KR20190048445A - The extraction method of micro vesicles from milk - Google Patents

Abstract

The present invention relates to a method for extracting micro-vesicle from milk, which extracts micro-vesicle including microRNA of high purity and high yield, comprising the steps of: centrifuging milk for an hour; removing a pellet of the milk; and additionally ultra-centrifuging a supernatant of the milk.

Description

The extraction method of micro vesicles from milk is a method for extracting micro-

The present invention relates to a method for extracting micro vesicles from milk and a health functional food composition for enhancing immune function comprising a micro-vesicle extracted by the method.

Extracellular microvesicles are membrane-shaped nano-sized cell bodies that are naturally released by cells in vitro and in living organisms. Micro vesicles are present in a wide range of plasma, urine, breast milk, and amniotic fluid, and have important functions in vivo. In particular, extracellular microfilms containing functional proteins, RNA and antigens are used in new ways of communication within the cell.

Milk is the only nutritional source of new mammals, and its nutrients differ depending on the type of mammal, the age of the mammal, and the characteristics of the mother. Milk has excellent nutritional balance of protein, fat and carbohydrates. It is a health food containing a lot of essential nutrients such as minerals, vitamins, and riboflamine that are necessary for human survival. Recently, there has been an attempt to analyze the important microRNAs present in breast milk as the presence of microRNAs related to immune enhancement in body fluids has been reported. Recently, Kosaka et al. (2010) analyzed the distribution of miRNAs in breast milk (0-6 months vs. 6 months or more) during breastfeeding and found that they are present in breast milk such as miR-181a, miR-17, miR-155 and miR-92 Specific miRNAs were selected. The microRNAs present in the oil component do not exist alone but are coated with a micro-vesicle similar to exosomes, so they are resistant to various environments such as RNase and low pH when passing through a human digestive tract. , And it is reported that a specific immune enhancing activity appears. It is necessary to study the method of optimally separating the micro-vesicle covering the immunoreactive microRNA from milk.

Accordingly, the present inventors have made efforts to develop a method of extracting micro-endoplasmic reticulum from milk, and have found a method of separating micro-endoplasmic reticulum containing microRNA at a high yield in milk when ultracentrifugation is performed at 135000G , Thereby completing the present invention.

Accordingly, the present inventors have completed the present invention by confirming a method for separating micro-vesicles containing microRNAs having a high yield in milk when ultracentrifugation of milk at 135,000 G was performed.

Accordingly, it is an object of the present invention to provide a method for extracting microvesicles from milk containing the following steps.

i) centrifuging the milk at 100000 G for 1 hour;

ii) removing the pellet of the centrifuged milk; And

iii) further ultracentrifuging the supernatant of the pellet-removed milk at 130000 G to 140000 G;

Still another object of the present invention is to provide a health food composition for enhancing immune function comprising a micro-vesicle extracted by the above method.

In order to achieve the above object, the present invention provides a method for extracting micro-endoplasmic reticulum from milk containing the following steps.

i) centrifuging the milk at 100000 G for 1 hour;

ii) removing the pellet of the centrifuged milk; And

iii) further ultracentrifuging the supernatant of the pellet-removed milk at 130000 G to 140000 G;

In a preferred embodiment of the present invention, the milk may be milk or colostrum.

In addition, the present invention provides a health food composition for enhancing immune function comprising a micro-vesicle extracted by the above-mentioned method.

In a preferred embodiment of the present invention, the health food may be milk.

The method of extracting micro vesicles from the milk of the present invention has an effect of extracting micro-vesicles containing microRNAs of high purity and yield.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a method for separating microvesicles that have been performed in the present invention. FIG.
FIG. 2 is a graph showing the results of analysis of miRNAs of the micro-vesicles isolated in Example 1 of the present invention and Comparative Examples 1 to 5 by electric perrograph.
FIG. 3 shows the results of analysis of miRNA of micro-vesicles isolated in Example 1 of the present invention and Comparative Examples 1 to 5 using qRT-PCR.
FIG. 4 shows the results of detection of the marker protein CD9 of the microemboli using Western blotting in the microembroine separated in Example 1 and Comparative Examples 1 to 5. FIG.
FIG. 5 shows the results of SEM image analysis of the micro-vesicles isolated using Example 1 and the internalization of PKH-labeled micro-vesicles by RAW 264.7 cells.

