TWI769644B - Composite composition of clostridium butyricum, yeast and enzyme, oral composite including the same and application on liver protection - Google Patents

Abstract

The present invention relates to a composite composition of Clostridium butyricum, yeast and enzyme, an oral composite including the composite composition and its applications on liver protection and serum lipid regulation. The composite composition includes ingredients of Clostridium butyricum, at least one yeast, soybean powder fermented by Lactobacillus sp., at least one plant-based enzyme, lecithin and so on. The composite composition can be applied to preparation of the oral composition for protecting liver protection.

Description

Clostridium butyricum, yeast and enzyme complex composition, oral composition containing the same and application for liver protection

The present invention relates to a compound composition of Clostridium butyricum, yeast and enzyme, in particular to a compound composition of Clostridium butyricum, yeast and enzyme with liver protection effect and its application in oral composition.

Liver cancer, cerebrovascular disease and heart disease account for the top ten causes of death in China. The occurrence of liver cancer is highly correlated with chronic liver damage caused by chronic liver diseases such as cirrhosis, viral hepatitis and fatty liver, while cerebrovascular disease and heart disease are highly correlated with atherosclerosis. The content of various lipids or lipoproteins in serum can be used to assess the risk of atherosclerosis.

At present, the medical community uses ultrasonic testing, serum GPT and GOT concentrations, organ body weight ratio, liver lipids, liver enzyme activity, liver tissue sections, etc. to assess the degree of liver damage.

At present, there are many synthetic drugs on the market for the treatment of liver damage and hyperlipidemia, but synthetic drugs generally have disadvantages such as high biological toxicity, large side effects in clinical trials, and easy generation of drug resistance. Therefore, researchers try to screen the active ingredients or compound ingredients with liver-protecting effect from natural source ingredients, so as to provide liver-protecting effect.

Therefore, one aspect of the present invention is to provide a complex composition of Clostridium butyricum, yeast and enzyme, comprising Clostridium butyricum, at least one yeast, lactic acid bacteria fermented soybean powder, at least one plant-derived enzyme, and lecithin and other ingredients.

Another aspect of the present invention is to provide an oral composition with liver-protecting effect, comprising the above-mentioned complex composition of Clostridium butyricum, yeast and enzyme as active ingredients, and is administered to a subject via intestinal administration route.

According to the above aspect of the present invention, a composite composition of Clostridium butyricum, yeast and enzymes is provided, which comprises 20 to 32 wt% of Clostridium butyricum EC80, 20.4 to 25.6 wt% of at least 1 yeast, 20% to 25% by weight lactic acid bacteria fermented soybean powder, 20% to 25% by weight at least one plant-derived enzyme, 2.4% to 3.6% by weight lecithin, 1.6% to 2.4% by weight % taurine, 0.4 to 0.6 weight percent choline bitartrate, 0.4 to 0.6 weight percent inositol, and 2.8 to 4.2 weight percent lactose. The above Clostridium butyricum EC80 is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is February 15, 2019, and the deposit number is BCRC 910868.

According to an embodiment of the present invention, the above-mentioned Clostridium butyricum, yeast and enzyme composite composition is 100% by weight, and the above-mentioned at least one yeast may include, for example, 20% to 25% by weight of Saccharomyces cerevisiae and/or 0.4 weight percent to 0.6 weight percent selenium yeast.

According to an embodiment of the present invention, with the above-mentioned Clostridium butyricum, yeast and enzyme composite composition as 100% by weight, the at least one plant-derived enzyme may include 10% by weight to 13% by weight of starch liquefaction enzyme and/or 10% by weight percent to 13 percent by weight of pineapple enzyme.

According to an embodiment of the present invention, the above-mentioned lactic acid bacteria fermented soybean powder can be prepared by, for example, fermenting soybean milk with Enterococcus spp. at a temperature of 30° C. to 45° C. for 5 to 24 hours, and then performing a drying step. .

According to another aspect of the present invention, an oral composition with liver protection effect is proposed, comprising the above-mentioned complex composition of Clostridium butyricum, yeast and enzyme, and the complex composition of Clostridium butyricum, yeast and enzyme is an active ingredient, It can be administered to the subject via the enteral route, wherein the dosage of the aforementioned Clostridium butyricum, yeast and enzyme complex composition can be, for example, 0.07 to 0.70 grams per kilogram of body weight.

