carotene is a precursor of vitamin a and is partially converted to vitamin a in humans. Beta-carotene has the ability to scavenge free radicals, can reduce lipid peroxidation, has oxidation resistance, and simultaneously has the function of providing immune protection.
Vitamin A has wide physiological functions, and participates in vision process, cell proliferation and differentiation, information communication among cells, growth of organs and tissues, reproduction and immune system functions. Vitamin A in breast milk can influence the growth and development, reproductive function, immune function, hematopoietic function and the integrity of skin mucous membrane of infants, and serious deficiency of vitamin A can lead to xerophthalmia and nyctalopia.
Vitamin E exists mainly in the form of alpha-tocopherol, and is used as a strong antioxidant, and has the effects of protecting cell membranes from peroxidation damage, maintaining fertility and the like.
Most of the vitamin detection in breast milk adopts liquid chromatography or liquid chromatography-mass spectrometry, and for the breast milk, the vitamin content level in the breast milk can be known only by the breast milk detection by a relevant detection mechanism, so that time and money are consumed. Meanwhile, the vitamin content in the breast milk is low, and the breast milk is lost to different degrees in the processes of collection, storage, transportation and testing. Since the vitamin content in breast milk is affected by lactation period, dietary intake, etc., these factors can be an important means for evaluating the vitamin content in breast milk.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for simply and quickly evaluating the content of fat-soluble vitamins in breast milk.
In order to achieve the above object, the present invention provides a method for evaluating fat-soluble vitamins in breast milk and application thereof, comprising the steps of:
(1) Collecting a breast milk sample, and collecting breast milk information corresponding to the breast milk sample at the same time of collecting the breast milk sample, wherein the breast milk information comprises mother basic information, infant basic information, meal intake information and infant feeding information;
(2) Pretreating a breast milk sample to obtain a sample to be tested;
(3) Measuring the content of fat-soluble vitamins in a sample to be measured, combining the content with breast milk information, and establishing a model of the fat-soluble vitamins in the breast milk;
(4) And (5) calculating the content of the corresponding fat-soluble vitamin in the breast milk according to the model evaluation.
Preferably or alternatively, the fat-soluble vitamins include vitamin a, vitamin E, beta-carotene.
Preferably or alternatively, the pretreatment method in step (2) is: adding an ethanol aqueous solution of vitamin C into a breast milk sample, then adding a potassium hydroxide solution, carrying out water bath saponification, cooling, adding ethanol, centrifuging to obtain a supernatant, purifying by a column, fixing the volume by using methanol, and filtering by a microporous filter membrane.
Preferably or alternatively, the temperature of the water bath saponification is 55 ℃ and the saponification time is 45min.
Preferably or alternatively, the centrifugation is at 6000rpm for 5min.
Preferably or alternatively, the pore size of the microporous filter membrane is 0.22 μm.
Preferably or alternatively, the step (3) of determining the fat-soluble vitamin content in the sample to be measured is performed using an ultra-high performance liquid chromatograph with a PDA detector.
The invention also provides application of the assessment method of the breast milk fat-soluble vitamins in the fields of nutritional consultation and diet suggestion.
From the above, the method can effectively establish a dynamic regression model, and can effectively judge the content level of fat-soluble vitamins in breast milk according to the dietary condition of a mother, thereby realizing the evaluation of the fat-soluble vitamins in the breast milk so as to infer whether the fat-soluble vitamins in the breast milk meet the growth needs of infants, avoid excessive intake risks and deficiency, prevent certain diseases, feed back the deficiency or excessive types of the fat-soluble vitamins in the breast milk, and give related diet suggestions in diet suggestions.
Drawings
FIG. 1 is a chromatogram of beta-carotene obtained by the method described in example 2;
FIG. 2 is a chromatogram of vitamin A obtained by the method described in example 3;
fig. 3 is a chromatogram of vitamin E obtained by the method described in example 4.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
In this embodiment, a method for collecting and preprocessing a breast milk sample is provided.
The method for collecting the breast milk sample comprises the following steps:
the method is characterized in that the breast milk samples are collected at a uniform time point, the physical indexes of lactates from which all the breast milk samples are derived are normal, and the infants are delivered at full term (38-42 weeks of gestation) without congenital or genetic diseases.
Lactating women need to empty one breast in the morning between 6:00 and 7:00 a.m. after which whole milk is collected from one breast (previously empty) in the morning between 9:00 and 11:00 a.m.. Mixing whole milk, packaging into 1mL sterile freezing tube, and storing in-80deg.C ultra-low temperature refrigerator.
