KR20250032537A - Composition for preventing, improving or treating dyslipidemia comprising fermented oats - Google Patents

Abstract

The present invention provides a food composition comprising fermented oats fermented with a Lactiplantibacillus plantarum strain as an active ingredient, a health functional food composition for preventing or improving dyslipidemia, a pharmaceutical composition for preventing or treating dyslipidemia, or a feed composition for preventing or improving dyslipidemia.

Description

Composition for preventing, improving or treating dyslipidemia comprising fermented oats as an active ingredient

The present invention relates to a composition for preventing, improving or treating dyslipidemia, comprising fermented oats as an active ingredient, and more specifically, to a food composition comprising fermented oats fermented with Lactiplantibacillus plantarum strain as an active ingredient, a health functional food composition for preventing or improving dyslipidemia, a pharmaceutical composition for preventing or treating dyslipidemia or a feed composition for preventing or improving dyslipidemia.

The prevalence of dyslipidemia (blood total cholesterol 240 mg/dL or higher and blood triglyceride 200 mg/dL or higher) in Korea has been increasing since 1998, and the rate of increase has been rapidly increasing since 2005, indicating that cardiovascular disease is expected to become a serious national health problem in the long term.

The Ministry of Health and Welfare's Comprehensive Plan for the Management of Cardiovascular Diseases includes measures for the prevention and management of hypertension and diabetes, which are major causes of cardiovascular diseases, but there are no measures for dyslipidemia. Recently, as the level of public awareness of related diseases and symptoms such as hyperlipidemia has increased, sales of health functional foods and drugs related to dyslipidemia have increased. However, in the case of functional foods, the improvement effect and experimental evidence are still insufficient, and the treatment market is dominated by multinational pharmaceutical companies, so the burden of medical expenses for long-term use is high.

Oats are one of the most widely cultivated crops in Europe. They are known to contain more protein than regular rice and are rich in beta-glucan, a type of dietary fiber, at around 2-6%, so their consumption as a diet food is increasing.

In addition, oats contain bioactive substances such as polyphenols (protocatechuic, syringic, vanillic, gallic, caffeic acid, avenanthramide, etc.), and avenanthramides (AVNs) are bioactive substances found only in oats. They are known to reduce histamine, IgE, and IL-4, which are inflammatory substances produced by macrophages, and alleviate allergic reactions by inhibiting the adhesion of monocytes to vascular endothelial cells, have antioxidant functions, and help relieve itching of dry skin. In addition, research results on alleviating inflammatory bowel disease and reducing the occurrence of colon cancer have been reported.

As conventional technologies related to fermented oats, a method for producing a fermented oat extract having antioxidant, whitening, and wrinkle-improving effects (Patent Publication No. 10-2022-0057281), a cosmetic composition for skin protection containing fermented oat kernel extract using Saccharomyces cerevisiae J2K-413 strain as an effective ingredient (Patent Publication No. 10-2023-0027503), etc. are known.

However, the above conventional technologies do not disclose the use of fermented oats for preventing, improving, or treating dyslipidemia, and thus, there is an urgent need to develop new uses for such fermented oats.

Publication of Patent Publication No. 10-2022-0057281 Publication of Patent Publication No. 10-2023-0027503

Accordingly, the inventors of the present invention discovered that fermenting oats with a Lactiplantibacillus strain had an effect of improving dyslipidemia, and completed the present invention.

Accordingly, the object of the present invention is to provide a new use of fermented oats fermented with a Lactiplantibacillus strain.

However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above purpose, the present invention provides a health functional food composition for preventing or improving dyslipidemia, which contains fermented oats fermented with Lactiplantibacillus plantarum strain as an effective ingredient.

In one embodiment of the present invention, the oats may be sprouted oats.

As one embodiment of the present invention, the strain may be a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.

As one embodiment of the present invention, the fermented oats can have an increased content of β-glucan and avenanthramide compared to non-fermented oats.