According to an embodiment of the present invention, the above-mentioned enteral administration route may be, for example, an oral route.

According to an embodiment of the present invention, the above-mentioned subject is a mammal.

According to another aspect of the present invention, an oral composition with liver protection effect is proposed, comprising the above-mentioned complex composition of Clostridium butyricum, yeast and enzyme, and the complex composition of Clostridium butyricum, yeast and enzyme is an active ingredient, It can be administered to the subject via the enteral route, wherein the dosage of the aforementioned Clostridium butyricum, yeast and enzyme complex composition can be, for example, 0.14 grams to 0.42 grams per kilogram of body weight.

Using the complex composition of Clostridium butyricum, yeast and enzyme of the present invention, it also has the effect of protecting the liver, and can be used as an active ingredient and applied to an oral composition.

Continuing from the foregoing, the present invention provides a compound composition of Clostridium butyricum, yeast and enzyme, comprising Clostridium butyricum, at least one yeast, lactic acid bacteria fermented soybean powder, at least one plant-derived enzyme, lecithin and other components, and has Liver protection.

In other words, the “complex composition of Clostridium butyricum, yeast and enzyme” referred to herein in the present invention refers to a compound comprising a specific proportion of Clostridium butyricum, at least one yeast, lactic acid bacteria fermented soybean powder, and at least one plant-derived product. Enzymes, Lecithin, Taurine, Choline Bitartrate, Inositol and Lactose. In one embodiment, the aforementioned Clostridium butyricum, yeast and enzyme complex composition may include, but is not limited to, at least one of 20 to 32.4 weight percent of Clostridium butyricum EC80 and 25.4 to 35.6 weight percent. Yeast, 25% to 35% by weight of lactic acid bacteria fermented soybean powder, 24% to 36% by weight of at least one plant-derived enzyme, 2.4% to 3.6% by weight of lecithin, 1.6% to 2.4% by weight of taurine, 0.4 to 0.6 weight percent of choline bitartrate, 0.4 to 0.6 weight percent of inositol, and 2.8 to 4.2 weight percent of lactose. The above Clostridium butyricum EC80 is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is February 15, 2019, and the deposit number is BCRC 910868.

In the above embodiment, the above-mentioned Clostridium butyricum, yeast and enzyme composite composition is 100% by weight, the above-mentioned at least one yeast may include but not limited to, for example, 25% to 35% by weight of Saccharomyces cerevisiae and Saccharomyces cerevisiae. /or 0.4 weight percent to 0.6 weight percent selenium yeast.

In one example, the above-mentioned S. cerevisiae may include, but are not limited to, for example, BCRC 20262, BCRC 20263, BCRC 20270, BCRC 20271, BCRC 20405, BCRC 20496, BCRC 20497, BCRC 20498, and/or BCRC 21680, but only exemplified here , the present invention is not limited to this, and other brewer's yeast strains deposited by the Bioresource Collection and Research Center (BCRC), Hsinchu Food Industry Development Research Institute, Taiwan can also be used.

In other embodiments, with the above-mentioned Clostridium butyricum, yeast and enzyme complex composition as 100% by weight, the at least one plant-derived enzyme may include 12% by weight to 18% by weight amylase and/or 12% by weight To 18 weight percent of pineapple enzyme (or called bromelain).

The aforementioned lactic acid bacteria fermented soybean powder can be prepared by, for example, fermenting soybean milk with Enterococcus ( Enterococcus spp.; Accession No.: BCRC 80605) at a temperature of 30°C to 45°C for 5 to 24 hours, and then performing a drying step. . It should be noted here that, not a single ingredient containing Clostridium butyricum, yeast, lactic acid bacteria fermented soybean powder or plant-derived enzymes has liver protection effect. In addition, if the usage amount of each component of the above-mentioned Clostridium butyricum, yeast and enzyme complex composition is outside the above-mentioned range, it cannot be expected to have a liver-protecting effect without experimental confirmation.

