KR20220028755A - Additive composition for low fish meal feed comprising PPARγ antagonist - Google Patents

Abstract

The present invention relates to a technique for providing a composition, which includes a PPARγ antagonist as an active ingredient, as a low-fish meal feed additive for aquaculture fish. More specifically, it has been confirmed that expression of PPARγ has increased in tissues of halibut fed with low-fish meal feed containing soybean cake, which is vegetable protein, and the increased PPARγ increases expression of inflammatory factors to induce inflammation in the gastric and intestinal tissues of halibut, and thus it has been confirmed that an inflammatory response is suppressed in the intestinal tissue of halibut fed with the low-fish meal feed containing maslinic acid or indole which is an PPARγ antagonist.

Description

Additive composition for low fish meal feed comprising PPARγ antagonist as an active ingredient

The present invention relates to a technique for providing a composition containing a PPARγ antagonist as an active ingredient as an additive to aquaculture fish.

Today, the aquaculture industry is one of the fastest growing aquaculture industries, and its economic importance is recognized worldwide due to the rapidly increasing demand for aquatic products. In Korea, too, consumption of aquatic products is increasing every year, but in contrast to the rapidly increasing consumption of aquatic products, the amount of aquatic products that can be obtained through fishery is limited.

In order for the aquaculture industry to be carried out smoothly, aquaculture elements such as farms, seedlings, and feed must be balanced. Among them, feed is a factor that greatly affects the growth, health, and quality of aquaculture target fish, and accounts for about 30 to 60% of the total aquaculture production cost. Therefore, it is very important to use an appropriate feed for each fish species as it leads to the quality of the fish to be farmed, that is, the price.

Feed for aquaculture differs from animal feed, in that it has a higher protein content and higher feed efficiency than terrestrial animals. In particular, carnivorous seafood requires high protein because physiological availability for carbohydrates is low. For this reason, protein source in fish feed is an important raw material that accounts for more than 60% of feed. Protein content in feed is used for new protein synthesis, tissue synthesis and repair, enzyme/antibody/hemoglobin synthesis, etc., and accounts for 40~70% of feed cost. In other words, it is important to properly mix and use a protein feed source because the growth, feed price, and aquaculture unit price of the target fish are determined depending on the type of protein feed source.

Currently, the most used protein source in fish feed is fish meal, which has a high protein content, balanced amino acid and fatty acid content, and high palatability. However, as the catches of anchovies, sardines, and horse mackerel used as fishmeal have continued to decrease and stagnate due to climate change and overfishing, the price of fishmeal has skyrocketed. In order to solve this problem, research on alternative protein sources that can reduce the use of fishmeal within the range that does not affect the quality and growth of aquaculture target fish is being actively researched. Alternative protein sources currently studied include soybean meal, rapeseed meal, cottonseed meal, canola meal, palm meal, flax meal, lupin, and rouge meal for vegetable protein.

However, vegetable protein has anti-nutritional factors such as trypsin inhibitor, lectin, and soybean antigen protein, so the absorption and availability of nutrients is poor, and there is a problem in that it causes an inflammatory reaction in fish tissues, so it is used limitedly. However, as the use of fishmeal is high in flounder (float) and rockfish (roketail) feed, which account for more than 60% of fish culture, 40~80% of fishmeal is used. This is absolutely necessary.

Republic of Korea Patent Publication No. 10-2015-0092549 (published on August 13, 2015)

As an alternative source of fishmeal, stir-fry feed containing vegetable protein induces an inflammatory response in farmed fish due to a protein component derived from soybean and shows a problem that digestion and absorption efficiency is lowered. In order to solve the above problems, An object of the present invention is to provide an additive composition for fermented feed containing a PPARγ antagonist as an active ingredient.

The present invention provides an additive composition for jerk feed containing a PPARγ antagonist as an active ingredient.

In addition, the present invention provides a compound feed composition for fish comprising a PPARγ antagonist and a fish feed.