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

The present inventors have studied an optimal method for extracting micro-endoplasmic reticulum containing mircro RNA having high yield and purity from milk by separating micro-endoplasmic reticulum from milk by various methods.

1) Centrifugation at 100,000 g for 1 hour (Comparative Example 1) 2) Centrifugation at 100,000 g for 1 hour, supernatant was then centrifuged at 135,000 g (Example 1), 3) lactic acid, Centrifugation at 100,000 g for 1 hour, ultracentrifugation of the supernatant at 135,000 g (Comparative Example 2) 4) chemical immersion using EDTA, centrifugation at 100,000 g for 1 hour, supernatant was centrifuged at 135,000 g (Comparative Example 3 5) Centrifugation at 1,500 g for 30 minutes after chemical immersion using ExoQuick ™ (Comparative Example 4) 6) Ultracentrifugation at 135,000 g for 1 hour and separation using OptiPrep ™ density gradient solution (Comparative Example 6) , And a total of six methods were used to extract the microvesicles from milk (Fig. 1).

Although the small RNA patterns of the microembossed tissues extracted by the above method are almost the same, the method of ultracentrifugation rather than the chemical immersion method (lactic acid, EDTA, ExoQuick ™ and OptiPrep ™ density gradient solution) (Fig. 2). In addition, the ratio of RNA: protein is an indicator of the micro-vesicle function and the risk of high contamination of uncontaminated non-vaginal biomaterials in the form of a protein that is secreted during separation. 2) After centrifugation at 100,000 g for 1 hour, (Fig. 2). Fig.

In the microembolisms extracted from the above six methods, CD9 as a biomarker of microemboli was assayed by Western blotting, and as a result, all the methods except OptiPrep ™ were positive for CD9.

2) After centrifugation at 100,000 g for 1 hour, the supernatant was centrifuged at 135,000 g, and the cells were incubated with RAW264.7 cells for PKA labeling. It was found that the ER was effectively absorbed in RAW 264.7 (FIG. 5).

The present invention provides a method of extracting microvesicles from milk comprising the steps of:

i) centrifuging the milk at 100000 G for 1 hour;

ii) removing the pellet of the centrifuged milk; And

iii) further ultracentrifuging the supernatant of the pellet-removed milk at 130000 G to 140000 G;

In the step iii), the ultracentrifugation may be performed at a speed of 130000 G to 140000 G, and most preferably at a speed of 135000 G.

In a preferred embodiment of the present invention, the milk may be milk or colostrum.

In addition, the present invention provides a health food composition for enhancing immune function comprising a micro-vesicle extracted by the above-mentioned method.

In a preferred embodiment of the present invention, the health food may be milk. Examples of foods that can be added with the Mysite ER from the milk include various foods, dairy products, beverages, gums, tea, vitamin supplements, health supplements, and the like, most preferably dairy products. Powders, granules, tablets, capsules or beverages.

It may also be added to foods or drinks for the purpose of enhancing immune function. In this case, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, . The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned Codonopsis fruit extract as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the micro-vesicle extracted from the milk of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies such as cheese and chocolate, , Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. The proportion of such additives is not critical, but is generally selected in the range of from 0.1 to about 20 parts by weight per 100 parts by weight of the microvesicles of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

[ Example  1] Optimal method for extracting microfilament from milk Conventional  method 2)

10 ml of milk was centrifuged at 100,000 g for 1 hour. After removing the pellet of the centrifuged milk, supernatant was further centrifuged at 135,000 g, and the pellet was resuspended in PBS and washed twice under the same conditions.

[ Comparative Example  1] A method of extracting microfilament from milk Conventional method  One)

10 ml of milk was ultracentrifuged at 100,000 g for 90 minutes at 4 ° C. The obtained pellet was resuspended in PBS and again washed twice by ultracentrifugation twice under the same conditions.

[ Comparative Example  2] Lactic acid  A method for extracting a micro-vesicle using

10 ml of milk was lowered to pH 4.6 with lactic acid and reacted on ice for 15 minutes. And centrifuged at 100,000 g for 1 hour. After removing the pellet of the centrifuged milk, the supernatant was further separated by ultracentrifugation at 135,000 g for 1 hour. The resulting pellet was resuspended in PBS and the pellet was washed by ultracentrifugation twice at the same rate. The pellet was then resuspended in 200 [mu] l PBS and stored at -80 for further analysis. Since acidification has previously been shown to have no effect on microbubbles (Hata et al, 2010), there is no negative impact on microbubbles for this method.