The "liver protection function school" referred to in the present invention refers to the evaluation of the liver protection effect of the complex composition of Clostridium butyricum, yeast and enzymes using an animal test model of chemical liver injury. In one embodiment, the aforementioned liver-protecting effect can be evaluated with reference to the “Evaluation Method for Liver-protecting Health-Efficacy of Health Foods” amended by the Announcement No. 1031304063 of the Ministry of Health and Welfare of Taiwan, which is incorporated herein as a reference document of the present invention. In short, the evaluation method of the liver protection effect is to use carbon tetrachloride (CCl4) to induce chronic liver damage in the subject, and then different doses (one dose contains the converted human recommended intake) of Clostridium butyricum, yeast The complex composition with enzymes is administered to the subject through the enteral route for a period of time, and then the liver function of the subject is measured, such as the determination of the components or enzyme activities (such as GOT, GPT) related to liver injury in serum or plasma, and the observation of liver tissue. Pathological sections, the weekly body weight, liver weight/body weight (%), mortality, etc. of the subject during the experiment were determined.

The obtained compound composition of Clostridium butyricum, yeast and enzyme has been proved to have liver protection effect after being evaluated by animal test, and can be further applied to the composition of various dosage forms. Suitable compositions may include, but are not limited to, oral compositions. In one embodiment, the above-mentioned oral composition uses a complex composition of Clostridium butyricum, yeast and enzymes as active ingredients, so that the composition of the above-mentioned dosage form has a liver-protecting effect. It is explained here that the complex composition of Clostridium butyricum, yeast and enzyme obtained in the present invention, when used as an oral composition, exhibits dose- and time-dependent (dose- and time-dependent) effects on experimental animals with chemical liver injury. ) related protection.

Several embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch. Example 1: Preparation of Clostridium Butyricum, Yeast and Enzyme Complex

First, according to Table 1, the compound compositions of Clostridium butyricum, yeast and enzymes of experimental groups 1 to 4 were prepared.

Table 1

The Clostridium butyricum listed in Table 1 is Clostridium butyricum EC80, which is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China Consortium, No. 331, Shishi Road, Hsinchu, Taiwan. The deposit date is February 15, 2019, and the deposit number is is BCRC 910868. The lactic acid bacteria fermented soybean powder is a commercially available soybean milk, which is fermented with Enterococcus ( Enterococcus spp.; Accession No.: BCRC 80605) at a temperature of 30°C to 45°C for 5 to 24 hours, and then carried out produced in the drying step.

After the above components are uniformly mixed, the complex composition of Clostridium butyricum, yeast and enzyme is prepared, and subsequent evaluation is carried out. Example 2: Establishing an animal test model for evaluating the efficacy of protecting the liver

For the evaluation of the liver-protecting effect, please refer to the “Evaluation Method for the Liver-protecting Health Effect of Healthy Foods” revised by the Ministry of Health and Welfare of Taiwan, Fu Shi Zi No. 1031304063 (URL: https://www.google.com.tw/url?sa=t&rct =j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwiewL_miJvPAhXGj5QKHTGFCdQQFggaMAA&url=http%3A%2F%2Fwww.fda.gov.tw%2Ftc%2Fincludes%2FGetFile.ashx%3FmID%3D19%26id%3D51915%26chk%3D85ce2d86d-8bf2-4af5ce2d86d -9af2-6f8e450bcc4e&usg=AFQjCNF6mTQAU3qOHC-sRwdXJFpYJU5ZfQ&sig2=2VdirEAh6MEPzG_fIA4Bqw) for evaluation. 1. Experimental animals

68 6-week-old male mice in Imprinting Control Region (ICR) (purchased from Lesco Biotechnology Co., Ltd., Yilan, Taiwan) were taken and raised in accordance with the relevant experimental animal management guidelines announced by the National Institutes of Health. In an animal room with independent air conditioning (IVC system, TECNIPLAST, Italy), the temperature was maintained at 22±2°C, the humidity was maintained at 60±10%, and the light and dark cycle was switched for 12 hours, and the feed was not restricted during the feeding period (Laboratory Rodent Diet 5001) and water (reverse osmosis water). After one week of adaptation in ICR mice, various evaluations of the follow-up liver protection efficacy were carried out. 2. Establishment of an animal model for chemical liver injury experiments

The above 68 ICR mice were randomly divided into 5 groups with 12 to 15 mice in each group, namely the control group (N=15), the placebo group (N=12), and the positive control group (namely, the silymarin group, N=12). , Experimental group 4 (fed 0.07 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition, N=14), experimental group 4 (fed 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition material, N=15), as shown in Table 2.