According to the present invention, the expression of PPARγ was increased in the tissues of flounder fed with stir-fry diet containing plant protein, soybean meal, and the increased PPARγ increased the expression of inflammatory factors to induce inflammation in the stomach and intestinal tissues of flounder. As it was confirmed that the inflammatory reaction was inhibited in the intestinal tissue of halibut fed with stir-fry containing PPARγ antagonist Maslinic acid or Indole, the PPARγ antagonist was used as an active ingredient. The composition containing it may be provided as an additive for a stir fry feed or a compound feed composition for fish.

1 is a schematic diagram of gene expression analysis for 3 groups (6 samples: con_liver vs test_liver, con_intestine vs test_intestine, con_muscle vs test_muscle).
2 is a result of RT-PCR analysis confirming the expression change of immune and inflammatory response-related genes according to low fish meal feeding, SM is the low fish meal feeding group, and FM is the control group.
3 is an RT-PCR analysis result confirming the increase in PPARγ 1, PPARγ 2 and fatty acid synthase (FASN) expression in halibut tissue according to the low fish meal feeding, SM is the low fish meal feeding group, and FM is the control group.
4 is a general feed (F), 30% low-fish meal (S), a stir-fry feed (Ma) containing maslinic acid additive, and stir-fry feed (Indole) containing an indole additive for 7 halibut is the result of RT-PCR analysis confirming the increased level of TNFα expression in each flounder intestinal (intestine) tissue after feeding for 10 weeks.
5 is an RT-PCR analysis result confirming the inhibitory effect of indole on TNFα expression in halibut cells.

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

In the case of farmed fish fed with a stir-fry diet containing vegetable protein as an alternative source of fishmeal, the inflammatory response is increased and digestion and absorption efficiency is lowered compared to that of conventional raw feed due to the protein component derived from soybeans. As this occurs, while the inventors of the present invention were conducting research for improving the aerobic diet, PPARγ, which induces inflammation in the human intestinal tissue, was increased by PPARγ along with the fact that the expression amount increased by feeding the aerobic diet. It was confirmed for the first time that the expression of the regulated genes is increased and completed the present invention.

The present invention may provide a stir-frying additive composition containing a PPARγ antagonist as an active ingredient.

The stir-fry feed may contain 30 to 70 parts by weight of vegetable protein based on 100 parts by weight of the total feed.

The vegetable protein may be a protein derived from soybean meal, rapeseed meal, cottonseed meal, canola meal, palm meal, flax meal, lupine and rouge meal, but is not limited thereto.

The stir-fry food may increase the expression of PPARγ 1 and PPARγ 2 in fish tissue to induce inflammation.

The PPARγ antagonist may inhibit the expression of an inflammatory factor induced by PPARγ.

The inflammatory factor may be selected from the group consisting of TNFα, Toll-like receptor (TLR), IL-1β (Interleukin-1β), IRF (intra-retinal fluid), and IL8 (Interleukin 8).

The PPARγ antagonist may be one selected from the group consisting of maslinic acid, indole, GW9662, isorhamnetin, T0070907, G3335 and SR202, and more preferably maslinic acid or indole. However, the present invention is not limited thereto.

The present invention can provide a compound feed composition for fish comprising a PPARγ antagonist and a stir fry food.

The compound feed composition may contain 0.01 to 1 g of the PPARγ antagonist with respect to 100 g of the stir-frying feed. More preferably, 25 mg of masrnic acid or 40 mg of indole as a PPARγ antagonist may be included with respect to 100 g of the jerk feed, but is not limited thereto.

The PPARγ antagonist may be one selected from the group consisting of maslinic acid, indole, GW9662, isorhamnetin, T0070907, G3335 and SR202.

The fish may be selected from the group consisting of halibut, rockfish, mullet, red snapper and sea bass, and more preferably halibut, but is not limited thereto.

The feed additive may additionally contain a fish-acceptable carrier. In the present invention, the feed additive may be added as it is, or a known carrier, stabilizer, etc. may be added, and if necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added. And as the shape, it may be in a suitable state such as powder, granule, pellet, suspension, and the like. In the case of supplying the feed additive of the present invention, it can be supplied alone or mixed with the feed for fish.

