WO2007108583A1

WO2007108583A1 - Lactic acid bacterium for reducing cholesterol in blood and a method for producing cholesterol reduced egg using the bacterium

Info

Publication number: WO2007108583A1
Application number: PCT/KR2006/004581
Authority: WO
Inventors: Heuyn Kil Shin
Original assignee: Heuyn Kil Shin
Priority date: 2006-03-22
Filing date: 2006-11-03
Publication date: 2007-09-27

Abstract

Disclosed is Enterococcus mundtii HFI 58 strain deposited with accession number of KCTC 11007BP as one of lactic acid bacteria strains useful for reducing cholesterol in blood. The strain exhibits excellent effects including: reduction of total cholesterol (TC) in blood; increase of high density lipoprotein (HDL) level; and reduction of low density lipoprotein (LDL) level.

Description

[DESCRIPTION] [Invention Title]

LACTIC ACID BACTERIUM FOR REDUCING CHOLESTEROL IN BLOOD AND A METHOD FOR PRODUCING CHOLESTEROL REDUCED EGG USING THE BACTERIUM [Technical Field]

The present invention relates to a) Enterococcus mundtii HFI 58 strain as one of lactic acid bacteria strains for reducing cholesterol in blood, b) the feed for egg-laying chickens added with the same, c) a method for breeding the egg- laying chickens with the feed, and d) cholesterol-reduced eggs obtained from the chicken bred by the said method.

[Prior Art]

The leading causes of death in Korea are hemorrhagic cerebrovascular disease, traffic accident and cancer which respectively occupy the first, second and third ranks, of which the hemorrhagic cerebrovascular disease, in particular, is the leading cause of death of people in their seventies.

High cholesterol level in blood can result in death for a person and can also cause cardiovascular disease (CVD) such as heart attack, atherosclerosis, etc. Cholesterol is a waxy substance in lipid contained in cells and blood stream of the human body. Cholesterol has essential functions necessary for the human body, but also increases the chance of CVD if its level in blood is too high, and may be detrimental to human health. High cholesterol level usually means that the total level of cholesterol in blood exceeds 240mg/dl or the level of low density lipoprotein (LDL) cholesterol exceeds 160mg/dl. When the cholesterol level in blood reaches the high values, cholesterol may be deposited on the blood vessel inner wall.

It has been normally known that cholesterol is transported from and/or to cells by specific carriers called LDL and high density lipoprotein (HDL). HDL cholesterol removes cholesterol from the blood vessel wall, moves it to the liver and makes it to be easily metabolized. Therefore, HDL cholesterol is considered beneficial cholesterol, that is, good cholesterol. On the other hand, LDL cholesterol carries the cholesterol along the blood flow, accumulates it on the inner wall of the blood vessel and accelerates arteriosclerosis, hence, is called harmful cholesterol or bad cholesterol. Accordingly, it is desirable for the level of cholesterol in blood to be lower.

On the other hand, Kimchi dominant strains that are normally known as lactic acid bacterium in connection with fermentation of Kimchi, Korean spicy pickled vegetables, has been reported to possess beneficial features such as improved immunity, anti-microbial, anti-oxidation and/or anti-cancer effect, prevention of hypertension or constipation, etc. [See, Hivak P. Odrska J. Ferencik M. Ebringer L. Jahnova E. Mikes Z.] One-year application of probiotic strain Enterococcus faecium M-74 decreases serum cholesterol levels: Bratisl Lek Listy 2005; 106(2); 67-72; Agerholm-Larsen L. Bell ML. Grunwald GK. Astrup A.: The effect of a probiotic milk product on plasma cholesterol: a meta-analysis of short-term intervention studies: Eur J Clin Nutr 2000; 54(11); 856-860].

Examples of such Kimchi Lactic acid bacteria strain that have been known to have such a property at present include: L. Bulgaricus, L. Acidophilus, Bifidobacterium bifidum, Bif. Longum, Bif. Infantis, Leuconstoc, Lactobacillus Helveticus as anti-cancer bacteria; Lactobacillus Rhamnosus as anti-microbial bacteria; and Lactobacillus GG as anti-constipation bacteria.