Table 2

The compound composition of Clostridium butyricum, yeast and enzyme in experimental group 4 (0.07 g/kg body weight, N=14) of Example 1 is based on the recommended amount of human body (70kg), namely 5g/person/day (low dose, Equivalent to 0.07g/kg body weight/day), converted into mice (20g) is 0.013g/mouse/day (5×0.0026=0.013g/mouse/day, equivalent to 0.00065g/g body weight/day). In the evaluation of liver protection efficacy, the nasogastric tube (0.25mL/10g body weight/day) was fed once every morning for 8 weeks, and the concentration of Clostridium butyricum and yeast was 10^8 to 10 ^10 CFU/mL.

The compound composition of Clostridium butyricum, yeast and enzymes in experimental group 4 (0.70 g/kg body weight, N=15) is 10 times the recommended dosage for the human body, that is, 50 g/person/day (a high dose, equivalent to 0.70g/kg body weight/day), converted into mice (20g) is 0.130g/mouse/day (50×0.0026=0.130g/mouse/day, equivalent to 0.0065g/g body weight/day). In the evaluation of liver protection efficacy, nasogastric tube feeding (0.25mL/10g body weight/day) was administered once every morning for 8 weeks.

For various assessments of hepatoprotective efficacy, carbon tetrachloride (CCl4) was fed twice a week (eg Monday and Thursday afternoon) at 4 μL of 5% CCl4/g body weight each time. For silymarin, 40 μL of 10 mg/mL silymarin/g body weight was given once a day (eg, every morning).

The evaluation of the above-mentioned liver protection effect lasted for eight weeks. All experimental animals were measured weekly for body weight (Table 3), and were administered with Clostridium butyricum, yeast and enzyme complex composition at the 1st, 3rd, 5th, and 6th weeks respectively. Hourly, liver biochemical functions (eg, serum GPT, serum GOT) were detected by tail blood sampling (Tables 4 to 5, Figures 1 to 2). At the end of the 8th week, all experimental animals were collected by tail blood to detect liver biochemical functions (such as serum GPT, serum GOT, total cholesterol, triglyceride, albumin) (Table 6), and all of them were sacrificed, and the liver and spleen were weighed. Re-records (Table 7, Figures 3 to 4).

Liver lipid content was analyzed with reference to the method published by Folch et al. in 1957. First, take 0.2 g of the lower right largest lobe of the experimental animal liver, add 0.5 mL of extract (containing a mixed solution of chloroform and methanol with a volume ratio of 2:1), and use a commercially available homogenizer (Model: Micromot IB/E, Proxxon , Germany) after homogenizing at 10,000 rpm for about 20 seconds, add 1 mL of extract to rinse the residual tissue on the tube wall, and then add 3.5 mL of extract to obtain liver lipid extract. Take 250 μL of liver lipid extract and add 250 μL of Triton X-100 (Sigma) to mix evenly, analyze the content of liver tissue lipids (total cholesterol (TC), triglyceride (TG)], the results As shown in Table 8 and Figures 5 to 6.

The activity of liver tissue enzymes was analyzed by the following method. First, take 0.2 g of experimental animal liver tissue, add 9 times the volume of homogenate (containing 0.25M sucrose, 1mM EDTA, 10M Tris/HCl, pH 7.4), use a commercially available homogenizer (Model: Micromot IB) /E, Proxxon, Germany) after homogenization, centrifugation (13,000rpm, 20 minutes, 4°C) to take the supernatant to measure the activity of liver tissue enzymes, of which the activity of catalase can be obtained by using commercially available Biooxytech Catalase 520 assay kit (Model: 21042, OxisResearch), superoxide dismutase (superoxidase dismutase; SOD) activity can be measured using a commercially available SOD activity assay kit (Model: 295-55701, Wako, Japan) using nitro blue Measured by nitroblue tetrazolium (NBT) reduction method, the activity of reduced glutathione (glutathione; GSH) can be measured by commercially available Bioxytech GSH 420 assay kit (Model: 21023, OxisResearch), glutathione peroxidation The activity of the enzyme (glutathione peroxidase; GPx) can be measured using a commercially available Glutathione peroxidase cellular activity assay kit (Model: CBP-1, Sigma), and the results are shown in Table 9 and Figures 7 to 10 .