The feed composition of the present invention may be prepared in the form of a conventional feed composition such as powder and pellets, but is not limited thereto, and can be prepared in various types of feed known in the art.

The feed composition may include ingredients commonly added to feed, such as carbohydrates, proteins, fats, minerals, vitamins, minerals and water. There is no particular limitation in the type of each of these components, and any commonly used in the art may be used.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Confirmation of expression genes according to ingestion of general feed or stir-frying feed

The difference in the genes expressed in the muscles, liver and stomach tissues of halibut fed the conventionally used general feed and the low fish feed, which is a mixed feed, was confirmed.

Provided from the Nutrition Research Building of the Feed Research Center of the National Academy of Fisheries Research, two flounder (control group) farmed with a general feed containing 70% fishmeal and two flounder farmed with a stir-fry feed containing 40% fishmeal and 30% vegetable protein (experimental group) received.

Liver, intestine and muscle were extracted from the provided halibut, respectively, and total RNA was extracted from the tissue.

1. Identification of differentially expressed genes (DEGs) by feeding

For differential expression gene (DEGs) analysis, RNA sequencing (RNA-seq) was performed with total RNA extracted from the tissue, and the RPKM (Reads per kb per million reads) value analyzed first after RNA sequencing was obtained. was used to analyze gene expression changes for each control group vs. experimental group.

Six samples of the control group, which is a general feed, and the experimental group, which is an improved stir-fry feed, were divided into 3 pairs (con_liver vs. test_liver, con_intestine vs test_intestine, con_muscle vs test_muscle), and the rate of expression change in each tissue (RPKM stir-fry/RPKM conventional feed) ) was calculated. In more detail, as shown in Fig. 1, which is an analysis flow chart for the gene expression change experiment, analysis was performed on 32,407 genes obtained as a result of strategy 2 of halibut annotation, and 6 samples divided into 3 groups were analyzed within each group. By comparing RPKM values with control vs test, genes with altered expression were selected.

As a result, 4,442 >2x fold DEGs were identified in the liver, 6,101 DEGs in the intestine, and 7,250 DEGs in the muscle.

Among the genes, 24 differentially expressed genes including desaturase, elongation enzyme, phospholipid degrading enzyme, lipoprotein and cholesterol metabolizing enzyme in the liver are preferentially used as markers indicating lipid metabolism disorders in halibut with clear signs of essential amino acid or phospholipid deficiency. was recognized as Of these, five genes are unspecified markers of lipid metabolism disorders, and the rest are emerging as definitive specific markers for fatty acid or phospholipid deficiency.

Although the number of differentially expressed genes was reduced from 24 to 9, the same trend was confirmed in skeletal muscle and adipose tissue, and the nucleotide sequence in which PPARγ responds as a transcription factor in the gene promoter, which is the expression control region of these genes, was confirmed.

2. Immunity and inflammation-related gene expression according to the intake of stir-frying food

In order to confirm the expression of genes related to metabolism/immunity/growth in halibut fed with fish feed, Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using the primers shown in Table 1.

As a result, it could be confirmed that the mRNA expression of genes related to immunity and inflammation was remarkably increased when fed with low fish feed (SM) as shown in FIG. 2 . In addition, PPARγ regulatory regions were also identified in the promoters of these genes.

<Example 2> Confirmation of changes in expression of PPARγ 1/2 and FASN according to the feeding method

PPARγ has been reported to increase inflammation of gastric tissue in studies using human cells and tissues, and excessive production and accumulation of lipid components due to metabolic regulation in cells is known to induce tissue inflammation. It was confirmed whether the expression of fatty acid synthase (FASN), which plays an important role in cellular lipid synthesis, was changed in halibut tissue fed with feed, and PPARγ 1 and PPARγ, two subtypes of PPARγ fed with stir-fry diet. 2 It was confirmed by RT-PCR whether it affects the expression change.