In the process of collecting the breast milk sample, information collection is carried out simultaneously, and breast milk information corresponding to the breast milk sample is collected, wherein the breast milk information comprises mother basic information, infant basic information, meal intake information and infant feeding information.
Wherein, the mother basic information comprises name, age, delivery mode, milk time, height, weight, breast milk sample (front section, rear section, whole milk).
The infant basic information includes name, date of birth, length, weight, sex, length of birth and weight of birth.
The diet record information is filled in the intake conditions of various main and auxiliary foods by a diet review method, and the intake conditions of foods are recorded according to nine kinds of meat, fruits, vegetables, fish and shrimp, eggs, milk, cereals and potatoes, mixed beans, oil and fat, soybeans and nuts of the large kinds of livestock and poultry according to the second edition of Chinese Nutrition science.
The infant feeding information includes the number of breasts, the amount of breast milk intake, and the time of lactation.
The collected breast milk samples were stored at-80 ℃ and thawed at room temperature at the time of use.
A sample of 5g of breast milk from a single source was weighed and placed in a 50mL round bottom centrifuge tube, and 5mL of a 15g/L aqueous ethanol (9:1, v/v) solution of vitamin C was added and mixed well.
10mL of 1.25g/mL aqueous potassium hydroxide solution was added, mixed well, saponified in a 55℃water bath for 45min, taken out and cooled to room temperature, 10mL ethanol was added, mixed well, centrifuged at 6000rpm for 5min, and the supernatant was taken.
Adding 5mL of methanol and 5mL of water into a Cleanert Vitamin (1 g,12 mL) solid phase extraction column for activation, adding supernatant after the activation is completed, eluting with 10mL of ethanol water solution (prepared according to the volume ratio of 1:1) after the supernatant completely flows out through the column, discarding all effluent liquid, and pumping for 20min. Then eluting with 2mL of acetone and 8mL of ethyl acetate, collecting the eluent, then blowing nitrogen to dryness at 40 ℃, dissolving the residue with 1mL of methanol, fixing the volume, and filtering through a 0.22 mu m microporous filter membrane to obtain a sample to be detected.
Example 2
The sample to be tested prepared in the manner of example 1 was measured using an ultra performance liquid chromatograph with a PDA detector and the beta-carotene content of the breast milk sample was obtained.
In this example, the ultra high performance liquid chromatograph with PDA detector used was Waters ACQUITY UPLC H-Class, the chromatographic column was BEH C18 (2.1X10 mm,1.7 μm), the detection wavelength was 445nm, the column temperature was 35 ℃, the sample injection volume was 10. Mu.L, and the flow rate was 0.4mL/min.
The mobile phase A is methanol, the mobile phase B is acetonitrile, and the elution gradient is shown in the following table:
Time flow (mL/min) A% B% C%
0 0.4 20.0 76.0 4.0
9 0.4 80.0 19.0 1.0
10 0.4 20.0 76.0 4.0
13 0.4 20.0 76.0 4.0
Name of the name Principal forms English name Detection wavelength Retention time
Beta-carotene Beta-carotene β-carotene 445nm 6.75min
The results of the sample measurement are shown in FIG. 1.
After all 259 samples are measured, 127 of the samples are randomly selected to carry out correlation analysis on diet intake data, lactation time and measured beta-carotene content of the samples through SPSS statistical software, factors with the lowest significant difference at a 95% confidence level (P < 0.05) are selected, and a data model is established through single-factor analysis of variance and multiple linear regression analysis.
The SPSS analysis results are shown in the following table:
model B Sig.
Constant value 374.942 0.000
Lactation time -2.180 0.000
Vegetables 0.233 0.014
Fruits and the like 0.304 0.003
Milk -0.551 0.000
Soybean and nut -0.749 0.039
According to the table, the built data model is Y β = (-2.180×lactation time+0.233×vegetable intake+0.304×fruit intake-0.551×milk intake-0.749×soybean and nut intake+ 374.942)/100.
Wherein the food intake unit is gram of food intake of 24 hours, and the lactation time is days after the post-partum time of the lactating women.
Statistics of Y for each sample β The value sets the dimension and the results are shown in the following table:
dimension(s) Y β
0 -1.497
25% 1.029
50% 2.361
75% 3.334
100% 6.404
Y calculated by the model β The value is brought into the dimension table to obtain the level of the beta-carotene content of the breast milk sample in the whole lactating mother, and when the level is too low, such as lower than 25%, timely diet and nutrition intervention measures of the lactating mother can be suggested.
After measuring all 259 samples, the model was verified using the remaining 132 samples as a verification set, and Y was calculated as a model β The corresponding dimension of the value is consistent with the dimension of the actual measured content of the beta-carotene in the sample to be a correct result.
