KR101816855B1 - A composition comprising the Phellinus species extracts or β-glucan for odor reduction - Google Patents

Abstract

The Phellinus species extract of the present invention or beta-glucan has been shown to increase the expression of FMO3, particularly in the FMO gene group in the liver. Thus, the expression of FMO3 using the above-described mushroom extract or beta-glucan (Trimethylamine), which is a malodorous volatile component, is metabolized by TMAO (trimethylamine oxide), which is a non-volatile component, and thus can be usefully used as a feed additive for eliminating or reducing odor.

Description

A composition comprising the Phellinus species extracts or β-glucan for odor reduction

The present invention relates to a composition for reducing odor containing Phellinus species extract or beta-glucan as an active ingredient.

About 20,000 mushrooms are known around the world, of which about 2,000 can be developed for food. About 992 species of mushrooms distributed in Korea have been recorded, of which 100 are edible mushrooms and 50 are poisonous mushrooms, and more than 20 mushrooms with severe toxicity have been identified. More than 20 kinds of mushrooms can be cultivated in Korea, and anticancer, cholesterol lowering, and blood sugar lowering have been proven. Around the world, there are about 280 types of Pseudomonas mushrooms. Most of the mushrooms have a yellow color and grow by forming a ring pattern, and it is one of the most widely artificially cultivated mushroom varieties. Mushrooms are known to have great potential for the production of useful bioactive metabolites and for many materials as pharmaceuticals. Most of the polysaccharides contained in mushrooms are a deferent chemical composition belonging to the β-glucan group. Valuable sources of these mushroom biologically active compounds have anti-cancer, anti-platelet and anti-inflammatory properties.

Among the Phellinus species, the representative one is the wood mud mushroom, Phellinus. It is known by the scientific name linteus. However, in the case of Korea, the representative cultivar is Phellinus baumii , which accounts for more than 98% of all farms excluding the situation. In particular, it is known that Korea is the only place where Pseudomonas mushrooms Baumi is allowed for edible use and is used as a health promotion food. Until now, studies on the separation of various physiologically active substances from Pseudomonas mushrooms are ongoing, and Pseudomonas mushrooms according to extraction methods Changes in efficacy and evaluation of extracts are being reviewed. In addition to the fruiting body, in the mycelium, there are a variety of blood anticoagulant activity similar to that of the mushroom fruiting body, antioxidant effect, anticancer activity, gastric ulcer relief and anti-inflammatory activity, beta-secretase inhibitory activity for the treatment of Alzheimer's disease As physiological activity has been reported, many studies have been focused on culturing the mycelium of Pseudomonas mushrooms, but the effect of reducing the odor of Pseudomonas mushrooms or substances isolated therefrom has not been known.

Beta-glucan (β-glucan) is known as immune modulators obtained from bacteria, mushrooms, yeast and grain sources, and generates polysaccharides, and these glucose polymers can be used for certain pathogenic bacteria and fungi ( fungi) is a component of the cell wall. Beta-glucan is composed of beta-1,3-linked beta-D-glucopyranosyl units, regardless of the beta-1,6-linked side chain, and contains fungal cell wall components. It is known to be preserved and recognized as one of the major PAMPs (Brown, 2006).

From a long time ago, basidiomycetes higher fungi have been used as general medicines. It is believed that the medicinal effects of mushrooms, such as immune enhancing and antitumor activity, are due to beta glucan. Because alpha-glucan is a eukaryotic nutrient element, it is easily degraded by mammalian enzymes and has no immunostimulatory activity, but beta-glucan is derived from various fungi and is not degraded by human enzymes when administered orally. It is absorbed by stimulating immunity (Vos et al., 2007). Absorbed beta-glucans not only activate their anti-tumor activity, but also their ability to protect the host in the presence of fungal and bacterial infections in animals and humans. Although beta-glucan has a high molecular weight, it is absorbed in the intestine when administered orally and activates innate and acquired immunity.

Beta-glucan is a glucose polymer, and a linear beta(1,3)-linked D-glucose molecule as a backbone and a beta(1,6)-linked side chain of various sizes exist at different intervals from the backbone. Among the various beta (1,3), beta (1,4) and beta (1,6) beta-glucan-linked, only beta (1,3) activates immunity and exhibits antitumor activity. The first known function of beta-glucan is anti-tumor activity (Chihara et al., 1970), followed by antifungal, anti-infection (Onderdonk et al., 1992), and radioprotective (Gu). et al., 2005), cholesterol reduction (Wolever et al., 2011) and postprandial glucose metabolic activities (Battilana et al., 2001). . Beta-glucans are associated with Aspergillus (Torosantucci et al., 2005) and Candida vaginal (Pietrella et al., 2010) infection in animals and Vibrio infection in sea fish (Zhu. et al., 2006) as a candidate for vaccine or adjuvant with immune enhancing activity. Beta-glucans in plants such as oat and barley are mainly beta (1,4) linked, and beta-glucans in mushrooms and fungi are branched as short beta (1,6)-linked side chains to the beta (1,3) backbone. (Yan et al., 2005). It is known that differences in the structure, morphology and source of these glucans can affect biological activity (Brown and Gordon, 2001).