The lactic acid bacteria strain according to the present invention, a bacteria strain among the above examples that are known to reduce cholesterol, was incubated and added to chicken feed for egg-laying chickens, and the effect of lowering cholesterol contained in egg yolk by as much as 54 % was demonstrated. A patent for such lactic acid bacteria strain based on an earlier result was filed in April of 2006 and is pending in Korea. [Disclosure] [Technical Problem]

Therefore, a purpose of the present invention is to isolate and identify lactic acid bacteria strain that lowers cholesterol level in blood, to prove that the lactic acid bacteria strain can be used in manufacturing medicines or health food, and to provide a method for production of eggs with a reduced content of cholesterol that causes cardiovascular disease.

[Technical Solution] The aforementioned object of the present invention is accomplished through the steps of: isolating and identifying Enterococcus mundtii HFI 58 strain that was deposited with the accession number of KCTC 1107BP; introducing the strain to rats fed with high fat diet to reduce its total cholesterol(TC) level; determining whether HLD value was increased while LDL value was decreased in the rats; and adding the strain to chicken feed with a purpose to produce cholesterol-reduced eggs from the chickens raised by feeding the feed.

Accordingly, the present invention relates to Enterococcus mundtii HFI 58 strain KCTC 1107BP, that is one of the lactic acid bacteria strains that is beneficial to reduce cholesterol level in blood. Furthermore, the present invention provides the cholesterol-reducing feed for egg-laying chickens which is added with KCTC 1107BP strain.

The present invention also provides a method of breeding egg-laying chickens with a reduced cholesterol level by feeding the chicken feed containing KCTC 1107BP strain. The egg-laying chickens with reduced cholesterol level according to the present invention are raised by feeding the cholesterol-reducing feed to an egg- laying chicken in amount of 0.1 to 1Og at one time per day for at least three weeks. Preferably, the feed is introduced to an egg-laying chicken with 1.5g once per day for four weeks. In addition, the present invention relates to the eggs with reduced cholesterol level that are produced by the chicken breeding method described above.

Even if the present invention is described in detail with reference to the specific examples presented below, it should be obvious to those skilled in the art that the spirit and scope of the present invention are not limited to the specific examples shown.

[Advantageous Effects] The lactic acid bacteria strain according to the present invention for reducing cholesterol level in blood has excellent effect of reducing cholesterol level in egg yolk and can also lower the cholesterol level in a human body when the lactic acid bacteria strain-added feed is fed to egg-laying chickens, and the eggs laid by the chickens are taken by the person.

[Description of the Drawings]

The above and other objects, features and other advantages of the present invention will be more clearly understood from the detailed description below in conjunction with the accompanying drawings, in which: FIG. 1 is a graph depicting reduction of cholesterol by Enterococcus mundtii HFI 58 strain KCTC 1107BP according to the present invention;

FIG. 2 is a graph depicting reduction of cholesterol contained in egg after introducing Enterococcus mundtii HFI 58 strain KCTC 1107BP of the present invention to chicken feed; FIG. 3 is a graph depicting reduction of total cholesterol (TC) level;

FIG. 4 is a graph depicting raising of high density lipoprotein (HDL) level; and

FIG. 5 is a graph depicting lowering of low density lipoprotein(LDL) level. [Best Mode for the Invention]

Example 1 : Isolation and identification of cholesterol reducing bacteria strain of the present invention

The present inventor achieved in practice isolation of the bacteria strain with an excellent property of reducing cholesterol level in blood by several experimental steps. The strain, a lactic acid bacteria strain, was isolated based on morphological and cultural characteristics and was correctly identified by a genotypic identification of rep-PCR, RAPD-PCR and 16S-rDNA gene sequencing. Enterococcus mundtii HFI 58 strain with an excellent cholesterol reduction property identified as described above was deposited with Korea Biological Resources Center(KBRC) of Korea Institute of Biotechnology on October 23, 2006 and was accepted with the accession number, KCTC 11007BP. [Table 1] Investigation Result of Cholesterol Reducing Strain

[Mode for Carrying out the Invention]

Example 2: Investigation of survival of Enterococcus strain of the present invention in gastric juice and intestine In order to study survival of the lactic acid bacteria strain in gastric juice and intestine, resistance of the lactic acid bacteria strain to pH value 3.0 and 0.5%

Oxgall was examined respectively. It was found that HFI 58 was the strain with the strongest resistance among strains which were subjected to the examination.

