JP5634118B2 - Method for increasing polyunsaturated fatty acid content in ruminant livestock milk, and pharmaceutical agent or feed composition used in the method - Google Patents

Description

  The present invention relates to a method for efficiently increasing the content of highly unsaturated fatty acids in ruminant livestock milk. Specifically, ruminating livestock is fed or fed with nutrients that increase the content of polyunsaturated fatty acids in the milk, and a method for producing highly polyunsaturated fatty acid-rich milk without performing genetic manipulation, and the like. The present invention relates to a pharmaceutical agent or a feed composition to be used.

  In ruminant livestock milk such as milk, fatty acids having various physiological activities exist. One example is a highly unsaturated fatty acid having three or more double bonds in the molecule, such as n-6 and n-3 fatty acids. In recent years, metabolic diseases caused by accumulation of body fat, so-called metabolic syndrome, has attracted attention in humans, and as unsaturated fatty acids for improving metabolic syndrome, highly unsaturated fatty acids (for example, γ-linolenic acid, eicosapentaenoic acid (EPA)). , Docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), arachidonic acid, and the like). These have been reported to have strong physiological activity and are useful for humans and the like.

  One of the enzymes involved in the biosynthesis of these highly unsaturated fatty acids is fatty acid desaturase 2 (FADS2). FADS2 is an enzyme involved in the formation of a double bond at the delta 6 (Δ6) position, and is known to be a key enzyme when proceeding from linoleic acid or α-linolenic acid to a highly unsaturated fatty acid. .

  In recent years, studies have been conducted to increase the content of polyunsaturated fatty acids in milk such as cows, but it has been reported that EPA and DHA in rumen etc. have a very low rate of transfer to milk (non-patent literature). 1). In order to improve the results so far, it is necessary to control the biosynthesis of unsaturated fatty acids in the mammary cells themselves.

  Very recently, the gene sequence encoding bovine FADS2 has been published (National Center for Biotechnology Information (NCBI) Accession No. NM_001083444). However, although FADS2 is an important enzyme for biosynthesis of polyunsaturated fatty acids, little research has been conducted on FADS2 activity control and expression control of genes encoding the enzyme.

  In other words, it has not been reported so far to confirm the endocrine and gene expression responses to nutrients to the expression of the gene encoding FADS2 using mammary gland tissue cells such as bovine. There is no report on the acquisition technology of high fatty acid milk. As a method for increasing milk fat content in milk, there has been reported a method of feeding an acid derived from hexose such as gluconic acid (Patent Document 1), but this method is applicable to all saturated fatty acids and unsaturated fatty acids. It is intended to increase the content of fat components and is not intended for FADS2 and is not intended to increase only highly unsaturated fatty acids. Although it is technically possible to modify the gene by genetic manipulation, this is not a preferable method in terms of safety.

International Publication No. 02/058483

Lipid, 39: 1197-1206 (2004).

  In view of the importance of ruminant livestock milk as a food, the present invention increases the added value as much as possible, and contributes to the health of the nation. It was made for the purpose of providing a nutritional component capable of increasing the fatty acid content. Furthermore, an object of the present invention is to provide an efficient new system for producing functional milk having a high polyunsaturated fatty acid content using this nutritional component.

  In order to achieve the above-mentioned object, as a result of examinations from various directions, the present inventors have focused on fatty acid desaturase 2 (FADS2) involved in the production of highly unsaturated fatty acids and the gene encoding the enzyme. And if the expression of the gene encoding FADS2 in ruminant live organisms and the activation of the enzyme can be increased, it is expected that the ability to produce highly unsaturated fatty acids and the enhancement of biological functions can be expected. Nutritional components capable of activating enzymes were investigated.

