KR101655048B1 - Method for the assessment of mother's milk quality which exposed to trans fat - Google Patents

Method for the assessment of mother's milk quality which exposed to trans fat Download PDF

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KR101655048B1
KR101655048B1 KR1020150166290A KR20150166290A KR101655048B1 KR 101655048 B1 KR101655048 B1 KR 101655048B1 KR 1020150166290 A KR1020150166290 A KR 1020150166290A KR 20150166290 A KR20150166290 A KR 20150166290A KR 101655048 B1 KR101655048 B1 KR 101655048B1
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breast milk
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breast
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조경현
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영남대학교 산학협력단
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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    • G01—MEASURING; TESTING
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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Abstract

The present invention can be used to analyze the content of total cholesterol in breast milk, the content of triglyceride, the content of glucose, the content of protein, the content of active oxygen species, the content of lactalbumin, the expression of apoA-I or the molecular weight of apoA- This study provides a method for evaluating the quality of breast milk exposed to trans fats, which confirms the survival rate of embryos after microinjection into the zebrafish embryo. Through this, the quality of breast milk can be confirmed and the effect of trans fat intake in breast milk To contribute to healthy growth and development of newborn infants.

Description

A method for evaluating the quality of breast milk exposed to trans fats

The present invention relates to a method for evaluating the quality of breast milk exposed to trans fats.

Breastfeeding is known to be more beneficial than nutritional, immunological, and emotional nutrition. To date, breast-fed infants have a lower morbidity rate, including otitis media, gastrointestinal infections, urinary tract infections, Haemophilus influenza infection, and neonatal sepsis, compared with infants receiving artificial nutrition, The morbidity and mortality rates of infants aged 6 to 6 years were low. The World Health Organization (WHO) recommends that breastfeeding is the ideal nutritional source for the healthy growth and development of infants and breastfeeding until 6 months of age.

Breastfeeding is influenced by the duration of breastfeeding after feeding, the nutrient intake of the breastfeeding group and the amount and composition of body fat of the breastfeeding group. Recently, as the eating habit is westernized, the transfusion fat of the breastfeeding body is transferred to the newborn through breastfeeding It was reported to be transmitted and caused a great wave.

In the 1990s, a Canadian study found that trans fatty acids were consumed at 8.4 grams of trans fat per person on the basis of trans fat content in breast milk. This means that 3.7% of the total energy is consumed as trans fat, which is more than four times higher than WHO's recommendation for trans fat intake of less than 1% per day.

Toxicities caused by trans fats consumed by lactating women are transmitted to the fetus or newborn through breastfeeding, and research is needed to determine the quality of breast milk.

1. Korean Patent No. 10-0930996.

Accordingly, it is an object of the present invention to provide a method for evaluating the quality of breast milk exposed to trans fats.

In order to achieve the above object, the present invention provides a method for preventing breast cancer, comprising the steps of: obtaining breast milk from a trans fat- The content of total cholesterol in breast milk, the content of triglyceride, the content of glucose, the content of protein, the content of active oxygen species, the content of lactalbumin, the expression of apolipoprotein AI and the molecular weight of apolipoprotein AI Analyzing at least one indicator selected from the group consisting of the group; And comparing the measured value with the index of the breast milk obtained from the normal lactation part. The present invention also provides a method for evaluating the quality of breast milk exposed to trans fats.

The present invention also provides a method of treating breast cancer, comprising the steps of: obtaining breast milk from a trans fat- Microfilling the breast milk into a zebrafish embryo; Confirming the survival rate of the zebrafish embryo; And comparing the survival rate of the zebrafish infused with breast milk obtained from the normal breast-feeding section; The present invention provides a method for evaluating the quality of breast milk exposed to trans fats.

According to the present invention, the content of total cholesterol, triglyceride, glucose, protein, active oxygen species, lactalbumin, apoA-I expression, Breast milk quality can be confirmed by microinjecting breast milk into zebrafish embryos and confirming the survival rate of the embryo, thereby contributing to the healthy growth and development of newborn infants by detecting the effect of trans fat intake on breast milk .