To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating dyslipidemia, which contains fermented oats fermented with Lactiplantibacillus plantarum strain as an effective ingredient.

In one embodiment of the present invention, the oats may be sprouted oats.

As one embodiment of the present invention, the strain may be a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.

As one embodiment of the present invention, the fermented oats can have an increased content of β-glucan and avenanthramide compared to non-fermented oats.

In order to achieve the above purpose, the present invention provides a feed composition for preventing or improving dyslipidemia, which contains fermented oats fermented with Lactiplantibacillus plantarum strain as an effective ingredient.

In one embodiment of the present invention, the oats may be sprouted oats.

As one embodiment of the present invention, the strain may be a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.

As one embodiment of the present invention, the fermented oats can have an increased content of β-glucan and avenanthramide compared to non-fermented oats.

To achieve the above purpose, the present invention provides a food composition including fermented oats fermented with a Lactiplantibacillus plantarum strain.

In one embodiment of the present invention, the oats may be sprouted oats.

As one embodiment of the present invention, the strain may be a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.

As one embodiment of the present invention, the fermented oats can have an increased content of β-glucan and avenanthramide compared to non-fermented oats.

Fermented oats fermented with the Lactiplantibacillus plantarum strain according to the present invention have an excellent effect of improving dyslipidemia, and therefore can be used in various foods, health functional foods, feeds, and pharmaceuticals.