As a result of RT-PCR analysis, it was confirmed that the expression of PPARγ 1 and PPARγ 2 increased, and the expression of FASN was also increased in the tissues of flounder fed with low fish feed as shown in FIG. 3 . In addition, it was confirmed that the feeding of the stir-fry food induced a decrease in the digestive efficiency of farmed fish species such as halibut compared to the general diet, and caused inflammation in the gastric tissue.

<Example 3> Confirmation of the expression change of TNFα according to the feeding of the stir-fry

As it was confirmed from the previous experimental results that stir-fry feed causes inflammation, in order to check how the effects of fish meal substitute feed and additives change according to the feeding period, as shown in Table 2, the control and fish meal of 30% of the control and fish meal For 100 g of the replacement group (FM30) and the 30% fish meal replacement group, feed the compound feed containing 25 mg of maslinic acid (Maslinic acid, additive 1) or 40 mg of indole (Indole, additive 2) for 10 weeks to culture halibut And by extracting stomach, liver and muscle tissue, the expression change of inflammation-related factors was confirmed.

As a result, as it was confirmed that the expression of inflammation-related factors increased in halibut fed the fishmeal substitute feed compared to the fishmeal feed, pro-inflammatory cytokines that induce inflammation in the individual unit of the halibut fed the fishmeal substitute feed and the additive It was confirmed whether the expression of TNF (tumor-necrosis factor) α, a pro-inflammatory cytokine, was changed.

As shown in Table 2, a total of 4 types of mixed feeds were classified and fed to 7 halibut, respectively, and then RT-PCR was performed to confirm the change in the expression of TNFα in the intestinal tissue of halibut.

As a result, as shown in FIG. 4 , the expression of TNFα was significantly reduced by masrnic acid and indole acting as PPARγ antagonists, and it was confirmed that the expression of TNFα was further reduced by indole treatment rather than masrnic acid.

From the above results, it was confirmed that the PPARγ antagonist can be an effective additive in relieving intestinal inflammation induced by low-fish meal formulations containing soybean meal.

<Experimental Example 4> Confirmation of the inhibitory effect of PPARγ antagonists on inflammation

As confirmed in the previous experiment, in order to confirm the intestinal inflammation alleviation effect of indole, a PPARγ antagonist, 0, 0.1, 1, 10, 20 and 30 μg/ Indole at a concentration of ml was treated.

As a result, as shown in FIG. 5, cytotoxicity by LPS and indole was not confirmed, but it was confirmed that the expression of TNF increased by LPS was decreased by indole.

From the above results, it was confirmed that the PPARγ antagonist was suitable as a feed additive for suppressing the inflammation of halibut caused by low-eating feed.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

An additive composition for stir-frying feed containing a PPARγ antagonist as an active ingredient. The additive composition for a stir-fry feed according to claim 1, wherein the stir-fry feed contains 30 to 70 parts by weight of vegetable protein based on 100 parts by weight of the total feed. The additive composition of claim 1, wherein the stir-fry feed increases the expression of PPARγ 1, PPARγ 2 and fatty acid synthase (FASN) in fish tissue to induce inflammation. The additive composition of claim 1, wherein the PPARγ antagonist suppresses the expression of an inflammatory factor induced by PPARγ. The additive composition of claim 1, wherein the PPARγ antagonist is selected from the group consisting of Maslinic acid, Indole, GW9662, isorhamnetin, T0070907, G3335 and SR202. . A compound feed composition for fish comprising a PPARγ antagonist and a stir fry. The compound feed composition for fish according to claim 6, wherein the compound feed composition contains 0.01 to 1 g of the PPARγ antagonist with respect to 100 g of the stir-frying feed. The compound feed composition for fish according to claim 6, wherein the PPARγ antagonist is selected from the group consisting of maslinic acid, indole, GW9662, isorhamnetin, T0070907, G3335 and SR202. . The compound feed composition for fish according to claim 6, wherein the fish is selected from the group consisting of halibut, rockfish, mullet, red sea bream and sea bass.

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