FIG. 1 shows the results of measurement of the total cholesterol content of breast milk or milk powder in the breastfeeding portion of the normal breastfeeding unit,
FIG. 2 shows the results of measurement of the triglyceride content of breast milk or milk powder in the breastfeeding portion of the normal breastfeeding unit,
FIG. 3 shows the results of measurement of total glucose content of breast milk or formula of breast-fed breast milk exposed to normal breast milk,
FIG. 4 shows the results of measurement of the total protein content of breast milk or milk powder in the breastfeeding portion of the normal breastfeeding unit,
FIG. 5 shows the results of examining the amount and molecular weight of apolipoprotein AI (apoA-I) expressed in the breast milk of the breastfeeding portion of the normal breast-fed breast-fed or trans-fat exposed breast.
FIG. 6 shows the results of confirming the survival rate of zebrafish embryos after microinjection of breast milk or milk powder in the breastfeeding portion of the normal breastfeeding portion,
FIG. 7 is a graph showing the relationship between total cholesterol and the embryo survival rate of breast milk in the transgender exposure-risk-lactating women,
FIG. 8 is a graph showing the relationship between the triglyceride of breast milk and the embryo survival rate in the lactating women exposed to trans fat,
FIG. 9 is a graph showing the relationship between the apoA-I molecular weight of breast milk and the embryo survival rate in the lactoferrin exposed to trans fat,
FIG. 10 shows the results of analysis of the protein composition in the breast milk of the female exposed to trans fat.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention discovered the differences in the respective components of the breast milk during the analysis of the breast milk components of the breast milk or the trans fat-fed breast milk of the normal breast milk, thereby completing the present invention.

According to the embodiment of the present invention, the contents of the triglyceride, glucose and reactive oxygen species in the breast milk of the normal breast milk and the trans fat breast milk of the transgender fat feeding group were compared with those of the normal breast milk group, Respectively.

According to another embodiment, the content of total cholesterol, protein or lactalbumin in the breast milk and the expression and molecular weight of apolipoprotein AI (apoA-I) in the breast milk of the normal breast feeding section and the trans fat feeding breast feeding section are The results of the measurement were lower in the breast milk of transfused lactating women than in normal breast milk.

Thus, the present invention provides a method of treating breast cancer, comprising: obtaining breast milk from a trans fat-fed rats; Selected from the group consisting of the total cholesterol content, the triglyceride content, the glucose content, the protein content, the active oxygen species content, the lactal albumin content, the expression of the apolipoprotein AI and the molecular weight of the apolipoprotein AI in the prepared breast milk Analyzing one or more indicators; And comparing the measured value with the index of the breast milk obtained from the normal lactation part. The present invention also provides a method for evaluating the quality of breast milk exposed to trans fats.

The triglyceride may be at least one selected from the group consisting of triglyceride, monoglyceride and diglyceride, more preferably triglyceride.

When the content of triglyceride, glucose, and reactive oxygen species is measured in the step of comparing with the index of the milk obtained from the normal lactating part, it can be judged that the trans fat is exposed to high risk.

In addition, in comparison with the index of breast milk obtained from normal breastfeeding, when any one of the indicators selected from the group consisting of total cholesterol, protein or lactalbumin content and expression of the apolipoprotein AI is measured to be low, It can be judged.

Also according to another embodiment, the apoA-I molecular weight in the breast milk of trans fat-fed rats is measured to be less than the breast milk of normal rats.

Therefore, when the molecular weight of the apoA-I is 20 to 26.5 kDa, it can be judged to be a high-risk group of trans-fat exposure, and more preferably 25.5 ± 0.6 kDa.

According to another embodiment of the present invention, when the breast milk of the normal breast feeding breast milk and the breast milk of the trans fat feeding breast feeding unit are microinjected into the zebrafish embryo, the survival rate is lower than that of the normal breast feeding breast milk, The speed was also confirmed to be late.

Thus obtaining breast milk from a trans fat-fed rats feeding unit; Microfilling the breast milk into a zebrafish embryo; Confirming the survival rate of the zebrafish embryo; And comparing the survival rate of the zebrafish infused with breast milk obtained from the normal breast-feeding section; The present invention provides a method for evaluating the quality of breast milk exposed to trans fats.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Sample preparation from breast milk or milk powder

Five breast milk transfusions in the 30s and a control group of 30 healthy breast - fed meals containing adequate trans fat were used as milk samples with breast milk using a breast pump. Collected breast milk was stored frozen and thawed for each experiment. In addition, there are five kinds of milk powder (milk powder 1: Nanyang Iem Mother 3: Milk Milk 2: Namyang Imperial Dream XO 3: Milk Milk 3: Milk Milk 3: Milk Milk 4: Milk powder 5: Pasteur Premium-Weed Mom) was purchased and 5.6 g was diluted in 40 mL of water according to the recommended dosage to prepare a sample. The results of each of the following experiments were calculated as mean value for each experimental group.