  Since FADS2 is an enzyme that serves as a base point of the arachidonic acid cascade, the enzyme is also involved in the production of prostaglandins that cause an inflammatory response. Therefore, it is presumed that moderate increase in gene expression (Mild induction) without rapid increase in gene expression, etc. is suitable for obtaining milk containing a high amount of highly unsaturated fatty acids based on the enzyme. The Therefore, it is considered that expression induction using nutrients such as amino acids, vitamins, minerals, and fatty acids is the most suitable method, not a method using genetic manipulation or a method using a special chemical agent.

  From such a viewpoint, as a result of further intensive studies, the present inventors have provided fatty acid desaturase in mammary tissue cells of ruminant livestock by providing nutrients such as acetic acid, menaquinone-4, and arachidonic acid among various nutritional components. The present inventors have found that the expression of a gene encoding 2 (FADS2) and the enzyme activity thereof are enhanced, thereby efficiently increasing the content of highly unsaturated fatty acids in milk while maintaining milk yield and quality. It was.

That is, the embodiment of the present invention is as follows.
(1) A ruminant livestock is fed (administered or fed) with a nutrient that enhances the expression of a gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme in mammary tissue cells of the ruminant livestock. A method for increasing the content of highly unsaturated fatty acids in ruminant livestock milk.
(2) A ruminant livestock is characterized by giving (administering or feeding) a nutrient that enhances the expression of the gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme in mammary gland tissue cells of the ruminant livestock. A method for producing (manufacturing) ruminant livestock milk having an increased content of highly unsaturated fatty acids.
(3) Supplying a nutrient that enhances the expression of the gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme to mammary gland tissue cells of ruminant livestock A method for enhancing expression and / or activity of the enzyme.
(4) Supplying a nutrient that enhances the expression of the gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme to mammary tissue cells of ruminant livestock, To increase the content of polyunsaturated fatty acids.
(5) The method according to any one of (1) to (4), wherein the nutrient is at least one selected from acetic acid, menaquinone-4, and arachidonic acid.
(6) The method according to any one of (1) to (5), wherein the ruminant livestock is a cow (particularly a dairy cow).

(7) Ruminating as an animal medicine, characterized by comprising as an active ingredient a nutrient that enhances the expression of a gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme in breast tissue cells of ruminant livestock An agent for increasing the content of highly unsaturated fatty acids in livestock milk.
(8) Expression of a gene encoding fatty acid desaturase 2 (FADS2) in a mammary gland tissue cell of ruminant livestock and / or a nutrient that enhances the activity of the enzyme as an active ingredient, Gene expression and / or the enzyme activity promoter.
(9) In ruminant livestock milk, comprising a nutrient that enhances the expression of the gene encoding fatty acid desaturase 2 (FADS2) and / or the activity of the enzyme in mammary gland tissue cells of ruminant livestock A feed composition for ruminant livestock that increases the content of highly unsaturated fatty acids.
(10) The agent or feed composition according to any one of (7) to (9), wherein the nutrient is at least one selected from acetic acid, menaquinone-4, and arachidonic acid.
(11) The agent or feed composition according to any one of (7) to (10), wherein the ruminant livestock is a cow (particularly a dairy cow).

  According to the present invention, expression of a gene encoding fatty acid desaturase 2 (FADS2) in vivo and / or without affecting the milk yield and quality of ruminant livestock without genetic manipulation. Enhancing enzyme activity and enhancing its ability to produce highly unsaturated fatty acids. Then, it becomes possible to efficiently increase the highly unsaturated fatty acid content in the mammary gland tissue cells of ruminant livestock and to provide an efficient new system for producing functional milk having a high highly unsaturated fatty acid content.

