KR100769299B1 - Lactobacillus fermentum and dairy products and health-promoting food containing the same - Google Patents

Abstract

The present invention relates to lactic acid bacteria having alcohol degrading ability and dairy products, health functional foods and food additives containing the same, and more particularly, to select lactic acid bacteria having excellent alcohol resistance and degradability, and adding the lactic acid bacteria to alcohol after drinking The present invention relates to a lactic acid bacterium having an alcohol degradable property and a dairy product, a health functional food, and a food additive containing the same, which can be widely used in the manufacture of foods such as a hangover-relieving effect and liver detoxification, which can prevent alcoholic liver disease.

Lactobacillus fermentum, probiotics, alcoholic liver disease, alcohol degrading enzyme, aldehyde degrading enzyme

Description

Lactobacillus fermentum and dairy products and health-promoting food containing the same}

1 is a photograph confirming the enzyme through the SDS-PAGE.

Figure 2 is a photograph examining the effect of liver function through hepatocyte culture.

The present invention relates to a new lactic acid bacterium Lactobacillus (Lactobacillus) having excellent alcohol resolution, and dairy products, health functional foods and food additives containing the same, and more particularly, by selecting lactic acid bacteria having excellent alcohol resistance and resolution. , Lactic acid bacteria by adding lactic acid bacteria to help the hangover and the detoxification effect of alcohol after drinking alcohol to prevent alcoholic liver disease in the future can be widely used in the production of foods such as alcohol lactic acid bacteria and dairy products containing them , Health functional foods and food additives.

The liver manages energy metabolism in our body as a whole and has the ability to store or distribute various metabolites and compounds such as bilirubin, bile acids, cholesterol, and phospholipids throughout the body, detoxifying toxins or serving as a major immune organ. It is an important organ.

Currently, the cause of mortality caused by disease in the country, the rank of death from liver disease is ranked 4th with 22.9 people per 100,000 population (as of 2001), and the liver disease due to alcohol consumption can not be ignored. According to the World Health Organization's alcohol consumption survey in 153 countries in 1996, Korea's adult alcohol consumption per capita was 14,4ℓ per year, ranking 2nd in the world, and 23,000 people died of accidents and diseases caused by alcohol every year. It also announced that the economic loss amounted to 16 trillion won. In addition, the proportion of alcoholics among Korean adults is 21.6%, the highest in the world, and one out of five adults is suffering from severe national illness as an alcoholic.

Alcoholic liver disease is acute / chronic liver disease with hepatocellular damage caused by persistent and excessive drinking. In the liver, there are enzymes that break down alcohol and decompose alcohol through aldehydes. Acetaldehyde is toxic to liver cells. It will be damaged.

These alcoholic liver diseases are classified into alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. The mechanism of liver damage may be due to alcohol itself, metabolites such as acetaldehyde, or immune response. Peroxidation of fat, binding to cytoplasm, disruption of electron transport systems in mitochondria, disruption of microtuble, formation of proteins and binding substances, increased collagen synthesis, etc.

Alcohol is absorbed by about 20% in the stomach and most of the rest is absorbed in the small intestine, 80-90% of the metabolism in the liver, so much of the liver's alcohol metabolism can be treated in the small and large intestine, less burden on the liver, further It may be able to suppress liver damage.

Therefore, because alcohol is not stored in the body, it must be metabolized, which is mostly done in the liver, and this metabolism is done by alcohol-degrading enzymes present in the liver. Alcohol is broken down through acetaldehyde by these enzymes, which are toxic and damage liver cells. And, as a result of alcohol metabolism, many fatty acids are made and fat is accumulated in the liver, which is called alcoholic fatty liver. The alcoholic fatty liver is often progressed to chronic liver disease, alcoholic hepatitis is said to occur in 10 to 35% cirrhosis in 8 to 20%.

