KR101357663B1 - Hepatoprotection fermented food comprising fermented rice bran and method of manufacturing the same - Google Patents

Abstract

The present invention relates to a fermented food comprising rice bran fermentation as an active ingredient and a method for producing the same, and more particularly, to a method for preparing a liver-protective fermented food comprising inoculating fermented soybean paste powder on rice bran medium. Liver protection rice bran fermented food according to the present invention can provide a liver protective effect through improved liver function and antioxidant activity of liver tissue when ingested.

Description

Liver-protected fermented foods using rice bran fermentation as an active ingredient and a method of manufacturing the same {HEPATOPROTECTION FERMENTED FOOD COMPRISING FERMENTED RICE BRAN AND METHOD OF MANUFACTURING THE SAME}

The present invention relates to liver protection using the rice bran fermentation as an active ingredient and a method for producing the same.

Rice, which has a long history as a food source for mankind, is obtained in the form of brown rice or white rice, depending on the degree of planting, and rice bran, a by-product of rice husk, rice bran, rice snow, and crab, which remain except the brown rice and white rice. You get

In the case of brown rice, it is recommended to eat it as a healthy food in modern times. However, when it is consumed with rice, there is a disadvantage in that the texture touched by the mouth is rough and the preference for taste is low and the intake rate is low. , It is more delicious than brown rice and the texture is softer. In case of modern people, since it is already used for a long time to eat white rice, it is a reality that it is consuming white rice with high preference. Problems that are not happening continue to arise.

In general, it is known that about 95% of bioactive substances such as gamma aminobutyric acid (GABA), lecithin, octacosanol, oryzanol, tocopherol, and ferulic acid are distributed in rice bran. Gamma aminobutyric acid (GABA) is a neurotransmitter of the central nervous system. It is also known to have a blood pressure lowering effect, suppression of blood cholesterol and triglyceride increase, and alcohol metabolism enhancement. Tocopherol is an oil-soluble vitamin E having antioxidative function. Abnormal beta-lipoproteinemia, and chronic cholestatic liver disease. Ferulic acid is known to play a role in the inhibition of active oxygen. Since there are many biologically active substances in rice bran, they are produced during the process even though they have various physiological activities such as heavy metal detoxification activity, anti-constipation activity, anti-cholesterol activity, skin beauty activity, antihypertensive activity, antihypertensive activity, hangover relieving activity, etc. Rice bran, a by-product, is mostly used for livestock feed.

Accordingly, the present inventors have found that fermenting the Cheonggukjang powder under the rice bran medium can produce a more effective liver protection composition, and have led to the present invention.

An object of the present invention is to prepare a rice bran fermentation product prepared by inoculating the rice bran soup in rice bran medium to provide a liver functional food.

In one embodiment, there is provided a method for producing a liver-protected fermented food comprising the step of inoculating the fermented soybean paste powder to the rice bran medium, the liver protection characterized in that the cheongukjang powder of the embodiment is 1 part by weight relative to the total weight ratio It provides a method for producing a fermented food, the step of fermenting the embodiment provides a method for producing a liver protective fermented food, characterized in that the fermentation at a temperature of 20 to 30 ℃, before the step of fermentation of the embodiment Prefermentation at a temperature of 75 to 90 ℃, and after the fermentation step provides a method for producing a liver protective fermented food, characterized in that the fermentation at a temperature of 75 to 90 ℃, the step of fermenting the embodiment Method for producing a liver-protective fermented food, characterized in that the fermentation under conditions of 55 to 65% relative humidity Provided, the fermentation step of the embodiment provides a method for producing a liver-protective fermented food, characterized in that performed for 40 days.

In one embodiment, provides a liver-protected fermented food comprising a rice bran fermentation inoculated with rice bran powder on rice bran medium, the chungkukjang powder of the embodiment provides a liver-protected fermented food, characterized in that 1 parts by weight relative to the total weight ratio do.

In one embodiment, there is provided a composition for the treatment of liver protection comprising a rice bran fermentation inoculated with the rice bran culture in the rice bran medium, wherein the composition for the treatment of liver protection of the embodiment powders, granules, premises, capsules, suspensions, emulsions, Oral formulations, including syrup or aerosol forms, are external preparations, suppositories, sterile injectable solutions.

In one embodiment, the present invention provides a composition for treating metabolic disease comprising a rice bran fermentation inoculated with the rice bran powder to the rice bran medium, wherein the composition for treating metabolic disease is powder, granules, premises, capsules, suspensions, emulsions, Oral formulations, including syrup or aerosol forms, are external preparations, suppositories, sterile injectable solutions.