Liver tissue was measured for its total protein concentration with a commercially available Bio-rad Protein assay kit (Bio-rad, U.S.A). Take 1 volume of Dye Reagent Concentrate and add 4 volumes of deionized water to prepare Dye Reagent working solution. Take BSA standard substance of different concentrations (0.05mg/mL to 0.5mg/mL) or 10 μL of liver tissue homogeneous solution, add Dye Reagent working solution respectively, and react at room temperature for 5 minutes, use ELISA reader to measure the absorbance at 595nm, and use ELISA reader to measure the absorbance at 595nm. The total protein concentration in relation to Table 9, Figures 7 to 10 was measured.

The largest right lobe of the liver of all experimental animals was cut into two pieces (blocks about 1 cm × 1 cm in size), fixed in 10% neutral formalin, encapsulated in paraffin, and then organized by a pathologist. The results are shown in Table 10 and Figures 11A to 11E.

Relevant values are expressed as mean ± standard error of mean (SEM; standard error of mean), and statistical analysis is performed using commercially available statistical software (eg, SPAA version 10.0), and Mann-Whitney U test is used to test the difference between the two groups Happening. In addition, since the number of samples in each group was less than 30, the Kruskal-Wallis H test was used to analyze the differences between groups using nonparametric statistics.

table 3

Table 4

table 5

Table 6

Table 7

Table 8

Table 9

3. 評估對體重的影響 Table 10

3. Assess the effect on body weight

The results in Table 3 show that one week after the administration of carbon tetrachloride to the ICR mice, although the body weight of all groups increased with the increase of the age of the mice, except for the 5th week, the average weight of the placebo group was higher than that of the placebo group. Body weight was higher than the rest of the groups in the same weeks. However, there was no significant difference in body weight between the ICR mice fed the C. butyricum, yeast and enzyme complex for 8 weeks compared with the body weight of the placebo group. 4. Assess the effect on serum GPT and serum GOT

Chemical liver injury caused by carbon tetrachloride will release GPT and GOT in hepatocytes into serum. Therefore, measuring the activity of serum GPT and serum GOT can be used as one of the biochemical indicators of liver injury. Organ specificity. The results from Tables 4 to 5 and Figures 1 to 2 show that the serum GPT and serum GOT values of the placebo group after the first week were higher than those of the control group (p<0.05), which represented that carbon tetrachloride did induce liver cancer. damage. The serum GPT value of the placebo group reached the highest value at the 8th week, while the serum GOT value of the placebo group was higher than that of the control group at the 3rd, 5th, 6th, and 8th weeks, and the serum GOT value of the placebo group was at the 6th week. reaches the highest value. Compared with the placebo group, there was no significant difference in the activities of serum GPT and serum GOT in the positive control group (ie, the silymarin group), which means that the liver protection effect of silymarin is not significant.

However, in the results in Tables 4 to 5 and Figures 1 to 2, two groups of ICR mice were fed 0.07 g/kg body weight and 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition respectively, Its serum GPT was significantly lower than the placebo group from the 5th week to the 8th week, and its serum GOT was significantly lower than that of the placebo group from the 6th week to the 8th week. As for the two groups of ICR mice that were fed 0.07 g/kg body weight and 0.70 g/kg body weight of the complex composition of Clostridium butyricum, yeast and enzyme respectively, there was a significant difference in serum GOT between them until the 8th week, that is, The serum GOT of ICR mice fed 0.70 g/kg body weight of the complex composition of Clostridium butyricum, yeast and enzymes at week 6 was significantly lower than that of the ICR mice fed 0.07 g/kg body weight of the complex composition of Clostridium butyricum, yeast and enzymes ICR mice of the object. 5. Assess the effect on serum albumin, serum total cholesterol, and serum triglycerides

Chronic liver disease, cirrhosis and other diseases can cause hypoalbuminemia. Therefore, the detection of serum albumin concentration can indirectly understand the effect of carbon tetrachloride on the ability of the liver to synthesize protein. According to the results of serum albumin in Table 6, the serum albumin concentration of the two groups of ICR mice fed with the complex composition of Clostridium butyricum, yeast and enzyme has no significant difference compared with the placebo group and the control group. It has been shown that chemical liver injury caused by carbon tetrachloride has not significantly affected the ability of the liver to synthesize protein.