Recently, probiotics, which have been spotlighted as enteric beneficial bacteria, have been widely applied to the prevention and treatment of diseases, and are known to be effective in inhibiting proliferation and decay activity against intestinal rot and pathogens. (Schrezenmeir J, De Vrese M: Am J Clin Nutr. 73 (2001), P 361S-4S) Probiotics inhibit the growth of harmful bacteria that produce ammonia, amines, etc. It has also been reported that the amount of nitrogen decreases to improve symptoms of liver disease. In addition, these useful bacteria have been reported to protect the liver from alcohol and improve its specific function. (Raibaud, P: The 3rd International Synposium on Lactic Acid Bacteria and Human Health. (1983), P116-126). Research has been reported of the possibility of actively promoting metabolism to rapidly decompose alcohol and to metabolize and detoxify acetaldehyde, which is harmful to the human body produced in this process. (Nosava. T. et al: Alcohol and Alcoholism. 35 (2000), P561-568)

Accordingly, the present inventors select the intestinal habitat probiotics having alcohol resistance among probiotics lactic acid bacteria, having alcohol degrading enzymes, and having the ability to decompose alcohol, thereby reducing the mechanism of alcohol degradation and reducing the blood alcohol concentration. As a result of confirming through animal experiments, Lactobacillus permanent (KCTC 10713BP) was found to have a very good alcohol resolution to complete the present invention.

Accordingly, it is an object of the present invention to provide Lactobacillus percentage which is excellent in alcohol resolution. Still another object of the present invention is to provide dairy products, health functional foods and food additives containing the Lactobacillus fermentum.

In order to achieve the above object, the present invention is an alcohol-degradable lactic acid bacteria, including Lactobacillus fermentum (KCTC 10713BP) that has the ability to decompose alcohol having a resistance to alcohol, alcohol degrading enzymes to provide.

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

Lactobacillus fermentum is obtained by separating the lactic acid bacteria present in kimchi and incubating for 15 to 24 hours at 37-42 ℃ temperature in the active medium containing milk as a main component to separate the lactic acid bacteria that can be applied to fermented milk lost.

Microscopy, gram staining, and catalase production were examined according to the characteristics of Lactobacillus strains in the Bergy's manual, and carbohydrates were identified using API 50 CHL medium (Kit). As a result of measuring the magnetization, it was confirmed that it is a new strain of Lactobacillus pertumtum that is gram positive, anaerobic, no spore, and catalase negative. It was deposited on November 2, 2004 and was given accession number KCTC 10713BP.

The results of evaluating 10% EtOH resistance, acid resistance, bile resistance, alcoholase activity, and aldehyde degrading enzyme activity of Lactobacillus percentage and selected Lactobacillus strains and other clinical strains selected in the present invention are shown in the following table. 1 is shown.

Experimental strain 10% EtOH resistant Acid resistance Bile resistance Alcohol degrading enzyme Aldehyde degrading enzyme Lactobacillus brevis MG 19al + + + - - Lactobacillus Planter Room MG 208AL + + + - - Lactobacillus casei MG 311AL + ++ ++ - - Lactobacillus rhamnosus MG 315AL ++ ++ ++ - - Lactobacillus axidophilus MG 501AL + ++ ++ - - Lactobacillus axidophilus MG 503AL + ++ ++ - - Lactobacillus luster MG 5073AL +++ + + ++ ++ Lactobacillus luster MG 507AL +++ + + ++ + Lactobacillus gassery MG 570AL + + + - - Lactobacillus permanent AL +++ +++ +++ +++ +++ Lactobacillus sp AL-GT-4 ++ + + - - Lactobacillus sp AL-GT-5 + + ++ - - Lactobacillus sp AL-MI-4 + ++ + - - Lactobacillus sp AL-MI-5 + + + - - Lactobacillus sp AL-MI-6 + ++ + + + Lactobacillus sp AL-MI-28 + ++ ++ - - Lactobacillus sp AL-MI-34 ++ +++ +++ +++ ++ Lactobacillus sp AL-MI-36 ++ + + - - Lactobacillus sp AL-MI-37 + + + + + Lactobacillus sp AL-MI-42 ++ + + - -

In Table 1, +++ is very good, ++ is good, + is slightly good, and-is not good.