It is a figure which shows the fluctuation | variation of FADS2 gene expression at the time of Lactogenic harmone complex, insulin, IGF-1, and GH addition in a bovine mammary epithelial cell. (A): The graph which compared the time of no addition (Basic control) and the time of addition of Lactogenic harmone complex. * Indicates P <0.05 in Student t test compared to the case of no addition (Mean ± SD, n = 5). (B): When added to the differentiated bovine mammary epithelial cells in stages with varying insulin concentrations (Mean ± SD, n = 4). (C): When added to differentiated bovine mammary gland epithelial cells in a stepwise manner (Mean ± SD, n = 4). (D): When added to differentiated bovine mammary gland epithelial cells in a stepwise manner (Mean ± SD, n = 4). In any graph, the vertical axis represents the ratio of FADS2 / RPS9 (the same applies to FIGS. 2 to 5). It is the figure which compared the fluctuation | variation of FADS2 gene expression at the time of the addition of various amino acids, minerals, and uridylic acid (U acid) in the differentiated bovine mammary epithelial cells compared with the addition of lactogenic hormones complex (control), Mean ± SD, n = 4). It is the figure which compared the fluctuation | variation of FADS2 gene expression at the time of the various vitamin addition in the differentiated bovine mammary gland epithelial cell with only the addition of lactogenic harmone complex (control). * Indicates P <0.05 in Student t test compared to control (Mean ± SD, n = 4). In the figure, VA represents vitamin A, and VC represents vitamin C. It is the figure which compared the fluctuation | variation of FADS2 gene expression at the time of various fatty acid addition in the differentiated bovine mammary gland epithelial cell with only Lactogenic hormone complex addition (control). * Indicates P <0.05 in Student t test compared to control (Mean ± SD, n = 4). In the figure, C2: 0 is acetic acid, C3: 0 is propionic acid, C8: 0 is octanoic acid, C10: 0 is decanoic acid, C18: 1 is oleic acid, C18: 2 is linoleic acid, C18: 3 Represents α-linolenic acid, C20: 4 represents arachidonic acid, and C22: 6 represents docosahexaenoic acid. It is a figure which shows the concentration dependence response of FADS2 gene expression at the time of arachidonic acid (A) and menaquinone-4 (B) addition in the differentiated bovine mammary epithelial cells. The different code indicates P <0.05 in the Duncan's multiple range test (Mean ± SD, n = 4). It is a figure which shows the fluctuation | variation of the FADS2 enzyme activity at the time of the addition of arachidonic acid and menaquinone-4 in the differentiated bovine mammary epithelial cells (unit is U / mg protein).

  Hereinafter, the present invention will be described in detail.

  The livestock subject to administration or feeding of nutrients according to the present invention is an enzyme that converts polyunsaturated fatty acids (such as linoleic acid and α-linolenic acid) into highly unsaturated fatty acids in vivo (for example, mammary gland tissue). 2 (fatty acid desaturase: FADS2) is a ruminant livestock (cow, goat, sheep, deer, buffalo, etc.) that accumulates highly unsaturated fatty acids in its milk. In particular, it is effective for dairy cows for the production of milk, and hereinafter, dairy cows will be exemplified and described as target ruminant livestock, but the present invention is not limited to dairy cows, and various ruminant livestocks are widely used. It is intended.

  Dairy cows convert linoleic acid, α-linolenic acid, and the like into highly unsaturated fatty acids in the mammary gland tissue by FADS2, which is an unsaturated enzyme. Here, the highly unsaturated fatty acid means an unsaturated fatty acid having three or more double bonds in the molecule. For example, γ-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaene. Examples include acid (DPA) and arachidonic acid. However, the conversion efficiency into highly unsaturated fatty acids by the enzyme is usually not so high. In the present invention, a nutrient capable of increasing the gene expression and enzyme activity of the enzyme is simply administered or fed to the cow, and it is efficient without adversely affecting milk yield or milk quality (flavor, milk fat percentage, etc.). It also increases the content of highly unsaturated fatty acids in milk.

  The nutrient to be administered or fed is not limited as long as it has an action of enhancing the expression of the gene encoding FADS2 and / or the activity of the enzyme, but preferable examples include acetic acid, menaquinone-4, and arachidonic acid. The These may be administered or fed alone, or may be a mixture of two or more of these.