Figure 1 shows the results of comparing the beta-glucan content of regular oats and sprouted oats.
Figure 2 shows the increase in the avenanthramide content of sprouted oats and the increase in the beta-glucan content of fermented sprouted oats.
Figure 3 shows the results of (A) pH and (B) log CFU/ml measurements during the oat fermentation process, where O42 is oats fermented with L. plantarum CR42 (green) and OW is oats fermented with L. plantarum WCFS1 (red).
Figure 4 compares the beta-glucan and avenanthramide contents of unfermented and fermented oats, wherein the contents of (A) β-glucan and (B) avenanthramide are compared. Data are expressed as mean ± SEM, and the P-value was determined using one-way analysis of variance, and **p < 0.01 and ***p < 0.001 compared to the oat group, and O42 represents the results of oats fermented with L. plantarum CR42, and OW represents the results of oats fermented with L. plantarum WCFS1.
Figure 5 shows the cholesterol-lowering effect in a high-cholesterol diet mouse model.
Figure 6 shows the changes in body weight of mice after feeding a high-cholesterol diet and fermented oats, where (A) the change in body weight, (B) the liver weight, and (C) the liver weight-to-body weight ratio are shown. In Figure 6, ND is the normal diet fed group; HCD is the high-cholesterol diet fed group; Oat, non-fermented oat group; O42, oats fermented with L. plantarum CR42; OW, oats fermented with L. plantarum WCFS1, and the data are expressed as the mean ± SEM, and the P-value was determined using one-way analysis of variance, and *p<0.05 and ***p<0.001 compared with the HCD group, and ###p<0.01 compared with the ND group.
Figure 7 shows the results of observation of the mouse liver tissue surface, showing the effect of fermented oat representative images of histological liver sections after H&E staining. The scale bar is 100 μm, lipid droplets (blue arrows), inflammation (red arrows), and the results of ND, normal diet group; HCD, high-cholesterol diet group; Oat, unfermented oat group; O42, oats fermented with L. plantarum CR42; OW, oats fermented with L. plantarum WCFS1 are shown.
Figure 8 shows the results of analysis of liver damage indicators in mice, namely (A) alanine aminotransferase (ALT), (B) aspergillus oryzae aminotransferase (AST), (C) total bilirubin, and (D) thiobarbituric acid (TBA). ND, normal diet fed group; HCD, high-cholesterol diet fed group; Oat, non-fermented oat group; O42, oats fermented with L. plantarum CR42; OW, oats fermented with L. plantarum WCFS1. Data are expressed as mean ± SEM. P-values were determined using one-way analysis of variance. *p<0.05 and **p<0.01 compared to the HCD group, and #p<0.05, ##p<0.01, and ###p<0.001 compared to the ND group.
Figure 9 shows the results of analysis of blood cholesterol content in mice: (A) total cholesterol (TC), (B) triglyceride (TG), (C) high-density lipoprotein cholesterol (HDL-C), (D) low-density lipoprotein cholesterol (LDL-C), and (E) LDL- C/HDL-C ratio. The data are expressed as the mean ± SEM, and the P-value was determined using one-way analysis of variance. Compared with the HCD group, *p<0.05, **p<0.01, and ***p<0.001, and compared with the ND group, ##p<0.01 and ###p<0.001.
Figure 10 shows the results of comparing the expression of cholesterol metabolism genes in the mouse liver, showing the effect of fermented oats on the expression levels of genes related to cholesterol metabolism in the liver of hypercholesterolemic mice. The data are expressed as the mean ± SEM, and the P-value was determined using one-way analysis of variance and is *p<0.05, **p<0.01, and ***p<0.001 compared to the HCD group, and #p<0.05, ##p<0.01, and ###p<0.001 compared to the ND group.
Figure 11 shows the results of comparing the expression of cholesterol metabolism genes in the mouse ileum, showing the effect of fermented oats on the expression levels of genes related to cholesterol metabolism in the ileum of hypercholesterolemic mice. The data are expressed as the mean ± SEM, and the P-value was determined using one-way analysis of variance and is *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the HCD group, and #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the ND group.
Figure 12 shows the results comparing the fecal cholesterol and bile contents of mice, showing the effect of fermented oats on the fecal cholesterol and bile acid excretion of hypercholesterolemic mice. Fecal cholesterol (A) and fecal bile acid (B) were measured by an enzyme assay kit, and the data are expressed as the mean ± SEM, and the P-value was determined using one-way analysis of variance, and compared with the HCD group, **p<0.01 and ***p<0.001, and compared with the ND group, ##p<0.01 and ###p<0.001.
Figure 13 shows the results of comparing the contents of short-chain fatty acids in mouse feces, showing the concentration of short-chain fatty acids (SCFA) in the feces. ND, normal diet group; HCD, high-cholesterol diet group; Oat, unfermented oat group; O42, oats fermented with L. plantarum CR42; OW, oats fermented with L. plantarum WCFS1. Data are expressed as mean ± SEM, and the P-value was determined using one-way analysis of variance. *p<0.05 and ***p<0.001 compared with the HCD group, and #p<0.05 and ###p<0.001 compared with the ND group.
Figure 14 shows the results of analyzing the intestinal microbiota within the mouse cecum, showing the alpha diversity of the cecal samples. ND, normal diet-fed group; HCD, high-cholesterol diet-fed group; Oat, unfermented oat group; O42, oats fermented with L. plantarum CR42; OW, oats fermented with L. plantarum WCFS1. Data are expressed as mean ± SEM, and the P-values were determined using one-way analysis of variance and are **p < 0.01 and ***p < 0.001 compared with the HCD group, and #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the ND group.
Figures 15A and 15B show the results of the analysis of the gut microbiota between groups in the mouse cecum, showing the beta diversity of the cecal samples. (A) Multidimensional scaling (MDS), (B) Nonmetric multidimensional scaling (NMDS) plot, and (C) Phylogenetic diagram. ND (red), HCD (purple), oat (yellow), O42 (green), and OW (blue).
Figure 16 shows the results of analyzing the correlation between cholesterol metabolism and intestinal microorganisms, showing the relative abundance and Pearson correlation between bacterial genera and cholesterol metabolism characteristics.

Hereinafter, the present invention will be described in detail.

The first embodiment of the present invention relates to a health functional food composition for preventing or improving dyslipidemia, which comprises fermented oats fermented with a Lactiplantibacillus plantarum strain as an effective ingredient.