As shown in Table 1, the selected Lactobacillus permanentum in the present invention in 10% EtOH resistance, acid resistance, bile resistance, alcohol degrading enzyme activity and aldehyde degrading enzyme activity compared to conventional known strains and various clinical strains All were well balanced.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are provided to facilitate the understanding of the present invention, but the scope of the present invention is not limited to these examples.

Example 1 Resistance Test According to Ethanol Concentration

The resistance test method according to the ethanol concentration is briefly described. First, 10% ethanol sterilized with a membrane filter is added to a pre-sterilized MRS liquid medium, and then a predetermined amount of the lactic acid bacteria activated in the MRS liquid medium is added. Add and incubate for 24 hours at a temperature of 37 ℃. And the cell mass of lactic acid bacteria was measured by absorbance.

The test results measured in the same manner as shown in Table 2 below.

 Strain Weight (g / l) Alcohol concentration (%) 0% 10% 15% 20% 25% Lactobacillus brevis MG 19al 0.4205 0.0740 0.1030 0.1010 0.0820 Lactobacillus Planter Room MG 208AL 0.5275 0.1040 0.0930 0.0960 0.0870 Lactobacillus casei MG 311AL 0.6400 1.3340 0.2380 0.1760 0.1170 Lactobacillus rhamnosus MG 315AL 1.5200 2.2790 0.0330 0.3010 0.3220 Lactobacillus axidophilus MG 501AL 1.0425 1.1540 0.2130 0.1660 0.1770 Lactobacillus axidophilus MG 503AL 0.9325 1.2640 0.2730 0.1660 0.1320 Lactobacillus luster MG 5073AL 3.3200 2.0940 0.6680 0.5110 0.4820 Lactobacillus luster MG 507AL 3.3400 2.4290 0.9130 0.5310 0.4370 Lactobacillus gassery MG 570AL 0.5475 0.0890 0.0580 0.1060 0.0820 Lactobacillus permanent AL 3.1230 1.0210 0.4930 0.2860 0.3470 Lactobacillus sp AL-GT-4 3.2903 1.5350 0.5730 0.4030 0.2985 Lactobacillus sp AL-GT-5 3.3243 1.2922 0.5263 0.3283 0.3219 Lactobacillus sp AL-MI-4 3.5190 1.2790 0.7483 0.3414 0.3079 Lactobacillus sp AL-MI-5 2.4213 1.2882 0.6106 0.2305 0.2274 Lactobacillus sp AL-MI-6 2.3168 1.0373 0.4610 0.2520 0.2235 Lactobacillus sp AL-MI-28 2.7165 1.0608 0.8880 0.2909 0.3011 Lactobacillus sp AL-MI-34 3.4408 1.3993 0.6500 0.4390 0.4046 Lactobacillus sp AL-MI-36 3.2535 1.4900 0.5155 0.3671 0.3269 Lactobacillus sp AL-MI-37 2.7133 0.9415 0.4150 0.2693 0.3074 Lactobacillus sp AL-MI-42 3.4125 1.3788 0.4450 0.3545 0.2895

Example 2: Alcohol Degrading Enzyme (ADH) Activity and Aldehyde Degrading Enzyme (ALDH) Activity Test

The alcohol degrading enzyme and aldehyde degrading enzyme activity test method was first cultured in MRS liquid medium to recover only the lactic acid bacteria cells, resuspended in 10 mM sodium phosphate buffer (pH 7.5), and then ice bath (Ice bath) The cells were crushed by using an ultrasonic grinder for 3 minutes. The pulverized cell solution was centrifuged for 60 to 90 minutes at a temperature of 4 ° C. at 10000 to 16000 rpm to harvest the cytoplasm of the lactic acid cells.

Alcohol degrading enzyme was measured for NAD reduction using alcohol as a substrate, and aldehyde degrading enzyme was measured for acetaldehyde as NAD reduction.