Secondly, the results of serum total cholesterol and serum triglyceride concentrations in Table 6 show that the positive control group (namely the silymarin group), the two groups of ICR mice fed with Clostridium butyricum, yeast and enzyme complex composition were at the 8th Weekly serum total cholesterol and serum triglyceride concentrations were not significantly different from those in the placebo group, among which the serum three levels of the ICR mice fed 0.07 g/kg body weight of the complex composition of Clostridium butyricum, yeast and enzymes. The acid glyceride concentration was significantly lower than that of the control group. 6. Assess the effect on the liver and spleen

Organ weight is one of the indicators for evaluating organ damage. When the liver is damaged, it will activate the regeneration function, so the liver weight will increase, but the severely damaged liver will shrink. When the spleen is inflamed, the weight of the spleen increases, so the ratio of spleen to body weight (referred to as the spleen-to-body ratio) can be regarded as one of the indicators of spleen inflammation and safety. The results from Table 7 and Figure 3 to Figure 4 show that the weight of the liver and spleen in the control group is the lowest, and the weight of the liver and spleen in the placebo group is greater than that in the control group, indicating that carbon tetrachloride does cause enlargement and weight of the liver and spleen. Increase. However, the positive control group (i.e. silymarin group) and the two groups of ICR mice fed with Clostridium butyricum, yeast and enzyme complex could significantly improve the liver enlargement caused by carbon tetrachloride (p<0.05), but the positive control Group (namely silymarin group) and feeding Clostridium butyricum, yeast and enzyme complex composition did not significantly improve spleen enlargement.

The liver tissue lipid results in Table 8 and Figure 6 show that the liver total cholesterol concentration of ICR mice fed with 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition was significantly lower than that of the placebo group (p< 0.05). 7. Assess the effect on liver enzymes

The chemical liver damage caused by carbon tetrachloride is mainly caused by the free radicals generated by the metabolism of the liver cytochrome P450 enzyme system, resulting in oxidative damage. Therefore, the GSH, GPx, catalase, SOD of the liver were measured. The activity of Clostridium butyricum, yeast and enzyme complex can be evaluated for the liver protection effect.

The results of liver enzyme activity in Table 9 and Figure 7 to Figure 10 show that carbon tetrachloride can cause a decrease in SOD activity, but the SOD of ICR mice fed with 0.07 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition The activity was significantly higher than the placebo group (p<0.05). Secondly, carbon tetrachloride can cause a decrease in catalase activity. The hydrogen peroxide of ICR mice fed with 0.07 g/kg body weight and 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition The enzyme activity was higher than the placebo group, but the difference was not statistically significant. Furthermore, carbon tetrachloride can cause the GSH concentration to increase. The GSH concentration of the positive control group (i.e. the silymarin group) and the ICR mice fed with 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition is lower than placebo group, but there was no statistically significant difference. 8. Assessing the effect on liver tissue

The results of hematoxylin-eosin (H&E) staining of the liver tissue sections from Figures 11A to 11E show that, compared with the normal and healthy liver tissue sections of the control group (Figure 11A), carbon tetrachloride The resulting chemical liver injury can be clearly observed from liver tissue sections of the placebo group (Fig. 11B), and liver vacuolization, inflammatory cell aggregation, fibrosis, loss of cell boundaries, and even multinucleation can be observed. Liver of positive control group (ie silymarin group; Fig. 11C), ICR mice fed with 0.07 g/kg body weight (Fig. 11D) and 0.70 g/kg body weight (Fig. 11E) of the complex composition of Clostridium butyricum, yeast and enzyme In tissue sections, it was observed that liver vacuoles, inflammatory cell aggregation, and fibrosis were reduced. Among them, ICR mice were fed with 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex composition (Fig. 11E). decrease is even more pronounced.