Rice bran fermented food according to the present invention may provide a liver protective effect through improved liver function and antioxidant activity of liver tissue when ingested.

1 is H & E staining result (40X) of liver tissue. (A. normal, B. CCl ₄ control, C. GR (0.4%) + CCl ₄ , D. GF (0.4%) + CCl ₄ , E. positive control)
Figure 2 is Masson Trichrome staining results (X40) of liver tissue. (A. normal, B. CCl ₄ control, C. GR (0.4%) + CCl ₄ , D. GF (0.4%) + CCl ₄ , E. positive control)
3 shows the Sirius red staining result (X40) of liver tissue. (A. normal, B. CCl ₄ control, C. GR (0.4%) + CCl ₄ , D. GF (0.4%) + CCl ₄ , E. positive control)
Figure 4 is a graph showing the collagen concentration of liver tissue.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example  One. Rice bran Fermented  Produce

Cheonggukjang powder (Product No. 157, Binmax Korea, Muan) was added to Migang (Korea, Seoul, Ilsan Sanghoe) powder at 1% ratio, and 40% of the total weight was supplied, followed by 42.5 at 82.5 ± 2.5 ℃. Primary fermentation was performed for a time. The fermentation was then continued for 40 days at a temperature of 25 ° C and a humidity of 60%. After 2 hours of primary drying at 82.5 ± 2.5 ° C., hot air drying was performed at 87.5 ± 2.5 ° C. for 2 hours to prepare a final product having moisture of 3% or less.

Solid feed was prepared using AIN93G formula (Dyets, Inc. US) based on the concentration of 0.4% of rice bran before fermentation and rice bran after fermentation. The positive control group was prepared by adding a commercial dietary supplement product containing shiitake mushroom mycelium extract at a concentration of 0.4% relative to the weight of the solid feed.

Example  2. Liver Protection Animal Experiment

2-1. Diet of Laboratory Animals

Experimental animals received male ICR mouse (27 ± 1 g), 5 weeks old, from Central Experimental Animal Co., Ltd., temperature cycles of 23 ± 1 ℃, relative humidity of 55 ± 15%, and 12-hour intervals (12 h light / Animal kennels with controlled dark cycles were used. After 1 week of acclimatization, the experimental groups were separated by total weight, and the feed and water were fed freely. Carbon tetrachloride was dissolved in corn oil at a concentration of 20% and administered orally at a dose of 1 ml / kg (by weight) twice a week. Normal control group was orally administered only corn oil twice a week, and negative control group was ingested general feed after administration of carbon tetrachloride. Positive control, test group non-fermented rice bran (GR) and fermented rice bran (GF) were fed feed containing each component after administration of carbon tetrachloride. Carbon tetrachloride was administered for 8 weeks and then fasted and sacrificed for 1 day. After collecting blood, serum was separated and liver tissues were washed in cold saline and frozen in liquid nitrogen. Liver tissues used for tissue staining were immediately fixed in 10% formalin solution.

2-2. ALT , AST , ALP , LDH , Cholesterol and Triglyceride  Level measurement

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and cholesterol and triglycerides in serum are assay reagents (Bayer, USA) It was measured by ADIVA 1650 (Bayer, Japan) using. Lactate dehydrogenase (LDH) was measured with ADIVA 1650/2400 (Siemens, USA) using analytical reagents (Siemens, USA).

2-3. Protein, lipid peroxidation in the liver, GSH  And measurement of collagen

Protein content of liver tissue was measured using Bradford reagent (Sigma, USA), and lipid peroxide was measured by MDA reacting with thibarbituric acid (TBA) using Thiobarbituric acid reactive substances (TBARS) measurement kit (Caymanchem, USA). The amount of (Malondialdehyde) was measured at an absorbance of 540 nm. Glutathione (GSH) was used as a glutathione measurement kit (Caymanchem, USA) to measure the product reacting with DTNB (5,5'-dithio-bis-2- (nitrobenzoic acid), Ellman's reagent) at an absorbance of 405 nm. The collagen content was measured by absorbance at 540 nm using Sircol collagen measurement (Biocolor.UK) after removing the protein in liver tissue.

2-4. Liver damage assessment

The left lobe of the liver was fixed with 10% neutral buffered formalin, embedded in paraffin, and embedded into 5 μm coronal sections. Paraffin was removed with xylene, hydrophilized with alcohol, and stained with H & E (Hematoxylin-eosin), Masson's trichrome, and Sirius red, respectively.