< Example  2> Total cholesterol content measurement

The total cholesterol content in the milk of each experimental group prepared in Example 1 was analyzed. The total cholesterol content of 0.02 mL of each sample was measured by cholesterol reaction color development reagent according to the method described in the prior art (J. Gerontol. A. Biol. Sci. Med. Sci. 2010, 65, 600-610) T-CHO, Wako Pure Chemical, Osaka, Japan) with a Victor 4 microplate reader at an absorbance of 490 nm.

As a result, control breast milk showed a high cholesterol content of 55 ± 25 mg / dL, but a significantly lower cholesterol level of 22 ± 9 mg / dL in trans fat-fed breast milk as shown in FIG. This was found to be due to the very low cholesterol level, similar to the level of powdered milk (12 ± 6 mg / dL), suggesting that intake of trans fats interferes with cholesterol secretion into breast milk.

< Example  3> Measurement of triglyceride content

The content of triglyceride in breast milk of each experimental group prepared in Example 1 was analyzed. 0.02 mL of the neutral fat content of each sample was measured with a Viktor 4 microplate reader at 540 nm in accordance with the manufacturer's instructions using a triglyceride (Cleantech TS-S Wako Pure Chemical, Osaka, Japan) .

As a result, as shown in Fig. 2, the triglyceride content of trans fat-fed breast milk increased more than twice as much as that of control breast milk. Control breast milk contained 1564 ± 786 mg / dL of neutral fat and 2841 ± 1107 mg / dL of breast milk trans fat. This is very similar to the amount of triglycerides in milk powder (3212 ± 463 mg / dL), which increases the intake of triglycerides and carbohydrates naturally through increased dietary intake of trans fats, It means that it is secreted.

< Example  4> Total glucose content measurement

The total glucose content in the milk of each experimental group prepared in Example 1 was analyzed. 0.02 mL of glucose content of each sample was measured with a Vitert 4 microplate reader at 490 nm according to the manufacturer's instructions using a glucose assay kit reaction reagent (AM201-K, Asan Pharmaceutical, Hwasung, Korea).

As a result, as shown in Fig. 3, the concentration of glucose in the breast milk of trans fat-fed group was 1.4 times that of the control breast milk. 39 ± 17 mg / dL was detected in the control breast milk and 53 ± 11 mg / dL in the breast milk trans fat. This concentration is significantly lower than that of powdered milk (190 ± 113 mg / dL), but it implies that the group that consumes trans fats more than the control group of home-based meals also increases glucose. This result can be interpreted to be related to the increase of triglycerides.

< Example  5> Measurement of protein content

The protein content in the milk of each experimental group prepared in Example 1 was analyzed. The protein content of 0.02 mL of each sample was measured with a Viktor 4 microplate reader at an absorbance of 450 nm along the well-known document (Anal Biochem. 1978, 87, 206-210).

As a result, as shown in FIG. 4, 9 ± 1 mg / mL of trans fat-fed breast milk was detected and 12 ± 3 mg / mL of control milk was detected. In the case of trans fat-fed group, Was significantly reduced.

< Example  6> apoA -I expression and molecular weight analysis

The expression of apoA-I in breast milk of each experimental group prepared in Example 1 was confirmed by Western blotting.

After the apoA-I protein was transferred to the membrane, 5% non-fat dry milk was dissolved in tris-buffered saline (TBST) containing 0.1% tween 20 After that, it was added to the membrane and blocked for 1 hour at room temperature. Then, apoA-I poliyclonal antibody diluted in TBST containing 5% skim milk was added and reacted overnight at 4 ° C. After the reaction, the membrane was washed three times with TBST at room temperature for 10 minutes and reacted with the secondary antibody at room temperature for 1 hour. The size of the apo-A-I band was then analyzed with Image Lab software (Bio-Rad) using a protein quantification kit (Chemi-doc XRS +, Bio-Rad, Hercules, CA, USA).

As a result, as shown in Fig. 5, the expression of apoA-I was decreased and the molecular weight of apoA-I was also changed in trans fat-fed group. While the control group showed a molecular weight of 27.5 kDa, trans fat - fed group milk showed a molecular weight of 25.5 kDa and the protein expression amount by the band intensity was also decreased.

< Example  7> Survival rate and development process of embryo

The embryo survival rate and the developmental process were compared after microinjection of breast milk or powdered milk of each experimental group prepared in Example 1 into zebrafish embryos, and the results are shown in FIG.