In addition, the severity of chronic hepatitis in the above groups can be assessed by pathologists according to histological activity index (HAI), which is divided into cell swelling and degeneration, hepatocyte necrosis, and liver cirrhosis. The results are shown in the table below. 10 shown. According to the HAI evaluation results in Table 10, feeding 0.70 g/kg body weight of Clostridium butyricum, yeast and enzyme complex has better liver protection effect, followed by positive control group (ie silymarin group) and feeding 0.7 g/kg Body weight of the two groups of Clostridium butyricum, yeast and enzyme complex, while the placebo group had the most severe liver inflammation and necrosis.

In conclusion, Example 2 proves that the complex composition of Clostridium butyricum, yeast and enzyme has liver protection effect, and can be used as an active ingredient and applied to an oral composition.

Secondly, the compound composition of Clostridium butyricum, yeast and enzyme in experimental group 4 was administered at various doses, such as the low dose (0.07 g/kg body weight) or high dose (0.70 g/kg body weight) in the evaluation of liver protection efficacy, or the liver protection The low-dose (0.14 g/kg body weight) or high-dose (0.42 g/kg body weight) dose at the time of efficacy evaluation was administered orally to experimental animals (mice, hamsters, at least three replicate data) by nasogastric tube, The 6- to 8-week trial did not cause death or adverse clinical signs. There were no significant dose-related changes in daily body weight gain and water intake, feed consumption, morphology, behavioral activity, and organ tissue weight. The results of blood analysis and biochemical index detection showed that there was no significant difference between the compound composition of Clostridium butyricum, yeast and enzyme in the experimental group 4 and the comparative example or the control group, which confirmed that there was no obvious subchronic toxicity, which met the food safety standards ( Figure not shown), and estimated that the no-observed-adverse-effect-level (NOAEL) of the complex composition of Clostridium butyricum, yeast and enzyme in experimental group 4 was 0.70 g/kg weight/day.

In conclusion, it is confirmed by the above-mentioned several examples that the complex composition of Clostridium butyricum, yeast and enzyme of the present invention can reduce serum GOT and serum GPT concentrations, reduce liver total cholesterol content, and inhibit liver body when taken as an oral composition. The ratio increases, the activity of SOD and catalase in liver cells is increased, and it has dose- and time-dependent protective effects on experimental animals with chemical liver injury. It does have liver protection effects and can be used in preparation Oral composition or pharmaceutical composition with liver protection.

It should be added that, although the present invention takes a specific process, a specific analysis method or a specific instrument as an example to illustrate the complex composition of Clostridium butyricum, yeast and enzyme of the present invention, the oral composition containing the same and its use in the protection Liver application, but anyone with ordinary knowledge in the technical field to which the present invention belongs will know that the present invention is not limited to this. Other processes, other analytical methods or other instruments may also be used.

As can be seen from the above examples, the present invention has the advantage of using a specific proportion of Clostridium butyricum, yeast and enzyme composite composition, oral composition containing the same and its application for liver protection. Bacteria, lactic acid bacteria fermented soybean powder, at least one plant-derived enzyme, lecithin and other ingredients as active ingredients can be used to prepare oral compositions or pharmaceutical compositions with liver-protecting effects.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

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In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows: [Fig. 1] is a graph showing the serum GPT concentration curve of the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. [Fig. 2] is a graph showing the serum GOT concentration curve of the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. 3 is a graph showing the ratio of liver to body ratio of experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Fig. 4 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Spleen-to-body ratio graph. Fig. 5 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Bar graph of liver total triglyceride (triglyceride; TG) content. Fig. 6 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Bar graph of liver total cholesterol content. Fig. 7 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Bar graph of hepatic catalase activity. Fig. 8 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Bar graph of hepatic superoxide dismutase (SOD) activity. Fig. 9 shows the experimental animals with chemical liver injury according to Example 2 of the present invention after being fed the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks. Bar graph of hepatic reduced glutathione (GSH) content. Fig. 10 shows the liver glutathione peroxidase of experimental animals with chemical liver injury according to Example 2 of the present invention after being fed with the complex composition of Clostridium butyricum, yeast and enzymes of experimental group 4 for 8 weeks Bar graph of (glutathione peroxidase; GPx) activity. [FIG. 11A] to [FIG. 11E] show the liver tissue sections of experimental animals with chemical liver injury according to Example 2 of the present invention after being fed with the complex composition of Clostridium butyricum, yeast and enzymes in experimental group 4 for 8 weeks Figure 11A represents the liver tissue section of the control group, Figure 11B represents the liver tissue section of the placebo group, Figure 11C represents the liver tissue section of the positive control group, and Figure 11D represents the experimental group 4 (0.07 g Figure 11E represents the liver of experimental group 4 (0.70 g/kg body weight) of Clostridium butyricum, yeast and enzyme complex composition of liver tissue section Tissue slice diagram.