A second embodiment of the present invention relates to a pharmaceutical composition for preventing or treating dyslipidemia, which comprises fermented oats fermented with a Lactiplantibacillus plantarum strain as an active ingredient.

A third embodiment of the present invention relates to a feed composition for preventing or improving dyslipidemia, which comprises fermented oats fermented with a Lactiplantibacillus plantarum strain as an effective ingredient.

In addition, the fourth embodiment of the present invention relates to a food composition comprising fermented oats fermented with a Lactiplantibacillus plantarum strain.

In the compositions according to the first to fourth embodiments of the present invention, it is preferable that the oats are sprouted oats, but is not limited thereto.

In the compositions according to the first to fourth embodiments of the present invention, the strain is preferably, but not limited to, a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.

The above Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain may exhibit the following characteristics: (a) no resistance to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, and tetracycline, (b) the ability to lower blood cholesterol by directly decomposing and metabolizing cholesterol or by externally producing a metabolite that directly decomposes and metabolizes cholesterol, and (c) a 2-hour survival rate of 99% or more at pH 3.

The above Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain was confirmed to meet the minimum inhibitory concentration (MIC) value according to the 2012 EFSA standard as it was not resistant to the above antibiotics.

The above Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain exhibits cholesterol assimilation activity. Unlike conventional Lactobacillus plantarum strains that reduce cholesterol in a culture medium by adsorbing or absorbing cholesterol into the cell membrane or interior, the strain directly decomposes and metabolizes cholesterol or externally produces metabolites capable of such action, thereby showing a difference in the mechanism of action in terms of cholesterol-lowering functionality.

The above Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain exhibits at least one activity selected from bile resistance, acid resistance, and auto-aggregation ability, and also has the characteristics of not exhibiting hemolytic activity and biogenic amine production activity.

In the composition according to the first to fourth embodiments of the present invention, the fermented oats may have an increased content of β-glucan and avenanthramide compared to non-fermented oats. β-glucan may be increased 2 to 3 times compared to non-fermented oats, and avenanthramide may be increased 20 to 25 times compared to non-fermented oats.

The food composition or health functional food composition of the present invention may additionally contain, in addition to the effective ingredients, auxiliary ingredients or additives such as nutrients, vitamins, electrolytes, flavoring agents, coloring agents, thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages.

In addition, the carrier, excipient or diluent may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and saline solution, but is not limited thereto.

Health functional foods that can be added to the food composition or health functional food composition of the present invention include, for example, various foods, beverages, gum, candy, tea, vitamin complexes, functional foods, etc.

In addition, in the present invention, the food composition or health functional food composition may include, but is not limited to, special nutritional foods (e.g., formulated milk, infant/toddler food, etc.), processed meat products, fish products, tofu, starch jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks), processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi types, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, ice cream, etc.), natural seasonings (e.g., ramen soup, etc.), vitamin complexes, cereals, alcoholic beverages, liquors, and other health supplements.

The pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent commonly used in the art for the manufacture of pharmaceutical compositions. Preferably, the pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermal absorbents, gels, lotions, ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages and suppositories, and may be in various oral or parenteral dosage forms, but is not limited thereto.

When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used, but are not limited thereto.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included, but are not limited thereto.

Preparations for parenteral administration may include, but are not limited to, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include, but are not limited to, witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of the disease, the drug form, the route of administration, and the period of administration, but can be appropriately selected by those skilled in the art. However, for more effective prevention or treatment, the puffed ginseng root concentrate of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg, and the administration may be administered once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

The feed composition of the present invention can be used without limitation as long as it is a component and method commonly used in the art. For example, it can be produced by mixing corn, soybean meal, wheat, beef tallow, molasses, calcium phosphate, limestone, salt, and the fermented product of Hong Kyung-Cheon of the present invention, but is not limited thereto.