  Acetic acid is an important energy source comparable to glucose in ruminants. In addition, there are naturally occurring and synthetic products of vitamin K. As natural products, there are phylloquinones (vitamin K1) contained in plants and menaquinones (vitamin K2) which are microbial products. Products include menadiones (vitamin K3) and related derivatives. Vitamin Ks absorbed from ruminant gastrointestinal tracts are said to be converted into active menaquinone-4 in vivo and exhibit functionality in tissues.

  Arachidonic acid (20: 4, n-6) is considered to be an essential fatty acid in most mammals and is synthesized from linoleic acid and linolenic acid in liver microsomes. It is released into the cytoplasm by the action of phospholipase on the cell membrane and is converted into various prostaglandins to exert physiological activity.

  Nutrients or the like that increase the blood concentration of at least one of the acetic acid, menaquinone-4, and arachidonic acid, and increase the expression of the gene encoding FADS2 and / or the activity of the enzyme in the mammary gland tissue (for example, Administration or feeding (and feeding conditions thereof) of vitamin Ks (particularly vitamin K2s, linolenic acid, etc.) are also encompassed in the present invention.

  As the above nutrients, not only purified pure products can be used, but also crude purified products (pasted products, diluted products, emulsified products, suspended products, etc.) can be used. In addition, it is also possible to use those that have been granulated by adding excipients such as starch and dextrin (granule), or tableted, etc. (tablets, capsules, etc.). Furthermore, the thing coated with fats and oils etc. so that it may not be decomposed | disassembled with a lumen | rumen etc. can be used.

  The above nutrient can be used as an active ingredient, an animal medicine or a promoter (enhancing agent) formulated as it is or together with various commonly used pharmaceutical ingredients. Moreover, the said nutrient can also provide a feed (feed composition) by adding, mixing, and using the usual feed component. The nutrients themselves can be fed directly to livestock as feed, or can be added to and mixed with other feed ingredients as feed additives.

  There is no particular limitation on the administration or supply amount of nutrients, but for example, 10 mg to 500 mg / day (preferably 100 mg to 300 mg / day) for menaquinone-4 as nutrients, and 0.5 g to 10 g / day for acetic acid and / or arachidonic acid. It is preferable to feed the milking period continuously on a day (preferably 1 g to 5 g / day).

  By adopting these constitutions, the expression of the gene encoding FADS2 in vivo and / or the enzyme activity thereof is enhanced while maintaining the milk quality and milk yield of dairy cows, thereby enhancing the ability to produce highly unsaturated fatty acids. Not only is this possible, it can provide an efficient new system for producing functional milk with an efficient high polyunsaturated fatty acid content.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

  The effects of various nutrients on the expression of the gene encoding fatty acid desaturase 2 (FADS2) related to the production of highly unsaturated fatty acids in mammary epithelial cells derived from bovine mammary gland tissue were confirmed by in vitro tests.

  Bovine mammary epithelial cells are described in J. Org. Endcrilinol. , 169: 381-388 (2001). That is, the mammary gland parenchyma is taken out from bovine mammary gland tissue, and after collagenase treatment, cells are obtained with a nylon mesh, several to several tens of cells are trypsinized to disperse the cells, and then subjected to hormone treatment. The cloned differentiable bovine mammary epithelial cells were subjected to the test.

The bovine mammary epithelial cells were seeded on a culture dish and cultured in a medium containing 10% Fetal bovine serum (FBS) under 5% CO ₂ conditions. In the test, 1 × 10 ⁴ / cm ² cells were seeded on a 12-well plate coated with collagen type I, and lactogenic hormones complex was added to the sub-confluent state. After 48 hours of culture and differentiation, the cells were used as a control. did. In addition, mammary epithelial cells that were not differentiated without the addition of lactogenic homone complex were used as basic control samples.

(1) Confirmation of influence of various hormones on expression of gene encoding FADS2 In order to observe the influence of various hormones on the expression of gene encoding FADS2, the following tests were conducted.