FMO (Flavin-containing monooxygenase) is a group of enzymes that promote the oxidation of substrates mainly as soft nucleophiles through the cofactor flavin. In contrast to the cytochrome P450 enzyme, FMO is generally not induced or inhibited by xenobiotic substances. Human FMO exhibits tissue characteristics: FMO1 is present in human fetal liver and adult kidney, FMO2 is present in lung, and FMO3 is present in adult liver.

FMOs are the second group of microsomal oxidative enzymes with broad and overlapping specificities. The main reactions of FMO are nitrogen, sulfur, or phosphorus heteroatoms where the hetero-atoms are N-oxide, S-oxide, or P-oxide, respectively. Promotes the nucleophilicity of the compound. Cytochrome P450 and functional part overlap, but the mechanism of action is different. FMO binds and activates molecular oxygen before the substrate binds to the enzyme. In addition, it requires flavin adenosine dinucleotide (FAD) as a cofactor. Unlike the cytochrome P450 enzyme, FMO is sensitive to heat and can be usefully used by researchers studying metabolism to differentiate enzyme systems.

TMA (trimethylamine) is one of the nitrogen compounds, and it is a tertiary aliphatic amine that gives off a stinging odor like rotten fish even at extremely low concentrations. Originally, TMA is produced by decomposing precursors such as choline among ingested foods by intestinal microbes. In normal cases, the generated TMA is converted into TMAO (trimethylamine N-oxide, melting point 220 to 222°C), which is an odorless non-volatile component by the action of the FMO3 enzyme in the liver, and is mostly discharged through urine, but pets, pigs, ducks, and chickens In the case of livestock such as cattle and cattle, it is not significantly converted to TMAO, and remains in urine and feces, which causes odors, and this odor is the cause of complaints and complaints in apartment houses or farms. . In particular, since the boiling point of TMA is 3 to 7°C, it always volatilizes in the form of gas at room temperature, so livestock farms where a large number of animals are raised in groups exhibit severe odors even in winter, reducing odors of animals and livestock without side effects. The technique of letting go is in a desperate situation.

Accordingly, the present inventors have researched to solve the above problem, it was confirmed that when the expression of the FMO3 gene increases, TMA, which is a malodorous volatile component, is converted to TMAO, which is an odorless nonvolatile component, thereby reducing the odor. When beta-glucan isolated from mushrooms is administered orally to an animal model, it is confirmed that the expression of FMO3 in the liver is significantly increased, so that Pseudomonas mushroom extract and beta-glucan can be usefully used as feed additives for reducing odor in pets and livestock. The present invention was completed by revealing.

It is an object of the present invention to provide a feed additive for removing or reducing odor containing extracts of Phellinus species or beta-glucan as an active ingredient.

In order to achieve the above object, the present invention provides a feed additive for removing or reducing odor containing an extract of Phellinus species as an active ingredient.

In addition, the present invention provides a feed additive for removing or reducing odor containing beta-glucan (β-glucan) as an active ingredient.

In addition, the present invention provides a blended feed composition for removing or reducing odor, containing a mushroom extract or beta-glucan as an active ingredient.

In addition, the present invention provides a method for removing or reducing odor comprising administering a Pseudomonas mushroom extract or beta-glucan to an individual other than humans.

By confirming that the Phellinus species extract or beta-glucan of the present invention increases the expression of FMO3 especially among the FMO gene groups in the liver, the expression of FMO3 using the Phellinus extract or beta glucan is By increasing the metabolism of TMA (trimethylamine), an odorless volatile component, to TMAO (trimethylamine oxide), an odorless nonvolatile component, it can be usefully used as a feed additive for removing or reducing odor.