Examples of animals targeted by the feed composition of the present invention include, but are not limited to, cattle, pigs, chickens, sheep, goats, horses, dogs, cats, and poultry (domestic birds).

The method for manufacturing the food composition, health functional food composition, pharmaceutical composition and feed composition of the present invention can be manufactured by a method widely known in the technical field to which the present invention belongs, and therefore a detailed description thereof is omitted.

Hereinafter, in order to help understand the present invention, examples and the like will be described in detail. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

<Example 1> Analysis of beta-glucan content and avenanthramide content in oats

This experiment was conducted using sprouted oats. Oats ( Avena sativa L.) are cereals belonging to the Gramineae family, have high nutritional value, and contain 2-6% beta-glucan. Beta-glucan is a polysaccharide that is heat-resistant and has low toxicity.

In addition, there are research results showing that avenanthramide (Avn), a polyphenol component found only in oats, has antioxidant, anti-inflammation, and dementia prevention effects. Depending on the residue of N-cinnamoyl anthranilic acid, avenanthramide is mainly composed of avenanthramide A, which is combined with p-coumaric acid, avenanthramide B, which is combined with ferulic acid, and avenanthramide C, which is combined with caffeic acid.

Beta-glucan was analyzed using an analysis kit from Megazyme (Ireland), and for avenanthramide, the standard materials, avenanthramide A, B, C Standard reagents (Sigma, USA) and oat samples were analyzed at the National Institute of Basic Analysis (NICEM), College of Agriculture and Life Sciences, Seoul National University.

As shown in Fig. 1, the beta-glucan contents of regular oats and sprouted oats were 4.48±0.15% and 4.14±0.05%, respectively, and the beta-glucan content was slightly lower in sprouted oats than in regular oats. On the other hand, as shown in Table 1 below, the avenanthramide content was found to increase by about 2-4 times in sprouted oats compared to regular oats, and in particular, all forms of avenanthramides were greatly increased.

In general, Lactobacillus strains also contain 5-10% of beta-glucan as a cell component, so it is expected that an increase in beta-glucan due to fermentation microorganisms and an increase in avenanthramide content due to germination can be expected in fermented sprouted oats.

Figure 2 shows the increase in the avenanthramide content of sprouted oats and the increase in the beta-glucan content of fermented sprouted oats.

<Example 2> Confirmation of cholesterol-lowering functionality of oats fermented with selected strains

1. Verification of the suitability of selected strains for oat fermentation and establishment of fermentation conditions

Oat fermentation was performed by suspending the strain in 0.85% NaCl at 1x10 ⁷ CFU/10 μl and inoculating 1% of the sterilized oat suspension (20%, W/V) at 37°C for 24 hours. The CFU measured during the fermentation process rapidly increased until 12 hours of fermentation, reached the highest at 24 hours, and then decreased until 36 hours (Fig. 3). As a result, 24 hours, when the strain activity was the highest, was determined to be the optimal fermentation time.

The daily intake of oats that can contribute to improving serum cholesterol levels as specified in the Standards and Specifications for Health Functional Foods of the Ministry of Food and Drug Safety of Korea is at least 3g of oat dietary fiber. The average body weight of Koreans is 66.55kg, and the average body weight of mice in this experiment is 18g. In comparison, the amount of oat dietary fiber per 18g should be at least 0.77mg.

According to the food composition table provided by the Rural Development Administration, the total dietary fiber content of oats is 24.1 g per 100 g. In this experiment, 1 g of sterilized oat powder was mixed with 5 ml of DW, and since the oat content was 4 mg/20 μl, the amount of oat dietary fiber contained in 4 mg of oats was 0.964 mg/20 μl. This value exceeds the reference value of 0.77 mg/18 g, providing a sufficient amount of oat dietary fiber to the mice.