See Table 2. [Table 2]

Investigation Result of Survival of Enterococcus strain of the present invention in Intestine

Strains HFI 58

** Excellent proliferation

* Good proliferation

Example 3: Investigation of growth performance of Enterococcus strain of the present invention in intestine of animal

In order to determine whether the Enterococcus strain isolated and identified according to the present invention proliferates in the intestine of animal, survival of the strain at pH value 3.0 and in Bile salt was examined respectively. From the examination, it was found that the present invented strain survived and had growth performance while typical lactic acid bacteria were all dead. See Table 3.

[Table 3]

Investigation Result of Survival of Lactic acid bacteria Strain of The Present Invention at pH2 and in Bile Salt

Example 4: Investigation of cholesterol decomposition performance of Enterococcus strain according to the present invention

Bile salt hydrolase activity means the extent of Bile salt decomposition performance. It is normally known that the amount of Bile salt to be decomposed is increased and the decomposition of cholesterol as source of Bile salt is accelerated when the amount of Bile salt hydrolase to be secreted is increased. In other words, Bile salt hydrolase has activity in close connection with reduction of cholesterol; therefore, in this example, the extent of Bile salt decomposition was determined by examining the activity of Bile salt hydrolase. According to the result of the test, most general strains showed no BSH activity while HFI 58 exhibits 1.1cm of circle size, as shown in Table 4 below.

[Table 4]

Investigation Result of Circle Size (diameter, cm) of MRS Agar Plate of

Enterococcus strain of the Present Invention

Example 5: Investigation of the cholesterol reducing effect of Enterococcus strain of the present invention

In order to determine cholesterol decomposition effect on the

Enterococcus strain of the present invention, the cholesterol reduction effect was examined in vitro by inoculating and incubating the strain to THIO-MRS. It was found that the present invented strain HFI 58 has an effect of lowering cholesterol by about 50%. See FIG. 1.

Example 6: Production of eggs with reduced cholesterol content In this example, analysis of cholesterol level in eggs was conducted by taking egg yolk out of the eggs laid by the adult chickens raised with typical chicken feed to which the present invented Enterococcus strain HFI 58 was added.

As a preferred embodiment of the present invention, the present invented Enterococcus strain was added to a general chicken feed available from Woo Jin B&G, Seoul, Korea at a rate of 10⁹ cells per gram of feed. After mixing Enterococcus strain well with the feed, the feed mixture was fed to each of egg- laying chickens in the amount of 1.5g once per day per chicken for four weeks. hi the above breeding experiment, another group of egg-laying chickens was also raised as a control with the same general feed without the present invented Enterococcus strain added. For both the test and the control groups, fifty egg-laying chickens were raised, the control group by feeding the regular chicken feed and the test group by feeding the regular feed added with the present invented HFI 58 strain. Both the test and the control groups were continuously fed with the respective feeds for three weeks as described above.

For the first group, thirty eggs were collected from the egg-laying chickens after breeding the chickens with the regular chicken feed, and then, the egg yolks were extracted from the eggs and subjected to measurement of cholesterol value (referred to as 'the control group'). For the second group, also thirty eggs were collected from the egg-laying chickens after breeding the chickens with the feed containing the present invented lactic acid bacteria strain HFI 58, and egg yolks were extracted from the eggs and subjected to the same measurement of the cholesterol value as the control group (referred to as 'the test group'). Afterwards, the measured cholesterol values from the two groups were compared each other. The cholesterol levels for each of the control and the test groups as the result of investigating are shown in Table 5 and FIG. 2. [Table 5] Investigation Result of Cholesterol Value in Egg of the Present Invention

As can be clearly seen in Table 5 and FIG. 2, the test group containing the present inventive Lactic acid bacteria strain showed marked reduction in the cholesterol level as compared to the control group. In terms of percent by weight, it was found that the cholesterol level for the test group using the present invented HFI 58 was 68.28% of that for the standard group by weight, with the cholesterol reduction rate of about 31.72% by weight for the cholesterol reducing chicken feed.