1A is a diagram showing the results of analyzing changes in liver gene expression in a single administration group of Phellinus species hot water extract (PBE) as a histogram.
1B is a diagram showing the results of analysis of changes in liver gene expression in a single administration group of Pseudomonas mushroom hot water extract (PBE) as a box plot.
Figure 1c is a diagram showing the result of analysis of the change in the expression of liver genes in a single administration group of hot water extract (PBE) of Pseudomonas mushrooms as a MA plot.
1D is a diagram showing the results of analyzing changes in liver gene expression in a single administration group of Pseudomonas mushroom hot water extract (PBE) as a correlation scatter plot.
Figure 2a is a diagram showing the results of hierarchical clustering in a single administration group of Pseudomonas mushroom hot water extract (PBE).
Figure 2b is a diagram showing the gene expression pattern in a single administration group of hot water extract (PBE) of Pseudomonas mushrooms.
Figure 3a is a diagram showing the results of analysis of changes in liver gene expression in the multi-administered group of hot water extract (PBE) of Pseudomonas mushrooms as a histogram.
Figure 3b is a diagram showing the results of analysis of changes in liver gene expression in a multi-administered group of hot water extract (PBE) of Pseudomonas mushrooms as a box plot.
Figure 3c is a diagram showing the result of analysis of the change in the expression of liver genes in the multi-administered group of hot water extract (PBE) of Pseudomonas mushrooms as a MA plot.
3D is a diagram showing a correlation analysis scatter plot of the results of analysis of changes in liver gene expression in a multi-administration group of Pseudomonas mushroom hot water extract (PBE).
Figure 4a is a diagram showing the results of hierarchical clustering in a multi-administration group of hot water extract (PBE) of Pseudomonas mushrooms.
Figure 4b is a diagram showing the gene expression pattern in the multi-administered P. mushroom hot water extract (PBE) group.
Figure 5 is a diagram confirming the RNA quality (quality) in the hot water extract (PBE) administration group:
Cont: control; And
PBE: Pseudomonas mushroom hot water extract treatment group.
Figure 6 is a diagram confirming the expression of the FMO (Flavin-containing monooxygenase) gene according to the mouse tissue:
Tissues: type of tissue;
Liver: liver tissue;
Lung: lung tissue; And
Kidney; Kidney tissue.
Figure 7 is a diagram confirming the change in the expression of the FMO gene according to the extract treatment for each mouse tissue:
Tissues: type of tissue;
Liver: liver tissue;
Lung: lung tissue;
Kidney; Kidney tissue;
Cont: control;
PBE: Pseudomonas mushroom hot water extract treatment group;
PBW: Pseudomonas mushroom water layer fraction treatment group;
PBG: Pseudomonas beta-glucan treatment group;
20: 20 mg/kg; And
100: 100 mg/kg.

Hereinafter, the present invention will be described in detail.

The present invention provides a feed additive for removing or reducing odor containing Phellinus species extract or beta-glucan as an active ingredient.

The mushroom extract is preferably prepared by a manufacturing method comprising the following steps, but is not limited thereto.

1) extracting by adding an extraction solvent to Pseudomonas mushrooms;

2) cooling the extract of step 1) and filtering; And

3) A step of drying after concentrating the filtered extract of step 2) under reduced pressure.

In the above method, the extraction solvent of step 1) is preferably extracted using water, C ₁ to C ₂ lower alcohol or a mixture thereof, and the lower alcohol is preferably ethanol or methanol, but limited thereto. It is not. The extraction solvent is preferably 10 to 100 times, and preferably 20 to 50 times, to the dried mushroom mushrooms, but is not limited thereto. The extraction temperature is preferably 70 to 140°C, and preferably 85 to 110°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 36 hours, preferably 9 to 24 hours, but is not limited thereto.

In the above method, the vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

Situation mushrooms above are Baumi (Phellinus baumii) Or Pseudomonas Linteus (Phellinus linteus) Is preferably, and more preferably, it is a mushroom baumi.

The beta-glucan is preferably isolated from any one selected from the group consisting of basidiomycetes, plants, and fungi, and Pseudomonas mushrooms, reishi mushrooms ( Ganoderma lucidum ), cordyceps ( vegetables) worms ), but is more preferably isolated from any one selected from the group consisting of basidiomycetes such as barley, plants such as barley, and fungi including yeast, but is not limited thereto.

The Pseudomonas mushroom extract or beta-glucan is characterized by increasing FMO3 (Flavin-containing monooxygenase3) gene expression, and the FMO3 gene is characterized by converting TMA (trimethylamine) to TMAO (trimethylamine N-oxide).

The FMO3 gene is characterized by having a nucleotide sequence represented by SEQ ID NO: 13 (GenBank registration number: NM_008030).

The odor refers to a substance that can cause discomfort to humans contained in the feces or urine of pets or livestock.

In a specific embodiment of the present invention, the present inventors prepared a hot water extract of Pseudomonas mushrooms, a water layer fractions of Pseudomonas mushrooms, and beta-glucan of Pseudomonas mushrooms, divided into single administration (24 hours) and multiple administrations (7 days), respectively, orally once a day. After administration, the change in the expression of liver genes in the single administration group and the multiple administration group was confirmed (see FIGS. 1A to 4B), and as a result of confirming the change in the expression of the FMO gene in mice to which PBE was administered, It was confirmed that FMO3 gene expression was increased in both single administration and multiple administration (see Table 4), and RNA quality in tissues was visualized and confirmed (see FIG. 5).

In addition, the present inventors confirmed the expression of the FMO gene according to the mouse tissue, it was confirmed that even in the same FMO gene family (see Fig. 6), there was a difference in the expression level according to the tissue (see Fig. 6), and hot water extract (PBE) ), As a result of confirming the change in the expression of the FMO gene in tissues by treatment of the Pseudomonas mushroom water layer fraction (PBW) and Pseudomonas mushroom beta-glucan (PBG), the expression levels in FMO3 in the liver and FMO4 in the kidneys depend on the concentration It was confirmed that it increased (see Fig. 7).