2. Analysis of functional substances in fermented oats

Cooking processes such as heat treatment of oats have been reported to improve the extraction rate of plant-derived β-glucans by changing the physicochemical properties of fiber. Analysis of the functional components of unfermented and fermented oats showed that oat fermentation by L. plantarum increased the β-glucan content. In addition, the avenanthramide content significantly increased in fermented oats (Fig. 4). This is expected to be the result of hydrolysis of oat components during the fermentation process, which resulted in the production of a large amount of peptides used as avenanthramide precursors.

3. Cholesterol-lowering effect in a high-cholesterol diet mouse model

The cholesterol-lowering functionality of oats fermented with CR42, which confirmed the cholesterol-lowering ability of L. plantarum CR42 strain from the above in vitro and in vivo experiments, was confirmed through animal testing. The experimental animal species were 4-week-old C57BL/6J female mice, divided into 5 groups. The hypercholesterolemia-inducing diet used was a product from Research Diet, USA.

The experimental group consisted of 8 mice per group, and food and water were supplied ad libitum . After one week of stabilization, the mice were fed a general diet and fermented oats for 2 weeks, and then the appropriate diet and fermented oats were fed to each group for 6 weeks. The diet was fed at 2 g/day per mouse. After 8 weeks of diet and strain intake, euthanasia was performed using _CO2 hyperinhalation, and autopsies were performed, and blood, liver, cecum, large intestine, and feces samples were collected (information about the groups and diets is presented below).

Figure 5 shows the cholesterol-lowering effect in a high-cholesterol diet mouse model.

There was no significant difference in the change in body weight according to the intake of a normal diet and a high-cholesterol diet, and the results were the same as the previous results. On the other hand, in the case of liver weight, it showed the characteristic of increasing the size and weight of the liver due to the increase in the amount of lipids in the liver and the inflammation induced by hypercholesterolemia (Fig. 6). The O42 group showed a tendency for the liver weight and the liver weight to body weight to decrease somewhat compared to the HCD group.

In addition, in microscopic observation of liver tissue, white lipids were observed in the inflammatory area and cytoplasm in the HCD group compared to the ND group, whereas in the O42 group, such liver damage was alleviated, showing a similar trend to the ND group (Fig. 7). This means that hypercholesterolemia caused by a high-cholesterol diet was suppressed.

Serum alanine aminotransferase (ALT) and asperate aminotransferase (AST) levels are known as representative liver damage indicators. ALT is an enzyme mainly found in hepatocytes, and when hepatocytes are damaged, ALT is released into the bloodstream, increasing the serum ALT level. An increase in the serum ALT level indicates liver damage or dysfunction. AST is another enzyme found not only in hepatocytes but also in other organs such as the heart, kidneys, and skeletal muscles, and an increase in the serum AST level can indicate liver damage, but it is less specific to the liver than ALT.

In hypercholesterolemia, an increase in total bilirubin levels indicates liver dysfunction or oxidative stress-related damage, which can occur when cholesterol accumulates excessively in the liver. Serum TBA levels increased in response to hypercholesterolemia when bile acid synthesis and secretion increased. The results of this study also confirmed that liver damage occurred in the high-cholesterol diet group significantly increased compared to the general diet group. On the other hand, a decreasing trend was observed in the O42 group (Fig. 8). This is consistent with the tendency for liver weight to decrease, indicating that hypercholesterolemia caused by a high-cholesterol diet was alleviated.

The results of quantitative analysis of total blood cholesterol in the normal diet group and the high-cholesterol diet group confirmed that the intended hypercholesterolemia model was well implemented. Compared to the normal diet group, all other experimental groups showed a significantly higher increase in cholesterol, but the O42 group showed a significant suppression of the increase in total cholesterol levels compared to the HCD group. The results of quantitative analysis of LDL blood cholesterol also showed a trend consistent with this (Fig. 9).

In the O42 group, the decrease in blood HDL cholesterol was the result of a decrease in total cholesterol, and there was no significant difference in the LDL/HDL-C ratio. As a result, O42 was found to have an effect of improving blood cholesterol increased due to a high-cholesterol diet.