2-4-1. Results of H & E staining of mouse liver tissues treated with carbon tetrachloride

1 shows the results of H & E staining of liver tissues for histopathological analysis of the effect of fermented rice bran in liver administered with carbon tetrachloride. The nuclei of cells are stained blue, and the rest of the tissues are colored red. Normal liver tissues show no liver damage, whereas carbon tetrachloride (CCl ₄ control) groups have hepatic vacuolization and necrosis. Some appeared (FIG. 1B). Nuclear cohesion, which occurs during cellular mitosis due to cell damage, was also observed. However, the fermented rice bran (GF) shows little such cellular mitosis and almost alleviates necrosis and saturation (FIG. 1D). In particular, FIG. 1E using commercially available dietary supplements prepared with shiitake mushroom mycelium, which is a positive control, showed severe cell destruction and saturation, and more severe hepatocellular damage than carbon tetrachloride (CCl ₄ control) group.

2-4-2. Of mouse liver tissue administered with carbon tetrachloride Falsity Trichrome  Dyeing results

Figure 2 shows the results of macular trichrome staining of liver tissues for observing the degree of fibrosis and the protective effect of fermented rice bran upon carbon tetrachloride administration. In masson's trichrome staining, all normal liver tissues are colored red and collagen is colored blue. In FIG. 2A, the normal group had no abnormalities, but the carbon tetrachloride (CCl ₄ control) group was found to have a lot of liver damage when observing the degree of collagen produced during the repair process for regenerating damaged liver (FIG. 2B). Fibrotic cells form a band at the edges of the hepatic lobules of the carbon tetrachloride (CCl ₄ control) group. This is similar to the non-fermented rice bran (GR) and positive control (Fig. 2C, 2E). However, fermented rice bran (GF) can be observed that the fibrotic cells are significantly reduced and the degree of collagen production is significantly reduced (Fig. 2D).

2-4-3. Sirius in mouse liver tissue administered carbon tetrachloride Red  Dyeing results

Figure 3 shows the results of sirius red staining of liver tissues to observe the histopathological changes of liver fibrosis in the carbon tetrachloride-induced hepatotoxicity model and the liver protective effect by fermented rice bran. As a result of Sirius red staining, collagen is stained red in liver tissue. In normal group, all the tissues are yellow, but carbon tetrachloride (CCl ₄ control) -administered group can be seen that the fibrotic cells are stained in red color and that the fibrosis is spread in most liver tissues (FIG. 3B). The non-fermented rice bran (GR) and the positive control group seemed to alleviate a little more cell damage than the carbon tetrachloride (CCl ₄ control) group, but many saturation phenomena appeared (Fig. 3C, 3E). Fermented rice bran (GF) can be seen that both the content of collagen and saturation was significantly lower (Fig. 3D).

2-4-4. Determination of collagen content in liver tissue

On the other hand, as a result of measuring the amount of collagen in the liver tissue, the indicator of liver fibrosis in tissue staining is quantitatively shown in FIG. Carbon tetrachloride (CCl ₄ control) group and positive control group were similar when considering the range of deviation. Non-fermented rice bran (GR) showed slightly lower collagen content. However, fermented rice bran (GF) showed a 20% decrease in collagen content compared to carbon tetrachloride (CCl ₄ control).

2-5. Statistical analysis

The experimental results of this study were SAS ver. The mean ± standard deviation was expressed using 9.1, and significance was tested at p <0.05 using Duncan's method using ANOVA.

2-5-1. Fermentation in a Carbon Tetrachloride-Induced Liver Toxicity Model Rice bran  Effect on weight, liver weight and liver protein content

process
(Treatment) Liver weight
(g) weight
(g) Liver / weight
(%) Liver protein
(mg / mL) Normal group 1.66 ± 0.20 ^b 45.77 ± 2.49 ^a 3.43 ± 0.37 ^b 48.67 ± 2.26 ^a CCl ₄ control 2.03 ± 0.12 ^a 43.88 ± 1.34 ^b 4.89 ± 0.35 ^a 45.34 ± 2.08 ^b Hepacellia + CCl ₄ 2.03 ± 0.16 ^a 44.03 ± 1.59 ^{a, b} 4.78 ± 0.24 ^a 48.61 ± 2.01 ^a GR (0.4%) + CCl ₄ 2.16 ± 0.11 ^a 44.53 ± 2.11 ^{a, b} 4.86 ± 0.10 ^a 46.60 ± 0.77 ^{a, b} GF (0.4%) + CCl ₄ 1.64 ± 0.21 ^b 47.32 ± 2.76 ^a 3.46 ± 0.54 ^b 47.14 ± 1.34 ^{a, b}

Mean ± Standard Deviation (n = 5)

GR: Non-fermented rice bran experiment group

GF: fermented rice bran experimental group

Carbon tetrachloride (CCl ₄ ) generates trichloromethyl radicals and trichloromethyl peroxy radicals through phase I metabolism, attacking the phospholipids of the cell membrane, causing lipid peroxidation, leading to hepatocellular necrosis, and in animals inducing similar liver fibrosis in humans It is known.