Thus measured results are shown in Table 3 below.

Experimental strain                                                                                                Alcoholase activity (unit / mg protein) Aldehyde degrading enzyme activity (unit / mg protein) 0% alcohol 10% alcohol 0% alcohol 10% alcohol Lactobacillus brevis MG 19al 0.0000 0.0157 0.0000 0.0000 Lactobacillus Planter Room MG 208AL 0.0000 0.0000 0.0000 0.0000 Lactobacillus casei MG 311AL 0.0000 0.0000 0.0000 0.0000 Lactobacillus rhamnosus MG 315AL 0.0000 0.0000 0.0000 0.0000 Lactobacillus axidophilus MG 501AL 0.0000 0.0117 0.0000 0.0000 Lactobacillus axidophilus MG 503AL 0.0000 0.0000 0.0000 0.0000 Lactobacillus luster MG 5073AL 3.1433 0.4297 1.4800 0.7520 Lactobacillus luster MG 507AL 3.6090 0.2308 0.6700 0.5210 Lactobacillus gassery MG 570AL 0.0000 0.0000 0.0000 0.0000 Lactobacillus permanent AL 3.9600 2.1832 2.3500 1.7600 Lactobacillus sp AL-GT-4 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-GT-5 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-4 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-5 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-6 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-28 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-34 3.2132 1.2008 0.5200 0.0500 Lactobacillus sp AL-MI-36 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-37 0.0000 0.0000 0.0000 0.0000 Lactobacillus sp AL-MI-42 0.0000 0.0000 0.0000 0.0000

Example 3: Enzyme Identification Test through SDS-PAGE

According to the Laemmli method using SDS-PAGE to determine whether alcohol degrading enzyme and aldehyde degrading enzyme are produced from lactic acid bacteria having high enzymatic activity shown in Table 3 as a result of Example 2 It was performed on a gel containing 15% acrylamide and 0.1% SDS.

Samples for the test were used in the same manner as in Example 2 after obtaining the cytoplasm.

Enzyme confirmation test results carried out in this embodiment is shown in FIG. In Figure 1, A is a marker, B is a standard ALDH (Baker's yeast), C is a standard ADH (Baker's yeast), D is a Lactobacillus permanent liquid medium added with 10% ethanol as an experimental group, E is Lactobacillus permanent medium as a control Liquid medium, F is Lactobacillus axidophyllus, G is Lactobacillus gasteria.

As shown in FIG. 1, the Lactobacillus permanentum 's ADH molecular weight is about 40 kDa, similar in size to standard ADH. In the case of Lactobacillus permanent liquid medium added with 10% alcohol (D), it can be seen that the ADH band is darker than that without adding 10% alcohol (E). This shows that the expression of ADH increased when alcohol was added. In addition, in the case of Lactobacillus axidophilus (F), which had inadequate activity in previous experiments, bands of the same size were weak, and in the case of inactive Lactobacillus gaseri (G), no band appeared at all. Did.

In case of ALDH, lactobacillus permanent liquid medium to which 10% alcohol was added was inadequate, but a band appeared.

Example 4: Alcohol Resolution Test

In this example, in order to confirm the degree of alcohol degradation by the lactic acid bacteria having high enzymatic activity in Example 2, 10% alcohol was added to the liquid medium after 4 hours of cultivation using gas chromatography. Then, the decomposition degree of alcohol was measured at 2 hour intervals.

At this time, the gas chromatography analysis method (Kim, JH et al., J. Microbial. Biotechnol. 13 (2003), 919-925) was used, the lactic acid bacteria were not added to the control group, Lactobacillus to the experimental group. Permanent was added.

Table 4 shows the results of testing the alcohol resolution according to the incubation time.

                Alcohol content in the medium 0 hr 2 hr 4 hr 6 hr Experimental group 7.5% 3.7% 2.9% 2.7% Control 7.5% 7.5% 7.5% 7.5%

As shown in Table 4, as time passes, the alcohol content of the experimental group decreases, while the alcohol content of the control group is found to be constant.