Therefore, by confirming that the hot-water extract or beta glucan of the present invention increases the expression of FMO3 in the FMO gene group in the liver, the odorous volatility by increasing the expression of FMO3 using the hot-water extract or beta-glucan of Pseudomonas mushrooms By metabolizing trimethylamine (TMA), which is an odorless, nonvolatile component, it was confirmed that it can be usefully used as a feed additive for odor removal or reduction.

In the present invention, the feed additive may be used as it is or a known carrier, stabilizer, etc. may be added, and various nutrients such as vitamins, amino acids, minerals, antioxidants, and other additives may be added as necessary. Powder, granules, pellets, suspensions, etc. may be in a suitable state. In the case of supplying the feed additive of the present invention, it may be supplied alone or mixed with the feed.

In addition, the present invention provides a blended feed composition for removing or reducing odor, containing a mushroom extract or beta-glucan as an active ingredient.

The active ingredient of the present invention is preferably composed of 0.01 to 10 parts by weight with respect to the feed to which the extract or beta-glucan is added, but is not limited thereto.

By confirming that the Pseudomonas mushroom extract or beta-glucan of the present invention increases the expression of FMO3 in particular among the FMO gene group in the liver, by increasing the expression of FMO3 using the Pseudomonas mushroom extract or beta-glucan, TMA, which is a malodorous volatile component By metabolizing to TMAO, which is an odorless nonvolatile component, it was confirmed that it can be usefully used as a blended feed composition for removing or reducing odor.

In addition, the present invention provides a method for removing or reducing odor comprising administering a Pseudomonas mushroom extract or beta-glucan to an individual other than humans.

The individual excluding humans refers to animals such as horses, sheep, pigs, goats, camels, antelopes, cows, chickens, ducks, dogs, and cats, excluding only humans.

Although the method for removing or reducing odor is a method for animals other than humans, it does not mean that this method is not effective in humans. In addition, in the case of humans, it can be sufficiently used to remove or reduce odor by the method for removing or reducing odor according to the present invention. In other words, in the case of a patient with trimethylaminuria (Fish odor syndrome) who cannot convert to TMAO because the expression of the FMO3 enzyme is unknown due to unknown cause, excessive TMA is secreted in the body through urine, sweat, and breath, resulting in a rotten smell of fish. Is severe, so it can be applied to such patients.

Pseudomonas mushroom extract or beta-glucan of the present invention is confirmed to increase the expression of FMO3 in the liver, especially in the FMO gene group, by increasing the expression of FMO3 by using the extract or beta glucan, odorous volatile TMA By metabolizing to TMAO, which is an odorless nonvolatile component, it was confirmed that it can be usefully used as a method of removing or reducing odor.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Statistical processing

Experimental results were expressed as mean ± standard deviation, and all measurements were performed at least three times. Intergroup comparison of mean values was performed by ANOVA one-way analysis using InStat 3.06 (GraphPad software Inc., USA). Significant p values were expressed as ≤0.05.

<Example 1> Pseudomonas mushroom ( Phellinus baumii ) Preparation of extract

<1-1> Hot water extract of Pseudomonas mushroom ( Phellinus baumii extract, PBE)

The present inventors have confirmed that in preparing Pseudomonas mushroom extract, the extraction yield varies depending on the extraction conditions such as temperature, amount of purified water, pH, extraction time, and Pseudomonas mushroom pieces. Usually, extraction temperatures are 85°C, 90°C, 95°C, 100°C, 105°C and 110°C, and extraction times are 9 hours, 18 hours and 24 hours. Thus, after drying the mushrooms to cut out pieces, 200 ml of purified water (20 times or 50 times) was added to 4 g or 10 g of the mushroom pieces, and the suspension was filtered, and the extraction yield was measured.

In addition, the extract was mass-produced using the above method. Finely chopped mushroom pieces and 20 ℓ purified water were added to a 200 liter extractor, extracted 20 times at 110° C. for 24 hours, filtered through a filtration device, and the filtrate was concentrated. Each of these was lyophilized under reduced pressure. The extraction yield is shown in Table 1 below.

Sample Scale Mushroom capacity extraction Lyophilized powder Extraction yield (%) Mushroom 400 ℓ 20 kg 350±10 ℓ 1050 g 5.3

In this study, the hot-water extract of Pseudomonas mushrooms was prepared by purchasing fruiting bodies of Pseudomonas mushrooms grown in Korea.

Specifically, 100 g of the fruiting body of Pseudomonas mushrooms was added to 2000 ml of distilled water and extracted for 24 hours in a 100°C bath. After filtration and storage of the extract, 2000 ml of distilled water was added to the residue and extracted for 24 hours in a 100℃ bath. The extract obtained by repeated extraction three times was concentrated under reduced pressure to 100 ml, and then freeze-dried to make hot water extract of Phellinus (Phellinus baumii extract, PBE) was obtained.