The significance value P of the model is less than 0.05, the correlation coefficient r=0.682 is obtained through correlation analysis, and the accuracy of the model is 73.21% through verification of a verification set. The results show that the model has good accuracy.
Example 3
The sample to be tested prepared in the manner of example 1 was measured using an ultra performance liquid chromatograph with a PDA detector and the vitamin a content of the breast milk sample was obtained.
In this example, the ultra high performance liquid chromatograph with PDA detector used was Waters ACQUITY UPLC H-Class, the chromatographic column was BEH C18 (2.1X10 mm,1.7 μm), the detection wavelength was 445nm, the column temperature was 35 ℃, the sample injection volume was 10. Mu.L, and the flow rate was 0.4mL/min.
The mobile phase A is methanol, the mobile phase B is acetonitrile, and the elution gradient is shown in the following table:
Time flow (mL/min) A% B% C%
0 0.4 20.0 76.0 4.0
9 0.4 80.0 19.0 1.0
10 0.4 20.0 76.0 4.0
13 0.4 20.0 76.0 4.0
Name of the name Principal forms English name Detection wavelength Retention time
Vitamin A Retinol Retinol 325nm 0.602min
The results of the sample measurement are shown in FIG. 2.
After all 240 samples were measured, 134 of them were randomly selected, and the dietary intake data, lactation time and measured vitamin a content of the samples were subjected to correlation analysis by SPSS statistical software, and the factors with the lowest significant difference at the 95% confidence level (P < 0.05) were selected, and a data model was established by single factor analysis of variance and multiple linear regression analysis.
The SPSS analysis results are shown in the following table:
model B Sig.
Constant value 50.754 0.000
Lactation time -0.231 0.000
Fish and shrimp 0.007 0.030
Eggs and eggs 0.037 0.001
According to the table, the built data model is Y A = -0.231 x lactation time +0.007 x shrimp intake +0.037 x egg intake +50.754.
Wherein the food intake unit is gram of food intake of 24 hours, and the lactation time is days after the post-partum time of the lactating women.
Statistics of Y for each sample A The value sets the dimension and the results are shown in the following table:
dimension(s) Y A
0 9.174
25% 26.306
50% 38.869
75% 48.044
100% 66.847
Y calculated by the model A The value is brought into the dimension table to obtain the level of the vitamin A content of the breast milk sample in the whole lactating mother, and when the level is too low, such as lower than 25%, timely diet and nutritional intervention measures of the lactating mother can be suggested.
After all 240 samples were measured, the model was validated using the remaining 106 samples as a validation set, and Y was calculated from the model A The corresponding dimension of the value is consistent with the dimension of the actual measured content of the vitamin A in the sample and is a correct result.
The significance value P of the model is less than 0.05 through correlation analysis, the correlation coefficient r=0.868, and the accuracy of the model is 71.05% through verification set verification. The results show that the model has good accuracy.
Example 4
The sample to be tested prepared in the manner of example 1 was measured using an ultra performance liquid chromatograph with a PDA detector and the vitamin E content of the breast milk sample was obtained.
In this example, the ultra high performance liquid chromatograph with PDA detector used was Waters ACQUITY UPLC H-Class, the chromatographic column was BEH C18 (2.1X10 mm,1.7 μm), the detection wavelength was 445nm, the column temperature was 35 ℃, the sample injection volume was 10. Mu.L, and the flow rate was 0.4mL/min.
The mobile phase A is methanol, the mobile phase B is acetonitrile, and the elution gradient is shown in the following table:
Time flow (mL/min) A% B% C%
0 0.4 20.0 76.0 4.0
9 0.4 80.0 19.0 1.0
10 0.4 20.0 76.0 4.0
13 0.4 20.0 76.0 4.0
Name of the name Principal forms English name Detection wavelength Retention time
Vitamin E Alpha-tocopherol DL-alpha Tocopherol 293nm 2.826min
The results of the sample measurement are shown in FIG. 3.
After all 248 samples are measured, 126 of the samples are randomly selected, correlation analysis is carried out on diet intake data, lactation time and measured vitamin E content of the samples through SPSS statistical software, factors with the lowest significance difference at a 95% confidence level (P < 0.05) are selected, and a data model is established through single-factor analysis of variance and multiple linear regression analysis.
The SPSS analysis results are shown in the following table:
model B Sig.