  Differentiated bovine mammary epithelial cells (control) were cultured in serum-free medium for 12 hours, and then insulin-like growth factor-1 (IGF-1), insulin, growth hormone (GH) (each 10-1000 ng / ml) and lactogenic A homone complex (10 μg / ml dexamethasone, 10 μg / ml insulin, 10 μg / ml prolactin) was added to the medium, and the cells were collected after culturing for 24 hours. The expression response of the gene encoding FADS2 was observed by real-time PCR. A gene encoding ribosomal protein S9 (RPS9; J. Dairy Sci., 90: 2246-2252 (2007)) was used as a house keeping gene. Hereinafter, all the results of gene expression are shown by the expression ratio of FADS2 / RPS9 (ratio of mRNA amount).

  The base sequences of the primers used for real-time PCR are shown in SEQ ID NOs: 1 to 4 and Table 1. Specifically, the forward primer for the expression analysis of the gene encoding FADS2 is encoded in RPS9 in SEQ ID NO: 1 and Table 1, and the reverse primer is encoded in SEQ ID NO: 2 and Table 2. The forward primer for gene expression analysis is shown in SEQ ID NO: 3 and the third row of Table 1, and the reverse primer is shown in SEQ ID NO: 4 and the fourth row of Table 1.

  The results of FADS2 gene expression response to IGF-1, insulin and GH in bovine mammary epithelial cells are shown in FIG. Expression of the gene encoding FADS2 in bovine mammary epithelial cells was increased about 2-fold by adding Lactogenic homone for 48 hours. On the other hand, even when IGF-1, insulin, and GH were added to the cell culture medium after differentiation induction, no change was observed in the mRNA expression level of the gene encoding FADS2, that is, the response of the gene to these hormones was I was not able to admit.

(2) Confirmation of influence of various nutrient additions on expression of gene encoding FADS2 In order to observe the influence of various nutrient additions on the expression of gene encoding FADS2, the following tests were performed.

  Differentiated bovine mammary epithelial cells (control) are cultured in a serum-free medium for 12 hours, and then amino acids (lysine, leucine, glutamic acid, arginine, methionine, glutamine, isoleucine), minerals (Mg, Mn, Ca), uridylic acid, Vitamins (vitamin A, vitamin C, biotin, pantothenic acid, menaquinone-4, menaquinone-7, 1,4-dihydroxy-2-naphthoic acid (DHNA)), fatty acids (acetic acid, propionic acid, octanoic acid, decanoic acid, olein 400 μM each of acid, linoleic acid, α-linolenic acid, arachidonic acid, and DHA) were added, and the cells were collected by culturing for 24 hours. The gene expression response of the gene encoding FADS2 was measured by the same method as in (1).

  The expression response results of the gene encoding FADS2 to various nutrients in bovine mammary epithelial cells are shown in FIGS. Expression of the gene encoding FADS2 in bovine mammary epithelial cells was significantly increased by the addition of arachidonic acid and menaquinone-4. Furthermore, high expression was observed even when acetic acid was added. However, linoleic acid and α-linolenic acid, which are precursors of highly unsaturated fatty acids, and menanquinone-7 and DHNA, which are the same vitamin K-related substances as menaquinone-4, did not change in this in vitro test.

Furthermore, in order to confirm the concentration-dependent response of the expression of the gene encoding FADS2 of arachidonic acid and menaquinone-4 in the differentiated bovine mammary epithelial cells, the above test was performed at each concentration of 0, 10, 100, 500 μM. In the same manner as described above, the gene expression after 24 hours of incubation in the medium was measured by real-time PCR.
The results are shown in FIG. 5, and arachidonic acid and menaquinone-4 increased the expression of the gene encoding FADS2 in a concentration-dependent manner.