Table 1 shows the weight gain, liver weight, and liver protein contents of rats fed oral administration of carbon tetrachloride twice a week for 8 weeks. It was. In the normal group and the fermented rice bran experimental group (GF), the liver weight was significantly the lowest while the liver weight was significantly the lowest, and the carbon tetrachloride-treated group (CCl ₄ control) showed the lowest weight and the liver weight. This is because liver damage and stress due to the administration of carbon tetrachloride affect weight loss, and the weight of swollen liver increases due to liver damage. In the liver weight to weight ratio, the carbon tetrachloride-treated group showed about 43% higher than the normal group. Fermented rice bran (GF) shows a similar value to that of the normal group, so it can be seen that the physical and physical characteristics of the fermented rice bran (GF) are recovered to near normal even when carbon tetrachloride is administered. The protein produced in the liver increased about 4% of the liver protein content of the fermented rice bran experiment group (GF) compared to the carbon tetrachloride administration group (CCl ₄ control), which is almost similar to that of the normal group.

2-5-2. Fermentation in Carbon Tetrachloride-induced Liver Toxicity Model Rice bran ALT , AST , ALP , LDH  And its effect on total cholesterol and triglycerides

process
(Treatment) ALT
(U / L) AST
(U / L) ALP
(U / L) LDH
(U / L) TC
(mg / dL) TG
(mg / dL) Normal group 58 ± 11 ^c 145 ± 28 ^c 64 ± 18 ^c 913 ± 52 ^c 146 ± 12 ^a 82 ± 24 ^b CCl ₄ control 28696 ± 6018 ^a 18640 ± 4945 ^a 138 ± 42 ^a 145068 ± 34993 ^a 130 ± 22 ^{a, b} 179 ± 20 ^a Positive control group 22428 ± 4305 ^b 13248 ± 2504 ^b 96 ± 20 ^{a, b, c} 118164 ± 21649 ^{a, b} 127 ± 20 ^{a, b} 145 ± 25 ^a GR (0.4%) + CCl ₄ 22700 ± 1188 ^b 14576 ± 1960 ^{a, b} 122 ± 52 ^{a, b} 129408 ± 9237 ^{a, b} 108 ± 18 ^b 155 ± 41 ^a GF (0.4%) + CCl ₄ 17536 ± 3630 ^b 10316 ± 2827 ^b 77 ± 13 ^{b, c} 98776 ± 20087 ^b 81 ± 2 ^c 100 ± 29 ^b

Mean ± Standard Deviation (n = 5)

GR: Non-fermented rice bran experiment group

GF: fermented rice bran experimental group

2-5-3. Enzyme level change rate measurement

process ALT
(%) AST
(%) ALP
(%) LDH
(%) TC
(%) TG
(%) Normal group - - - - CCl ₄ control 100 100 100 100 100 100 Positive control group 78 71 70 81 98 81 GR (0.4%) + CCl ₄ 79 78 88 89 83 87 GF (0.4%) + CCl ₄ 61 55 56 68 62 56

GR: Non-fermented rice bran experiment group

GF: fermented rice bran experimental group

The amino acid transferases alanine aminotransferase (ALT) and aspartate aminotransferase (AST), known as indicators of liver damage, are known to exhibit high activity as they are released into the blood as liver tissue breaks down. In particular, ALT is present in many livers, and AST is found in heart liver and skeletal muscle, so its specificity is recognized. The liver enzyme levels measured in plasma are shown in Table 2, and the enzyme levels in each experimental group were converted to% ratios based on carbon tetrachloride administered group (CCl ₄ control) and expressed in Table 3. The carbon tetrachloride treated group showed significantly higher enzyme activity than the normal group, indicating that carbon tetrachloride caused liver damage. Fermented rice bran (61) showed a 61% decrease in ALT enzyme levels compared to carbon tetrachloride treated group, which was 17% lower than 78% positive control group. Unfermented rice bran (GR) was less than 79%. Alkaline phosphatase (ALP) is known to increase levels when bile is poorly drained from hepatocytes or when bile ducts are blocked. Fermented rice bran (GF) group showed a 44% lower enzyme activity than the carbon tetrachloride treated group (CCl ₄ control) and showed similar values to the normal group and the positive control group (Table 2). Lactic acid dehydrogenase (LDH) also flows into the blood when the enzyme-containing tissues are destroyed, which leads to an increase in enzyme levels.In addition, the nonfermented rice bran (GR) group is 11% less than the carbon tetrachloride group (CCl ₄ control). ) Decreased by 32%. Total cholesterol (TC) and triglyceride (TG) balances the synthesis and use of normal liver cells, but when CCl ₄ is damaged by liver, CCC ₃ groups, which are highly reactive, produce hepatocellular function and lower cholesterol. It is reported that the content of and glycerides is increased. Total cholesterol (TC) was not significantly affected by carbon tetrachloride (CCl ₄ control) compared to the normal group, but the fermented rice bran (GF) group decreased by about 38% than the carbon tetrachloride group. Trigly