Constant value 269.666 0.000
Lactation time -1.316 0.002
Fish and shrimp 0.113 0.024
Eggs and eggs 0.377 0.042
Cereal potatoes and miscellaneous beans 0.030 0.035
According to the table, the built data model is Y E = -1.316 x lactation time +0.113 x shrimp intake +0.377 x egg intake +0.03 x cereal potato intake +269.666.
Wherein the food intake unit is gram of food intake of 24 hours, and the lactation time is days after the post-partum time of the lactating women.
Statistics of Y for each sample E The value sets the dimension and the results are shown in the following table:
dimension(s) Y E
0 32.631
25% 146.072
50% 206.622
75% 279.401
100% 486.108
Y calculated by the model E The value is brought into the dimension table to obtain the level of the beta-carotene content of the breast milk sample in the whole lactating mother, and when the level is too low, such as lower than 25%, timely diet and nutrition intervention measures of the lactating mother can be suggested.
After all 248 samples were measured, the model was validated using the remaining 122 samples as a validation set, and Y was calculated as a model E The corresponding dimension of the value is consistent with the dimension of the actual measured content of the vitamin E in the sample and is a correct result.
The significance value P <0.05 of the model is obtained through correlation analysis, the correlation coefficient r=0.888, and the accuracy of the model is 72.32% through verification of a verification set. The results show that the model has good accuracy.
From the above, the method can effectively establish a dynamic regression model, and can effectively judge the content level of fat-soluble vitamins in breast milk according to the dietary condition of a mother, thereby realizing the evaluation of the fat-soluble vitamins in the breast milk so as to infer whether the fat-soluble vitamins in the breast milk meet the growth needs of infants, avoid excessive intake risks and deficiency, prevent certain diseases, feed back the deficiency or excessive types of the fat-soluble vitamins in the breast milk, and give related diet suggestions in diet suggestions.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims (5)

1. A method for evaluating fat-soluble vitamins of breast milk, which is characterized in that the fat-soluble vitamins comprise vitamin A, vitamin E and beta-carotene, and the method comprises the following steps:
(1) Collecting a breast milk sample, and collecting breast milk information corresponding to the breast milk sample at the same time of collecting the breast milk sample, wherein the breast milk information comprises mother basic information, infant basic information, meal intake information and infant feeding information;
(2) Pretreating a breast milk sample to obtain a sample to be tested; the pretreatment method comprises the following steps: adding an ethanol aqueous solution of vitamin C into a breast milk sample, then adding a potassium hydroxide solution, carrying out water bath saponification, cooling, adding ethanol, centrifuging to obtain a supernatant, purifying by a column, fixing the volume by using methanol, and filtering by a microporous filter membrane to obtain the vitamin C-containing milk;
(3) Measuring the content of fat-soluble vitamins in a sample to be measured by adopting an ultra-high performance liquid chromatograph with a PDA detector, combining the content with breast milk information, and establishing a model of the fat-soluble vitamins in the breast milk;
beta-carotene model Y β = (-2.180×lactation time+0.233×vegetable intake+0.304×fruit intake-0.551×milk intake-0.749×soybean and nut intake+ 374.942)/100;
vitamin A model Y A -0.231 x lactation time +0.007 x shrimp intake +0.037 x egg intake +50.754;
vitamin E model Y E -1.316 x lactation time +0.113 x shrimp intake +0.377 x egg intake +0.03 x cereal potato intake +269.666;
wherein the intake unit of each type of food is the gram number of the intake of the food in 24 hours, and the lactation time is the number of days of post-natal time of the lactating women;
the ultra-high performance liquid chromatograph with the PDA detector is Waters ACQUITY UPLC H-Class, the chromatographic column is BEH C18, the column temperature is 35 ℃, the sample injection volume is 10 mu L, and the flow rate is 0.4mL/min;
the detection wavelengths for measuring beta-carotene, vitamin A and vitamin E are 445nm, 325nm and 293nm respectively; (4) Calculating the content level of the corresponding fat-soluble vitamin in the breast milk according to the model evaluation;
y calculated by each model β 、Y A And Y E The values are brought into each dimension table to obtain the content level of beta-carotene, vitamin A and vitamin E in the breast milk sample:
。
2. the method for evaluating a fat-soluble vitamin of breast milk according to claim 1, wherein the temperature of the saponification in the water bath is 55 ℃ and the saponification time is 45min.
3. The method for evaluating a fat-soluble vitamin in breast milk according to claim 1, wherein the centrifugation is at 6000rpm for 5min.
4. The method for evaluating a fat-soluble vitamin in breast milk according to claim 1, wherein the pore size of the microporous filter membrane is 0.22 μm.
5. Use of the method according to any one of claims 1-4 in the field of nutritional counseling and meal suggestion.
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