(3) Confirmation of effects on FADS2 activity when arachidonic acid and menaquinone-4 are added Arachidonic acid and menaquinone-4 are added to differentiated bovine mammary epithelial cells at a concentration of 500 μM, and the FADS2 activity of cell microsomes after 24 hours of culture is observed. Fatty acids were derivatized with phenacyl bromide and measured by a method of quantifying by high performance liquid chromatography (HPLC). The substrate used was linoleic acid, and the activity was defined as 1 U for producing 1 nmol of γ-linolenic acid per minute.

  The results are shown in FIG. The addition of arachidonic acid and menaquinone-4 tended to increase the FADS2 activity of bovine mammary epithelial cells. This result reveals that arachidonic acid and menaquinone-4 not only activate the expression of the gene encoding FADS2, but also enhance the FADS2 enzyme activity itself and promote the production of highly unsaturated fatty acids. It was.

  From these results, it was revealed that acetic acid, arachidonic acid, and menaquinone-4 have an action of increasing the expression of the gene encoding FADS2. It was also shown that the desaturase of bovine mammary epithelial cells is activated. Although this mechanism of action is not clear at this time, it has been speculated that it is related to inflammation and prostaglandins.

  It was also shown that the expression of the gene encoding FADS2 in the differentiated bovine mammary epithelial cells is hardly affected by hormones. Rodriguez-Cruz et al. (J. Lipid Res. 47553-560, 2006) reported that FADS2 fluctuates in synchrony with transcription factors SREBP-1 and PGC-1beta in the mammary gland of rats fed high corn oil. ing. However, in this test, even if the conditions under which SREBP-1 is thought to change (for example, addition of insulin or biotin) or linoleic acid, which is the main component of corn oil fatty acid, are added, the expression change of the gene encoding FADS2 Was not recognized. That is, the gene encoding FADS2 in bovine mammary epithelial cells is different from the gene encoding stearoyl-CoA desaturase (Japanese Patent Application No. 2009-228231), etc., and SREBP-1 and PGC-1beta function as main regulators. This suggests that there is no possibility (not controlled by the transcription factor).

  From the above results, it became clear that administration or feeding of acetic acid, arachidonic acid, and menaquinone-4 as a nutrient is particularly effective as a nutrient in a system that produces milk with a high content of highly unsaturated fatty acids based on FADS2. .

  The present invention is summarized as follows.

  That is, an object of the present invention is to provide a nutritional technique for efficiently increasing the content of polyunsaturated fatty acids (functional components) in ruminant livestock milk.

  Then, the expression of the gene encoding fatty acid desaturase 2 (FADS2) in the mammary gland tissue cells of ruminant livestock and / or the activity of the enzyme, an animal medicine or feed containing nutrients such as acetic acid, menaquinone-4, and arachidonic acid The above-mentioned problems are solved by increasing the composition by administering or feeding the ruminant livestock to increase the content of polyunsaturated fatty acids in the mammary gland tissue cells and milk.

Claims (9)

  A method for increasing the content of polyunsaturated fatty acids in ruminant livestock milk, characterized in that menaquinone-4 is given to ruminant livestock.   A method for producing ruminant livestock milk having an increased polyunsaturated fatty acid content, characterized in that menaquinone-4 is given to ruminant livestock.   The method according to claim 1, further comprising providing acetic acid and / or arachidonic acid.   The method according to claim 1, wherein the ruminant livestock is a cow.   An agent for increasing the content of polyunsaturated fatty acids in ruminant livestock milk, comprising menaquinone-4 as an active ingredient.   An agent for promoting the expression of a gene encoding fatty acid desaturase 2 (FADS2) in mammary gland tissue cells of ruminant livestock and / or an activity promoter for the enzyme, comprising menaquinone-4 as an active ingredient.   A feed composition for ruminant livestock that increases the content of highly unsaturated fatty acids in ruminant livestock milk, comprising menaquinone-4.   The agent or feed composition according to any one of claims 5 to 7, further comprising acetic acid and / or arachidonic acid.   Ruminant livestock is a cow, The agent or feed composition of any one of Claims 5-8 characterized by the above-mentioned.

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