Example 5 Examination of Hepatic Function Effect by Hepatocyte Culture

In this example, in order to confirm the effect of liver function through the hepatocyte culture of Lactobacillus fermentum excellent in alcohol resolution of the present invention, two-step-in of Seglen from 8-week-old Sprague-Dawley rats Hepatocytes were isolated by two-step in situ collagenase perfusion technique and added to DMEM (Dulbecco's Modified Eagle Medium) medium with 10% alcohol, 10% alcohol and lactic acid bacteria cytoplasm. After grouping the cells into 20% alcohol and lactic acid bacteria cytoplasmic addition group and incubating for 7 days, the state of the hepatocytes was observed under a microscope and shown in FIG. 2.

In Figure 2, A1 is the pre-experimental state of the control group to which nothing is added, A2 is the post-experimental state of A1, B1 is the pre-experimental state of the medium to which 10% alcohol is added, B2 is the post-experimental state of B1, C1 is 10 The pre-experimental state of the medium containing% alcohol and lactic acid bacteria cytoplasm, C2 is the post-experimental state of the C1, D1 is the pre-experimental state of the medium to which the 20% alcohol and lactic acid cytoplasm was added, and the D2 is the post-experimental state of the D1.

As shown in Figure 2, it was observed that hepatocytes (C2) containing cytoplasm of alcohol and Lactobacillus fermentum had more living cells than alcohol (B2), and mostly alcohol (B2). Hepatocytes were seen to come off the plate. However, it was found that the hepatocytes were maintained when the cytoplasm of Lactobacillus fermentum was added (C2, D2), and there was no significant difference when the concentrations of the Lactobacillus fermentum cytoplasm were changed (C2, D2).

Through the above experiments, it was confirmed that Lactobacillus permanentment reduces the damage of hepatocytes and maintains cell activity.

Example 6 Blood Alcohol Reduction Effect Test in Mice

In this example, 2 hours after oral administration of Lactobacillus fermentum lactobacillus culture medium and 22% alcohol to Sprague-Dawley rats in order to measure the effect of reducing blood alcohol concentration using rats Blood samples were collected and blood alcohol levels, GOT (glutamic-oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase) were measured.

Table 5 shows the test results of Example 6, where the control group was administered with 3 ml of 22% alcohol, and the experimental group was administered Lactobacillus fermentum lactic acid bacteria with 3 ml of 22% alcohol.

Blood alcohol level (%) GOT (Karmen unit) GPT (Karmen unit) Before intake 2 hours Before intake 2 hours Before intake 2 hours Control 0.000% 0.007% 20.5 71.9 31.3 75.4 Experimental group 0.000% 0.005% 21.6 17.9 27.7 14.3

As shown in Table 5, the experimental group administered Lactobacillus Permanent Lactobacillus was found to be significantly lower in blood alcohol concentration, GOT, GPT than the control group.

On the other hand, the production of dairy products, health functional foods and food additives containing Lactobacillus permentum lactic acid bacteria according to the present invention and the content thereof can be easily determined from conventional methods known in the art, lactic acid bacteria survival in food It may also contain other substances for maintaining the properties.

As described above, the Lactobacillus lactic acid bacterium selected from the present invention (Lactobacillus permentum (Accession Number: KCTC 10713BP)) is excellent in alcohol resistance, especially because of excellent acid resistance and bile resistance and milk culture, alcoholic liver When included in medicines, dairy products, dietary supplements, and food additives to prevent disease, it is possible to achieve hangover and liver detoxification.

Claims (4)

Lactobacillus fermentum with alcohol resolution KCTC 10713BP. Dairy products containing lactic acid bacteria having alcohol decomposability according to claim 1. Health functional food containing lactic acid bacteria with alcohol decomposability according to claim 1. A food additive containing lactic acid bacteria having an alcohol resolution according to claim 1.

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