<1-2> Mushroom Aqueous fraction ( Phellinus baumii water fraction , PBW ) And beta-glucan ( Phellinus baumii Preparation of β-Glucan, PBG)

The beta-glucan was purified using a known method by purchasing the fruiting bodies of Pseudomonas mushrooms grown in Korea.

Specifically, 100 g of the fruiting body of Pseudomonas mushrooms was added to 2000 ml of distilled water and extracted for 24 hours in a 100°C bath. After filtration and storage of the extract, 2000 ml of distilled water was added to the residue, extracted for 24 hours with a 100℃ bath, and the resulting extract was repeatedly extracted three times under reduced pressure to 100 ml, and then made to 4℃. 300 ml of ethanol (about 3 times) ice-cooled at 20° C. was added and left at 4° C. for 4 hours. After 4 hours, the precipitate was beta-glucan, and the isolated beta-glucan was confirmed using a Beta-glucan assay kit (Megazyme.co), which was freeze-dried and called Phellinus baumii β-Glucan (PBG). Called. On the other hand, the supernatant that does not precipitate in ethanol from the hot water extract of P. mushroom is filtered through filter paper, concentrated, and then freeze-dried to obtain a fraction of Phellinus (Phellinus). baumii water fraction, PBW).

<Example 2> Cell culture and animal model design

<2-1> cell culture

Raw 264.7 cells (ATCC, Rockville, MD), a mouse macrophage cell line, were used as penicillin (10,000 units/ml)-streptomycin (10,000 units/ml) and fetal bovine serum (FBS) (Gibco). -BRL, USA) was added to DMEM medium, and cultured in a 37°C, 5% CO ₂ incubator. When cultured in a T-25 flask and subcultured, a 10X trypsin-EDTA solution was diluted 10 times using DMEM and used. Add 1X trypsin-EDTA solution to Raw 264.7 cells cultured in a tissue culture flask, mix for 10 minutes at 37°C, and make single cells by trypsin reaction, and to terminate this reaction, DMEM medium containing 10% FBS After addition, it was washed twice. After that, it was centrifuged at a low temperature and then subcultured.

<2-2> Animal model design

A 5-week-old male MPF (murine pathogen free, outbred-mouse) ICR mouse weighing 25 to 30 g was purchased from Samtako Bio Korea and used as an orally administered experimental animal. Mice were allowed to eat freely with water and diet, and the lighting cycle was set to be bright for 12 hours and dark for 12 hours. The room was kept at a temperature of 22.5° C. and a humidity of 42.5%.

The hot-water extract of Pseudomonas mushrooms was lyophilized, dissolved in distilled water, and administered orally. The experimental group was divided into a control group (control, n=2) and a 100 mg/kg hot water extract treatment group (PBE-100, n=2), and was divided into a single dose (24 hours) and multiple doses (7 days). Divided and administered orally once a day.

In addition, Pseudomonas mushroom hot water extract (PBE), Pseudomonas mushroom water layer fraction (PBW) and Pseudomonas mushroom beta-glucan (PBG) were orally administered by dissolving them in distilled water using a lyophilized powder, and the experimental group was a control group (control, n =3), 20 mg/kg hot water extract treatment group (PBE-20, n=3), 100 mg/kg hot water extract treatment group (PBE-100, n=3), 20 mg/kg Pseudomonas mushroom water layer fraction treatment group (PBW-20, n=3), 100 mg/kg Pseudomonas mushroom water layer fraction treatment group (PBW-100, n=3), 20 mg/kg Pseudomonas beta-glucan treatment group ( It was divided into PBG-20, n=3) and 100 mg/kg of Pseudomonas beta-glucan-treated group (PBG-100, n=3), and was administered orally once a day for 7 days.

<Example 3> Analysis of changes in the expression of liver genes in a single administration group

Using the animal model designed in the same manner as in Example <2-2>, a sample was obtained from the liver, and changes in the expression of liver genes were analyzed through a microarray method.

Specifically, among the animal models designed in the same manner as in Example <2-2>, a single-administered mouse _{was euthanized with dichloromethan (CH 2} Cl ₂ ), and then 150 mg of tissue was obtained in a 2 ml tube, and RNA was used. Samples were stored below -20°C to obtain. A microarray was performed using a Mouse Gene 1.0 ST array (Affymetrix Inc), and the OD 260/280 ratio was confirmed. The result of this confirmation was analyzed using a histogram, a box plot, an MA plot, and a correlation scatter plot.

As a result, as shown in Figs. 1A to 1D, the liver gene expression pattern was confirmed (Figs. 1A to 1D).

In addition, based on the above results, major genes were analyzed and the expression pattern changes in the experimental group in which PBE was administered orally at a concentration of 100 mg/kg once compared to the control group were shown in Table 2 below.

Analysis design cutoff control Number of major genes The number of gene ontology information Number of route information Control vs PBE-100 1.3 down 1264 901 282 work 1184 925 349 1.5 down 292 184 75 work 328 226 93 2 down 61 31 14 work 45 36 18 3 down 22 One 0 work 9 8 7

In addition, by setting cutoff 2.0 based on the above <Table 2>, specific gene clustering was performed using hierarchical clustering.