To confirm the mechanism of the effect of O42 on cholesterol, genes related to cholesterol were analyzed in the liver and ileum tissues, where cholesterol absorption and metabolism occur, using qRT-PCR. First, in the liver tissue, the LDLR gene, which mediates exogenous cholesterol absorption, was significantly increased in the O42 group compared to the HCD group (Fig. 10). In the ileum tissue, the ABCG5/8 gene, which promotes cholesterol excretion, as well as the PPARa gene, which promotes beta-oxidation of fatty acids, were significantly increased in the O42 group compared to the HCD group (Fig. 11).

Accordingly, it is understood that exogenous cholesterol is absorbed and metabolized through LDLR within the O42 treatment time, and then cholesterol excretion is increased through ABCG5/8 in the ileum and beta-oxidation of fatty acids is increased to reduce blood cholesterol.

Among the methods by which blood cholesterol is excreted by probiotics, there is a direct method of excretion by being converted to cholesterol or bile acid and excreted, and an indirect method of regulating the production of short-chain fatty acids in the intestines that are involved in cholesterol production and excretion. Lipids were extracted from mouse feces collected 3 days before the end of the experiment using a method appropriate for each analysis, and then the experiment was conducted.

Fecal cholesterol was measured using a total cholesterol measurement kit (AM202, ASAN PHarm Co., Ltd, Seoul), and fecal bile acids were measured using a total bile acid measurement kit (Cell Biolabs, Inc., USA). Fecal short-chain fatty acids were analyzed using gas chromatography (GC system 7890B, Agilent technologies, USA) and Nukol (Capillary GC Column 24107, Supelco, USA).

In Fig. 12, the amounts of fecal cholesterol and bile acid excreted in the HCD group were significantly increased compared to the regular diet group, and although there was no significant difference in the amount of fecal bile acid, it is assumed that blood cholesterol was excreted in the feces in the form of cholesterol rather than bile acid through the tendency of increasing fecal cholesterol. As a result of measuring the content of short-chain fatty acids in the intestine, the content of acetate, which promotes cholesterol synthesis in the liver, was significantly reduced in the O42 group, and the content of butyrate, which promotes cholesterol excretion, was significantly increased (Fig. 13).

Within each group, the diversity of zOTUs was assessed using eight indices based on 16S amplicon sequencing data. The richness, as indicated by the number of zOTUs, was used to measure the diversity within each sample. The similarity of microbial communities in different environments was assessed via species evenness, which represents numerical similarity. The Shannon index quantifies ecological entropy, which provides a measure of diversity, and the Simpson index quantifies the probability that two individuals are randomly selected from the same data set, indicating evenness.

To analyze the intestinal microbiome, the cecum microbiome was analyzed. First, the richness, evenness, and Shannon, Simpson indices in alpha diversity, which compare the abundance of intestinal microorganisms within the group, significantly increased in the HCD group compared to the ND group. The Guilli and OW groups showed an increasing trend similar to the HCD group, but the O42 group showed a significant decrease, showing a similar trend to the ND group (Result 14).

These results imply that the intestinal microbiota composition of the O42 group changed in a similar trend to that of the ND group. Next, the beta-diversity results comparing the clusters of intestinal microbiota between the groups showed that the clusters of the ND group and the HCD group were significantly different, implying that the intestinal microbiota significantly changed when fed a normal diet and a high-cholesterol diet.

It was confirmed that the O42 group formed its own intestinal microorganism cluster, unlike the ND and HCD groups (Fig. 15a and Fig. 15b). As a result of analyzing the correlation between the intestinal microorganisms of the O42 group and indicators related to cholesterol metabolism, it was confirmed that Eisenbergiell , which shows a positive correlation with butyrate, which promotes cholesterol excretion, was specifically distributed in large quantities (Fig. 16). Eisenbergiell is known as a strain that can produce alpha-arabinosidase to decompose and utilize arabinose, a sugar component of oats, and promotes beta-oxidation.