Domestic Deposit Information Clostridium butyricum EC80 is deposited at the Biological Resource Conservation and Research Center, Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is February 15, 2019, and the deposit number is BCRC 910868 . Foreign storage information none

Claims (9)

A use of a Clostridium butyricum, yeast and enzyme complex composition for preparing an oral composition with liver protection effect, wherein the Clostridium butyricum, yeast and enzyme complex composition comprises: 20 to 32 weight percent of Clostridium butyricum Clostridium butyricum EC80, the Clostridium butyricum EC80 is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan, the deposit date is February 15, 2019, and the deposit number is BCRC 910868; 20.4 to 25.6 weight percent of at least one yeast, wherein the at least one yeast is Saccharomyces cerevisiae and/or selenium-enriched yeast; 20 to 25 weight percent of lactic acid bacteria yeast Soybean powder, wherein the lactic acid bacteria fermented soybean powder is fermented by a soybean milk with Enterococcus ( Enterococcus spp.; accession number: BCRC 80605) at a temperature of 30°C to 45°C for 5 to 24 hours, followed by a drying step and obtained: 20% to 25% by weight of at least one plant-derived enzyme, wherein the at least one plant-derived enzyme is starch liquefaction enzyme and/or pineapple enzyme; 2.4% to 3.6% by weight of lecithin; 1.6% by weight 0.4 to 0.6 weight percent of taurine; 0.4 to 0.6 weight percent of choline bitartrate; 0.4 to 0.6 weight percent of inositol; and 2.8 to 4.2 weight percent of lactose, and wherein the butyrate shuttle The bacteria, the yeast and the enzyme complex are an active ingredient, and the dosage of the Clostridium butyricum EC80, the yeast and the enzyme complex is 0.07 to 0.70 grams per kilogram of body weight. The use of the complex composition of Clostridium butyricum, yeast and enzyme as claimed in claim 1 for preparing an oral composition with liver protection effect, wherein the weight of the Clostridium butyricum EC80, the complex composition of yeast and enzyme is 100 wt. percentage, the at least one yeast comprises 20 to 25 weight percent of the brewer's yeast and/or 0.4 to 0.6 weight percent of the selenium yeast. The use of the complex composition of Clostridium butyricum, yeast and enzyme as claimed in claim 1 for preparing an oral composition with liver protection effect, wherein the weight of the Clostridium butyricum EC80, the complex composition of yeast and enzyme is 100 wt. percentage, the at least one plant-derived enzyme includes 12 to 18 weight percent of the starch liquefaction enzyme and/or 12 to 18 weight percent of the pineapple enzyme. The use of the complex composition of Clostridium butyricum, yeast and enzyme as claimed in claim 1 for preparing an oral composition with liver protection effect, wherein the complex composition of Clostridium butyricum, yeast and enzyme passes through an intestinal tract The route of administration administers to a subject. The use of the complex composition of Clostridium butyricum, yeast and enzyme as claimed in claim 4 for preparing an oral composition with liver protection effect, wherein the intestinal administration route is an oral route. Use of the complex composition of Clostridium butyricum, yeast and enzyme according to claim 4 for preparing an oral composition with liver protection effect, wherein the subject is a human. The use of the complex composition of Clostridium butyricum, yeast and enzyme according to claim 4 for preparing an oral composition with liver protection effect, wherein the object is an animal with chemical liver injury. Use of the complex composition of Clostridium butyricum, yeast and enzyme according to claim 1 for preparing an oral composition with liver protection effect, wherein the liver protection effect includes reducing serum GOT concentration, reducing serum GPT concentration, reducing liver Total cholesterol content and inhibition of liver-to-body ratio rise. Use of the complex composition of Clostridium butyricum, yeast and enzyme according to claim 1 for preparing an oral composition with liver protection effect, wherein the liver protection effect includes increasing liver superoxide dismutase activity and liver peroxidation Hydrogenase activity.

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