Serum (TG) decreased 44% in fermented rice bran (GF) group compared to carbon tetrachloride (CCl ₄ control) group (Table 3).

2-5-4. Fermentation in Carbon Tetrachloride-induced Liver Toxicity Model Rice bran GSH , MDA - TBA Effect on

process GSH (μmol / g wet weight) MDA-TBA (nmol / mg protein) Normal group 0.73 ± 0.20 ^{a, b} 0.66 ± 0.21 ^b CCl ₄ control 0.64 ± 0.18 ^b 1.32 ± 0.21 ^a Positive control group 0.97 ± 0.26 ^{a, b} 0.37 ± 0.10 ^b GR (0.4%) + CCl ₄ 0.52 ± 1.11 ^b 1.58 ± 0.45 ^a GF (0.4%) + CCl ₄ 1.11 ± 0.50 ^a 0.41 ± 0.22 ^b

Mean ± Standard Deviation (n = 5)

GR: Non-fermented rice bran experiment group

GF: fermented rice bran experimental group

Table 4 shows the measurement of glutathione content and MDA-TBA content in liver tissue. Glutathione (GSH) is known as a water-soluble antioxidant that is also involved in the detoxification of free radicals. Compared to the normal group, carbon tetrachloride (CCl ₄ control) decreased about 12% of GSH content due to liver damage, but it was higher in the fermented rice bran (GF) group than the normal group and 47% more significant than the CCl ₄ control group. Increased. MDA is a reaction product generated by attacking unsaturated fatty acids, which is a component of cell membranes, and can measure oxidative stress and peroxidation of cells when measured. The CCT ₄ control group showed a twofold increase in MDA-TBA content compared to the normal group, and the fermented rice bran group showed a 69% decrease compared to the CCl ₄ control. . This value was similar to that of the normal group, and was significantly different from the carbon tetrachloride (CCl ₄ control) group and the non-fermented rice bran (GR) group.

Compositions comprising the substances according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The extract of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once or several times a day. Can be. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (13)

Method of producing a fermented food for liver protection comprising the step of inoculating the fermented soybean paste powder in rice bran medium. The method of claim 1,
The Cheonggukjang powder is a method for producing a fermented food for liver protection, characterized in that 1 part by weight relative to the total weight ratio. The method of claim 1,
The fermentation step is a method for producing a liver protective fermented food, characterized in that the fermentation at a temperature of 20 to 30 ℃. The method of claim 3, wherein
Method for producing a fermented food for liver protection characterized in that the fermentation at a temperature of 75 to 90 ℃ before the step of fermentation. The method of claim 3, wherein
Method for producing a fermentation foods for liver protection, characterized in that after the fermentation step is a post-fermentation at a temperature of 75 to 90 ℃. The method of claim 3, wherein
Prefermentation at a temperature of 75 to 90 ℃ before the step of fermentation,
Method for producing a fermentation foods for liver protection, characterized in that after the fermentation step is a post-fermentation at a temperature of 75 to 90 ℃. The method of claim 3, wherein
The fermentation step is a method for producing a liver protective fermented food, characterized in that the fermentation under conditions of 55 to 65% relative humidity. The method of claim 3, wherein
The fermentation is a method for producing a liver protective fermented food, characterized in that is carried out for 40 days. Fermented food for liver protection comprising rice bran fermented product inoculated with rice bran powder on rice bran medium. The method of claim 9,
The fermented food for liver protection, characterized in that the Cheonggukjang powder is 1 part by weight based on the total weight ratio. A pharmaceutical composition for liver protection treatment comprising rice bran fermentation inoculated with rice bran powder on rice bran medium. The method of claim 11,
The liver protective therapeutic composition is a pharmaceutical composition for hepatoprotective treatment in the form of a powder, granules, premises, capsules, suspensions, emulsions, syrups or aerosols. The method of claim 11,
The liver protective treatment composition is a pharmaceutical composition for liver protection treatment in the form of an external preparation, suppository or sterile injectable solution.

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