[ Comparative Example  3] EDTA  A method for extracting a micro-vesicle using

Ten milliliters of EDTA was added to 10 ml of milk and reacted on ice for 15 minutes. And centrifuged at 100,000 g for 1 hour. After removing the pellet of the centrifuged milk, the supernatant was further separated by ultracentrifugation at 135,000 g for 1 hour. The resulting pellet was resuspended in PBS and the pellet was washed by ultracentrifugation twice at the same rate. The pellet was then resuspended in 200 [mu] l PBS and stored at -80 for further analysis.

[ Comparative Example  4] ExoQuick Method for extracting micro-vesicles using

10 ml of milk was mixed with 2 ml of ExoQuick ™ solution and reacted overnight at 4 ° C. The solution was then centrifuged at 1,500 g for 30 min at room temperature and the supernatant was removed. The pellet was centrifuged at 1,500 g for 5 minutes to remove traces of fluid. The pellet was then resuspended in 10 mL of PBS and ultracentrifuged at 135,000 g for 90 minutes.

[ Comparative Example  5] OptiPrep  ™ Density The gradient solution  A method for extracting a micro-vesicle using

Colostrum samples were separated by ultracentrifugation at 135,000 g for 1 hour to isolate microorganism samples. The pellet was resuspended in 500 L of PBS and the crude sample was placed on a sucrose gradient solution and centrifuged for 16 hours. The gradient was generated by diluting the stock solution of OptiPrep ™. 3 ml of a 40% iodixanol solution was added and carefully graded with 3 ml of 20% and 10% solution and 2.5 ml of 5% solution (Tauro et al., 2012). After centrifugation, 12 individual 1 mL gradient fractions were manually collected (density increased by top-bottom collection). Using this method, fraction 7 (corresponding to a density of 1.11 g / mL) found to be the most abundant of the microvesicles (Lobb et al., 2015) was collected in a separate microcentrifuge tube. It was then resuspended in PBS and washed 2 times at 135,000 g for 1 hour to obtain the final micro-vesicle pellet.

[ Experimental Example  One] RNA  analysis

The total RNAs of the microfilaments extracted by the method of Example 1 and Comparative Examples 1 to 5 were extracted using a miRNeasy Serum / Plasma Kit (QIAGEN). The absorbance (A260 / 280) was measured on a Nanodrop spectrophotometer (Optizen-NanoQ, Mecasys Co., Korea) to confirm the RNA yield and purity. Protein levels of the samples were measured by Bradford analysis (Bio-Rad). For analysis, a standard curve was obtained using bovine serum albumin. The yields of total RNA and protein of the microembossed tissues extracted by the method of Example 1 and Comparative Examples 1 to 5 are shown in Table 1 below.

Comparison of the levels of isolated microvesicle RNA and protein using the method of Example 1 and Comparative Examples 1 to 5 Way Total miRNA yield (ng) Protein level (mg / ml) Comparative Example 1 7800 5.142 Example 1 9120 4.669 Comparative Example 2 5660 3.042 Comparative Example 3 5840 0.1487 Comparative Example 4 9330 17.1777 Comparative Example 5 985 0.0603

[ Experimental Example  2] Bioanalyser  Used small RNA  analysis

The total RNA integrity and miRNA content of the micro-endoplasmic reticulum extracted by the method of Example 1 and Comparative Examples 1 to 5 were measured using Small RNA Kit (Agilent) and Bioanalyser. Several methods were compared using Bioanalyser's Electroherograms and miRNA concentration data. One microliter of RNA was analyzed with an Agilent 2100 Bioanalyzer using an RNA 6000 nano kit (Agilent Technologies, Santa Clara, Calif.) According to the manufacturer's protocol. The microvesicles extracted by the method of Example 1 and Comparative Examples 1 to 5 exhibited a similar pattern for small RNA (Fig. 2A). Comparative Example 1 and Example 1 have the highest small RNA yield. Although not shown in the figure, Example 2 has slightly lower% of small RNA because the initial pellet of 100,000 g centrifugation contains a considerable amount of microfibrils. As shown in FIG. 2B, microembodiment miRNA yields were higher in Comparative Example 1 and Example 1, which were subjected to ultracentrifugation, than in Comparative Examples 2 to 5. This shows that ultracentrifuges result in higher yields of microRNA microRNAs when compared to chemically based precipitation methods. The ratio of RNA: protein is an important factor in the application of micro-bodies in vivo and in vitro to assess micro-vesicle function, and the risk of high contamination of uncontaminated non- There is no. Therefore, the present inventors measured RNA / protein ratio of Example 1, Comparative Example 1 and Comparative Example 4, and found that Example 1 has a higher RNA: protein ratio than Comparative Examples 1 and 4 (FIG. 2C).