As a result, as shown in Figs. 2a and 2b, the hierarchical clustering results of the control group and the hot water extract of Pseudomonas mushrooms were confirmed, and their gene expression patterns were confirmed (FIGS. 2a and 2b ).

< Example 4> Analysis of changes in liver gene expression in multiple dose groups

Using the animal model designed in the same manner as in Example <2-2>, a sample was obtained from the liver, and changes in the expression of liver genes were analyzed through a microarray method.

Specifically, among the animal models designed in the same manner as in Example <2-2>, mice in the multi-dose group were analyzed by the same method as in Example 3>.

As a result, as shown in Figs. 3a to 3d, the liver gene expression pattern was confirmed (Figs. 3a to 3d).

In addition, based on the above results, major genes were analyzed, and the expression pattern changes in the experimental group in which PBE extract was administered orally for 7 days at a concentration of 100 mg/kg were shown in Table 3 below compared to the control group.

Analysis design cutoff control Number of major genes The number of gene ontology information Number of route information Control vs PBE-100 1.3 down 1153 864 300 work 1358 1092 349 1.5 down 304 219 86 work 368 300 93 2 down 51 38 17 work 87 81 26 3 down 13 13 5 work 22 22 8

In addition, by setting cutoff 2.0 based on the above <Table 3>, specific gene clustering was performed using hierarchical clustering.

As a result, as shown in FIGS. 4A and 4B, the hierarchical clustering results of the control group and the P. mushroom extract-treated group were confirmed, and their gene expression patterns were confirmed (FIGS. 4A and 4B ).

<Example 5> Confirmation of the expression change of FMO (Flavin-containing monooxygenase) gene

Changes in the expression of the FMO gene in the liver tissue of ICR mice to which PBE obtained by using the same method as in <Example 3> were orally administered was confirmed.

FMO type Gene Expression in Liver Tissues After Oral Administration of Hot Water Extract of Pakistan Mushroom Single dose Multiple dose FMO1 No change No change FMO2 No change 3.8 times increase FMO3 5.13 times increase 17.6 times increase FMO4 No change 3.14 times increase FMO5 No change No change

As a result, as shown in Table 4, in the case of single administration, only the FMO3 gene increased by 5.13 times, and in the case of multiple administration, it was confirmed that FMO2 increased by 3.8 times, FMO3 by 17.6 times, and FMO4 by 3.14 times (Table 4 ).

<Example 6> RNA quality check

RNA was obtained from the tissue obtained in the same manner as in <Example 3>, and 18S ribosomal RNA (rRNA) and 28S rRNA were visualized and confirmed using gel electrophoresis.

As a result, as shown in FIG. 5, in the case of single administration, the integrity of rRNA was 1.6 and 1.7, and in the case of multiple administration, the integrity of rRNA was confirmed to be 2.4 and 2.4 (FIG. 5).

<Example 7> Confirmation of expression of FMO gene according to mouse tissue

FMO gene expression was confirmed in mouse liver, lung and kidney tissue.

Specifically, after euthanizing mice with dichloromethane (CH ₂ Cl ₂ ), liver, lung and kidney tissues were obtained, and then frozen before the experiment and stored. The frozen tissue was cut and homogenized by adding 1 ml of Trizol reagent, and then the sample was stored at room temperature for 5 minutes. 0.2 ml of chloroform was added thereto, and the tube was vigorously vortexed for 15 seconds, and stored for 2 to 3 minutes. After that, when the sample is centrifuged at 12000 rpm for 15 minutes at 4° C., the mixture is divided into a layer of phenol-chloroform at the bottom and a layer of water at the top without color. The water layer was transferred to a new tube, 0.5 ml of isopropyl alcohol was added, homogenized, and stored at room temperature for 10 minutes, followed by centrifugation at 12000 rpm for 15 minutes at 4°C. The supernatant was removed, and the precipitated RNA pellet was washed once with 1 ml of 75% ethanol (100% EtOH 35 ml + DEPC water 15 ml), and then the sample was vortexed and mixed at 4°C. Centrifuged at 7500 rpm for 5 minutes. After drying the RNA pellet, 50 µl of RNase-free water was added and stored at 56° C. for 10 minutes. The concentration of RNA thus obtained was measured with a NanoDrop 2000 UV spectrophotometer. The obtained RNA was synthesized cDNA using M-MLV reverse transcriptase (Moloney Murine Leukemia Virus reverse transcriptase) (Promega, USA). For the RNA/primer mixture, 5 µg RNA, 1 µl of oligo(dT) (0.5 µg/µl), and up to 10 µl of DEPC-treated water were added to a sterilized PCR tube, and reacted at 70° C. for 10 minutes, and then put on ice. Let it sit for at least 5 minutes. The reaction mixture was mixed with 4 µl of 5 X reaction buffer solution, 2 µl of 10 mM dNTP mixture, 2.4 µl of distilled water, 0.1 µl of RNase inhibitor, gently mixed with each RNA/primer mixture, and collected by briefly centrifuging. After placing this at 42°C for 3 minutes, 1 µl M-MLV reverse transcriptase was added, mixed, and left at 42°C for 60 minutes. The reaction was terminated at 70° C. for 5 minutes, placed on ice to cool, and then simply centrifuged to obtain a reaction product. The reaction product thus obtained is directly PCR or stored at -20°C until the experiment.