 As a result, embryo survival rate of trans fat-fed group breast milk was 50%, which was significantly lower (66%) than control breast milk infusion (39%).

In addition, 48 hours after the embryo injection, the control group showed normal development rate when injected with control milk. When injected with trans fat, the embryo's vertebrae did not grow longer and the melanin pigment was less formed in the eyes and trunk And the development was slow.

To compare the production of reactive oxygen species in the embryos, the cells were stained with dihydroethidium (DHE, Cat # 37291; BioChemika) fluorescence reagent. After staining, the amount of red fluorescence was quantitated by fluorescence spectrophotometer and the embryo was observed by fluorescence phase contrast microscope. As a result, strong red fluorescence was observed in the trans fat - fed group and the powdered milk group, and it was confirmed that a lot of active oxygen species were produced. In contrast, almost no red fluorescence was detected in the control group.

< Example  8> Zebra fish  Analysis of association of embryo survival rate

1. Cholesterol and Zebra fish  Embryo survival rate relevance

The results of Examples 1 to 6 show that the trans fat-fed group breast milk has lower cholesterol, higher triglyceride and decreased molecular weight of apoA-I compared to the control group milk.

Therefore, the relationship between the concentration of each component in the breast milk and the zebrafish embryo survival rate confirmed in Example 7 was compared.

As a result, the survival rate decreased as the concentration of triglyceride in the milk increased as shown in FIG. 7, and the survival rate also increased proportionally as the concentration of cholesterol increased as shown in FIG. As shown in Fig. 9, the survival rate also increased proportionally with increasing molecular weight of apoA-I. It was confirmed that the growth and viability of embryos increased as apoA-I was fully preserved.

< Example  9> Analysis of Protein Composition in Breast Milk

FIG. 4 shows that the composition of the protein varies depending on the decrease in the protein concentration in the trans fat-fed group.

For this purpose, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by a conventional method in the art, and anti-alpha lactalbumin antibody (cat # ab178431 ), abcam (Cambridge, UK)) was used. Then, it was stained with Coomassie brilliant blue to confirm the protein composition in the milk.

As a result, it was found that the band of lactalbumin disappeared (14 kDa) in trans fat-fed group breast milk as shown in Fig. Lact albumin is an essential amino acid-rich protein, a precursor protein that makes hormones that promote the growth of embryos and newborns. It is a source of peptides that have anti-bacterial functions. It is a protein that kills cancer cells (human α-lactalbumin made lethal to tumor cells , HAMLET).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

Obtaining breast milk from a trans fat-fed group lactation;
Analyzing the molecular weight of the apolipoprotein AI in the breast milk as an indicator of breast milk; And
And comparing it to an indicator of breast milk obtained from a normal breastfeeding portion,
Wherein the amount of the trans fatty acids is 20 to 26.5 kDa, and the trans fat is exposed to a high-risk group. The method according to claim 1,
Wherein the indicator is at least one selected from the group consisting of total cholesterol content in breast milk, triglyceride content, glucose content, protein content, active oxygen species content, lactalbumin content and expression of apolipoprotein AI Gt; transfusion &lt; / RTI &gt; level of the trans fat. 3. The method of claim 2,
Wherein the neutral fat is at least one selected from the group consisting of triglyceride, monoglyceride, and diglyceride. 3. The method of claim 2,
Wherein the trans fat is judged to be a high-risk group when the content of triglyceride and glucose is measured in a step of comparing with the index of the breast milk obtained from the normal lactating part. 3. The method of claim 2,
In the step of comparing with the index of the milk obtained from the normal lactating part, when the index of total cholesterol, protein or lactal albumin and the expression of the apolipoprotein AI is low, The method comprising the steps of: determining the quality of breast milk exposed to trans fats.

















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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100930996B1 (en) 2007-10-10 2009-12-11 임병윤 Powdered milk and baby food for atopy containing green tea and lactic acid bacteria
KR20140120081A (en) * 2013-04-02 2014-10-13 (주)유투바이오 Method and Apparatus for Providing Breastfeeding Analysis Services

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100930996B1 (en) 2007-10-10 2009-12-11 임병윤 Powdered milk and baby food for atopy containing green tea and lactic acid bacteria
KR20140120081A (en) * 2013-04-02 2014-10-13 (주)유투바이오 Method and Apparatus for Providing Breastfeeding Analysis Services

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