Example 7: Effect of feed containing the present inventive lactic acid bacteria strain to mice

After nurturing mice (purchased from Hyo Chang Science Inc., 6 week old male, C57BL/6) for one week, the mice were divided into two groups. A high fat diet was applied to one group while the other was subjected to the regular diet, both of the diets being shown in Table 6 below which illustrates composition and calories thereof. Herein, the present invented strain was added to 2g of the high fat diet and the feeding was regulated to provide 10⁸ cfu of the present inventive strain to each of the mice per day every day. Whole blood assay was conducted for the mice from the both groups every week starting from the third week for a period of total six weeks during which time all of TC, HDL and LDL values were analyzed. The results of analysis can be seen in Table 7 below. [Table 6] Comparison of Compositions for High Fat Diet and Basal Diet

[Table 7]

As can be seen in Table 7, as the result from the experiment for the total six weeks, TC value went down by 20%, HDL value went up by 15 to 86% and LDL value went down by 92% for the high fat diet group with the present invented HFI 58 bacteria strain. From the result, it was judged that the present inventive strain has excellent cholesterol reduction effects such as increase of HDL and decrease of LDL levels.

More specifically, the measurement of the TC level reduction property of

HFI 58 bacteria strain showed that the difference in the cholesterol levels between the two groups was not so significant initially until the third week. However, from the fourth week, the difference between the groups started becoming significant as shown in FIG. 3.

Also for the HDL values, there was substantially little difference between the two groups until the third week. The difference between the cholesterol values became significant from the fourth week, and especially after six weeks, the difference became remarkable as shown in FIG. 4.

As for the LDL values, the difference in the levels between the two groups became evident already from the third week and, especially, the high fat diet group had the highest LDL value. Especially, the LDL values were 45.972mg/dl for the high fat diet group whereas the HFI 58 strain group had 4.126mg/dl, thereby indicating about 11 times of the difference in the LDL values between the two groups as shown in FIG. 5.

The present invention has shown and described with reference to certain preferred examples as described above, however, it will be understood by those skilled in the art that various changes in form and details for the invention may be made therein without departing from the spirit and scope of the invention.

[Industrial Applicability]

From the above results, it appears that the Enterococcus strain of the present invention effects reduction of cholesterol when the strain is employed in medicines and health foods. Furthermore, it is judged that if the present inventive strain is added to feed for chicken, eggs obtained from the chickens will have greatly reduce cholesterol level. Furthermore, it is thought that HFI 58 strain of the present invention should also be widely applicable in manufacture of health food and medicines.

Claims

[CLAIMS]

[Claim 1] Enterococcus mundtii HFI 58 strain with accession number of KCTC 11007BP as one of Enterococcus strains for reducing cholesterol in blood.

[Claim 2] A composition for reducing cholesterol level in blood, comprising combinations of the Enterococcus strain as defined in Claim 1 with other Enterococcus strains as active ingredient.

[Claim 3] A foodstuff for reducing cholesterol in blood, fabricated by comprising the Enterococcus strain as defined in Claim 1.

[Claim 4] A feed product for egg-laying chickens for reducing cholesterol, comprising the Enterococcus strain as defined in Claim 1 as active ingredient.

[Claim 5] A method of breeding cholesterol-reduced egg-laying chicken with reduced cholesterol, comprising feeding egg-laying chickens the feed product in amount of 0.1 to 1Og once per day for three weeks for reducing cholesterol as defined in Claim 4.

[Claim 6] The method according to Claim 5, comprising addition of other Enterococcus strains to the feed product and feeding the mixture to the egg- laying chickens.

[Claim 7] Eggs with reduced cholesterol, produced from the egg-laying chicken raised by the method as defined in Claim 5.