4. Conclusion

In this study, we investigated the cholesterol-lowering effect of oats fermented with L. plantarum CR42 in a diet-induced hypercholesterolemic mouse model. By analyzing serum cholesterol levels, gene expression related to cholesterol metabolism in the liver and ileum, short-chain fatty acids, and intestinal microorganisms, the effects of oats fermented with Lactiplantibacillus plantarum WCFS1 ( L. plantarum WCFS1), a widely studied cholesterol-lowering strain, and oats fermented with L. plantarum CR42 were evaluated as a control.

Results: Feeding only L. plantarum CR42 showed potential effects on cholesterol regulation through changes in intestinal microflora and increased expression of LDLR and ABCG5 genes in the liver. This increase was followed by an increase in serum HDL-C levels, which significantly decreased the LDL-C/HDL-C ratio.

In contrast, when oats were fermented with L. plantarum CR42, the beta-glucan and avenanthramide contents significantly increased compared to unfermented oats. When oats fermented with L. plantarum CR42 were fed to mice, the expression of LDLR, which absorbs exogenous cholesterol in the liver, and ABCG5/8, which excretes cholesterol in the small intestine, significantly increased.

As a result of the intestinal microbiota analysis, alpha diversity showed a similar trend to the ND group fed a general diet. In addition, among the intestinal microbiota, Eisenbergiella , a strain that increases butyric acid, a short-chain fatty acid with a cholesterol-reducing effect, increased, and butyrate significantly increased, and blood total cholesterol levels significantly decreased. In conclusion, it was confirmed that oats fermented with L. plantarum CR42 can be a candidate for food, health functional food, medicine, or feed that is effective in preventing and improving hypercholesterolemia.

While specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily utilized by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15318BP 20230209

Claims (16)

A health functional food composition for preventing or improving dyslipidemia, comprising fermented oats fermented with Lactiplantibacillus plantarum strain as an effective ingredient.
A health functional food composition for preventing or improving dyslipidemia, characterized in that in claim 1, the oats are sprouted oats.
A health functional food composition for preventing or improving dyslipidemia, characterized in that in claim 1, the strain is Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.
A health functional food composition for preventing or improving dyslipidemia, characterized in that in the first paragraph, the fermented oats have an increased content of β-glucan and avenanthramide compared to non-fermented oats.
A pharmaceutical composition for preventing or treating dyslipidemia, comprising fermented oats fermented with Lactiplantibacillus plantarum strain as an active ingredient.
A pharmaceutical composition for preventing or treating dyslipidemia, characterized in that in claim 5, the oats are sprouted oats.
A pharmaceutical composition for preventing or treating dyslipidemia, characterized in that in claim 5, the strain is Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.
A pharmaceutical composition for preventing or treating dyslipidemia, characterized in that in claim 5, the fermented oats have an increased content of β-glucan and avenanthramide compared to non-fermented oats.
A feed composition for preventing or improving dyslipidemia, comprising fermented oats fermented with Lactiplantibacillus plantarum strain as an effective ingredient.
A feed composition for preventing or improving dyslipidemia, characterized in that in claim 9, the oats are sprouted oats.
A feed composition for preventing or improving dyslipidemia, characterized in that in claim 9, the strain is Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.
A feed composition for preventing or improving dyslipidemia, characterized in that in claim 9, the fermented oats have an increased content of β-glucan and avenanthramide compared to non-fermented oats.
A food composition comprising fermented oats fermented with a Lactiplantibacillus plantarum strain.
A food composition according to claim 13, characterized in that the oats are sprouted oats.
A food composition according to claim 13, characterized in that the strain is a Lactiplantibacillus plantarum CR42 (Accession Number: KCTC 15318BP) strain.
A food composition according to claim 13, wherein the fermented oats have an increased content of β-glucan and avenanthramide compared to non-fermented oats.