For RT-PCR, 1 μg of running RNA and reverse primer were mixed in a PCR tube using primers designed as shown in Table 5 below, reacted at 70° C. for 5 minutes, and then placed on ice. To this, forward primer is added, and the reaction volume is filled with DEPC water so that the volume is 20 µl. This was performed under PCR conditions as shown in Table 6 below. The PCR result was confirmed by electrophoresis using 1.0% agarose gel, and the image was confirmed with LAS-3000.

purpose
gene primer name order direction Tissue used Sequence number FMO1 FMO1_F CAT TCC AAC TAC AAG GAC TCG Forward direction kidney SEQ ID NO: 1 FMO1_R TGT CTC TGG ACA GTG GGA AGT Reverse SEQ ID NO: 2 FMO2 FMO2_F CCG GGT CTT TAA GGG TTT CAG G Forward direction lungs SEQ ID NO: 3 FMO2_R AGG CTC CAT CTT CCC AAC CGT A Reverse SEQ ID NO: 4 FMO3 FMO3_F CAG CAT TTA CCA ATC GGT CTT C Forward direction liver SEQ ID NO: 5 FMO3_R TTG CTG TGA TGC ATG AAG TTG Reverse SEQ ID NO: 6 FMO4 FMO4_F TCC TGA GCC CAC ATT TAC CTC Forward direction kidney SEQ ID NO: 7 FMO4_R CCA GTG TTT CCA AGA CCA ACC Reverse SEQ ID NO: 8 FMO5 FMO5_F GCT TGC CTA CAC GGT TCA AG Forward direction liver SEQ ID NO: 9 FMO5_R ATC ACA CGG ATG CTC ACC TG Reverse SEQ ID NO: 10 GAPDH GAPDH_F AGC CTC GTC CCG TAG ACA AA Forward direction SEQ ID NO: 11 GAPDH_R CAC GAC ATA CTC AGC ACC GGC Reverse SEQ ID NO: 12

PCR cycle Temperature time 1 cycle initial denaturation 95℃ 2 minutes 30 cycle denaturation 95℃ 30 seconds annealing 55℃ 30 seconds elongation 72℃ 1 minute 1 cycle final elongation 72℃ 5 minutes

As a result, as shown in FIG. 6, even in the same FMO gene family, it was confirmed that the difference in expression levels appeared according to tissues (FIG. 6).

<Example 8> Confirmation of the expression change of FMO gene according to extract treatment for each mouse tissue

Changes in FMO gene expression according to extract treatment in mouse liver, lung and kidney tissue were confirmed.

Specifically, an animal model in which the extract was orally administered by the method described in Example <2-2> was used, and it was confirmed by RT-PCR in the same experimental method as in Example 7 above.

As a result, as shown in FIG. 7, with results similar to those of <Example 5>, hot-water extract (PBE), water layer fraction (PBW) and beta-glucan of Pseudomonas mushrooms from FMO3 in the liver and FMO4 in the kidneys ( PBG) was found to increase the expression level in a concentration-dependent manner compared to the non-treatment group, and the expression of FMO3 in the group treated with Pseudomonas beta-glucan was more evident than the hot water extract of P. It was confirmed that it appeared (FIG. 7).

Based on the results so far, it was confirmed that the expression of FMO3 in the FMO gene group in the liver was significantly increased by oral administration of Pseudomonas mushroom extract or beta-glucan, so that the expression of FMO3 by using Pseudomonas mushroom extract or beta-glucan of the present invention By increasing the metabolism of trimethylamine, which is an odorless volatile component, to trimethylamine oxide, which is an odorless, nonvolatile component, it can be used as an additive in pet and livestock odor reduction feed.