[ Experimental Example  3] found in isolated microemboli miRNA  Performing qPCR for relative expression of fractions

Total RNA extracted from the microembolisms extracted by the above six methods was subjected to miScript II RT kit to prepare cDNA according to the manufacturer's instructions (Qiagen). qPCR was performed using a StepOne Plus Real Time PCR system (Applied Biosystems, USA) with miScript SYBR Green PCR Kit. The critical period (CT value) is a relative measure of the individual miRNA concentration in the PCR reaction, with low CT values indicating high expression. The qPCR values for the four detectable microembolic miRNAs were normalized to Cel_miR_39 (spikes in the control miRNA) and the delta Ct values were plotted against the microvesicles in a different way. miR-200c had the lowest delta CT, and miR-155 consistently showed low CT values, but it was abundant in the microemic fraction of milk.

[ Experimental Example  4] Western Blat

To evaluate the purity of the isolated endoplasmic reticulum and the presence of the isolated endoplasmic reticulum, the CD9 protein, the biomarker of the extracellular microcellular body, was confirmed by Western blot. Western blotting was performed on the basis of known techniques (Cvjetkovic et al., 2014). Briefly, samples were homogenized using a radioimmunoprecipitation assay (RIPA) containing a protease inhibitor (Cell Biolabs, San Diego, Calif., USA). Were mixed with Laemmli buffer to reduce the volume containing the same amount of protein. Proteins were separated on 4-20% Tris-glyceride SDS polyacrylamide gel (Bio-Rad). Proteins were adsorbed onto polyvinylidene difluoride membranes (Pall Life Sciences). The membranes were blocked with 5% skim milk and incubated in primary antibody (anti-CD9 [1: 500], Invitrogen) for 1 hour at room temperature and the blots were incubated with TBS-T (20 for each wash in TBS buffer containing 0.05% Tween Min, 15 min) and incubated at room temperature for 1 h with HRP-conjugated antibody. Membranes were washed again three times in TBS-T and signals were visualized using an Amersham ™ enhanced chemiluminescence kit (GE Healthcare Companies) by imaging equipment (C-DiGit® Blot Scanner, Li-COR). All of the micro-vesicles isolated by the method of Example 1 and Comparative Examples 1 to 4 were positive for CD9 and contained micro-vesicles (Fig. 5).

[ Experimental Example  5] Cell phase  Absorption of micro-vesicles

The microvesicles were stained using a PKH67 green fluorescent cell linker kit for cell membrane labeling (Sigma Aldrich, St. Louis, Missouri). The labeled microvesicles were incubated with RAW 264.7 cells at 37. Cellular uptake of labeled microvesicles was measured using fluorescence microscopy. The cells were inoculated at a rate of 10 ⁴ cells / cm ² in a 24-well plate and treated with microvesicles equivalent to 20 μg of protein. For SEM imaging, the microvesicles were fixed in 2% paraformaldehyde at room temperature for 15 minutes. The droplets of the fixed sample were placed completely on the silicon chip and air dried before imaging. Figure 5 shows the micro-vesicles extracted using Example 2. Using a microscope, the PKE-labeled micro-vesicles in RAW264.7 cells were observed to show that the micro-vesicles were effectively absorbed (Fig. 5).

Claims (4)

i) centrifuging the milk at 100000 G for 1 hour;
ii) removing the pellet of the centrifuged milk; And
iii) further ultracentrifuging the pellet-removed milk supernatant at 130000 G to 140000 G;
≪ / RTI > The method of claim 1, wherein the milk is milk or colostrum. A health functional food composition for enhancing immune function comprising a micro-vesicle extracted by the method of claim 1. The health functional food composition according to claim 3, wherein the health functional food is milk.

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