<110> University of seoul Industry Cooperation Foundation <120> A composition comprising the Phellinus species extracts or beta-glucan for odor reduction <130> 2017P-08-046 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO1_F <400> 1 cattccaact acaaggactc g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO1_R <400> 2 cattccaact acaaggactc g 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO2_F <400> 3 cattccaact acaaggactc g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO2_R <400> 4 cattccaact acaaggactc g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO3_F <400> 5 cattccaact acaaggactc g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO3_R <400> 6 cattccaact acaaggactc g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO4_F <400> 7 cattccaact acaaggactc g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO4_R <400> 8 cattccaact acaaggactc g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO5_F <400> 9 cattccaact acaaggactc g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMO5_R <400> 10 cattccaact acaaggactc g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 11 cattccaact acaaggactc g 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 12 cattccaact acaaggactc g 21 <210> 13 <211> 2020 <212> DNA <213> Mus musculus <400> 13 attcggcacg agaaaggaag acaaagaaaa ggcacccatg aagaagaaag tggccatcat 60 tggagctggt gtcagtggcc tggctgccat caggagctgt ctggaggagg ggctggagcc 120 cacatgcttt gagaggagtg atgatgttgg gggcctgtgg aaattctcag accatataga 180 agagggcagg gccagcattt accaatcggt cttcaccaac tcttccaaag agatgatgtg 240 ttttccagac ttcccctatc ccgatgactt tcccaacttc atgcatcaca gcaagctcca 300 agaatacatc acttcatttg ccaaggaaaa gaacctcctg aaatacatac agtttgagac 360 acctgtaacc agtataaata aatgtcctaa tttctcaact actggcaaat gggaagtcac 420 cactgaaaag cacggtaaaa aagaaacagc tgtctttgat gctacaatga tttgttctgg 480 gcatcacata tttccccatg taccaaaaga ctcctttcca ggactgaacc gttttaaagg 540 caaatgcttc cacagcaggg actataagga accaggaata tggaagggaa aacgagtcct 600 ggtgattggc ctggggaact caggctgtga cattgctgca gaactcagcc atgtagctca 660 gaaggtcacc atcagctcta gaagtggttc ttgggtgatg agtcgagtct gggacgatgg 720 ctacccttgg gacatggtgg tgctcacacg gtttcaaact ttcctcaaaa acaacttacc 780 caccgccatc tctgactggt ggtacacaag gcagatgaat gccagattca agcacgaaaa 840 ctatggtttg gtgcctttaa acagaacact caggaaagag cccgtgttca atgatgagct 900 cccagcccgc atcctgtgtg gcatggtgac catcaagcct aatgtaaagg agttcacaga 960 gacgtccgct gtgtttgagg atgggaccat gtttgaggcc attgactgtg tcatctttgc 1020 cacaggctat ggttatgcct accccttcct ggatgactct attatcaaaa gcagaaataa 1080 tgaggtcact ttgtacaaag gtgtcttccc tcctcaacta gagaaaccaa ccatggcagt 1140 gattggcctg gtccagtccc tgggtgccac catccccata actgacctgc aggcacgctg 1200 ggcagcacaa gtaataaaag gaacttgcac tttgccttct gtaaacgaca tgatggatga 1260 cattgatgag aaaatggggg aaaagttcaa atggtatggc aatagcacca ccatccagac 1320 agattacatt gtttatatgg atgaactggc ctccttcatt ggtgcaaagc ccaatctcct 1380 atggctgttt ctcaaggatc ccaggttggc tgtagaagtg ttctttggcc cttgcagccc 1440 ctaccagttc cggctggtag gcccaggaaa gtggtcagga gcccggaacg ccatcctaac 1500 acagtgggac cgatcactga agcctatgaa gacgcgtgtc gtcagtaaag ttcagaagtc 1560 ttgctctcac ttctattccc gtttgctcag gctcctggct gttcccgttc tgctcattgc 1620 tttgttcctt gtgttgatct gatcgtgagt ccctctagaa tttctgagag tcactgacaa 1680 caaccagaca agaactttgc tatttaaaaa ttaagatttt tcacacctct ccctttcctg 1740 ttcaagatct tttgccagaa ctctagacgt taattagtaa gcaagacaat gttttgttct 1800 ggctttgtaa ctaagcccct ttcagaatca tgttgatctg cagtgggcat aagtcccttt 1860 ctctttaagt tccaccaaca atttgtaagg agaatggtat ttttatggac aacttgtatc 1920 atctcttgga acagttggac atgattcctt ttctctttta aacaatgctt gaaagatata 1980 cttcagattc agacagtgaa taaaatagag tatttagaat 2020

Claims (5)

1) preparing an extract of Phellinus species;
2) adding ethanol to the extract of step 1) to separate into a precipitate and an aqueous layer fraction; And
3) administering the precipitate or aqueous layer fraction of step 2) to an individual other than a human;
(Trimethylamine) to TMAO (trimethylamine N-oxide).
The method according to claim 1, wherein the mushroom is Phellinus baumii or Phellinus linteus, and converting TMA (trimethylamine) to TMAO (trimethylamine N-oxide).
The method according to claim 1, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent, and converting TMA (trimethylamine) into TMAO (trimethylamine N-oxide).
2. The method according to claim 1, wherein the mushroom extract increases FMO3 (Flavin-containing monooxygenase3) gene expression by converting TMA (trimethylamine) to TMAO (trimethylamine N-oxide).
1) preparing an extract of Phellinus species;
2) adding ethanol to the extract of step 1) to separate into a precipitate and an aqueous layer fraction; And
3) administering the precipitate or aqueous layer fraction of step 2) to an individual other than a human;
(Trimethylamine) to TMAO (trimethylamine N-oxide) by increasing expression of FMO3 (Flavin-containing monooxygenase3) gene.

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