Background Art CLA, which is a conjugated isomer of linoleic acid (hereinafter referred to as LA), an essential fatty acid, is a natural fatty acid found in a small amount in milk or muscle of ruminants.

CLA has conjugated double bonds at positions cis-9 and trans-11 or at positions trans-10 and cis-12, in intra and trans configuration. Particularly, by a conjugated double bond at cis-9 and trans-11 positions, physiological activities useful to human bodies are expressed.

CLA is known to show reduction in development of the artery sclerosis (Artery. 1997.22 : 266-277), enhancement of immune system (J. Nut. 1999.129 : 32- 38), anticancer effect (Anticancer research. 1997.17 : 969-973), growth promotion (J.

Nut. 2000.130 : 2981-2989) and excellent effects of treating some diseases including diabetes and to inhibit the obesity through reduction of body fat (Am. J. Physiol.

1998.275 : R667-R672). Owing to such properties, CLA may be usefully used as an effective ingredient of functional food and medicaments.

CLA is mainly contained in animal food, particularly at a large amount in ruminant animals. It is shown that beef contains 2. 9-4. 3 mg CLA/fat, lamb meat

contains 5.6 mg CLA/fat and marine products contain as little as 0. 3-0. 6 mg CLA/fat. For dairy products, milk contains 5.5 mg CLA/fat and cheese contains 3-7 mg CLA/fat. With respect to human beings'daily CLA intake, it is presumed that oriental people who principally keep a vegetable diet intake 0.1 g of CLA per day and western people who eat more meat intake 0.4 g of CLA per day.

So far, various methods have been proposed to mass-produce CLA. The conventional methods include methods for chemically synthesizing CLA from LA, such as urea addition, molecular distillation, HPLC, etc. , and there are several isolated microorganism able to convert LA into CLA.

However, the chemical synthesis methods have problems, such that they require expensive equipments or too much time is taken for processes. Also, since conventional chemical synthesis methods produce a kind of CLA along with various kinds of isomers, it is very inefficient to perform production of a kind of CLA by such chemical synthesis methods.

Therefore, the most efficient method for producing CLA is to produce CLA by a microorganism, followed by isolation. Representative microorganisms capable of producing CLA includes microorganisms in the bowels such as Lactobacillus, Propiotzibacterium, Butyrivibrio fibrisolvens etc. and are usefully used as an effective ingredient in functional food or medicaments such as probiotics and feed which are fed to animals in many countries.

However, the direct addition of CLA as an effective ingredient to food or medicaments is not allowed in Korea. Therefore, in order to employ CLA as an effective ingredient in food and medicaments, a method comprising directly adding a strain of a microorganism capable of producing CLA should be used, rather than a method comprising directly adding CLA isolated from a microorganism.

As known up to now, in the synthesis of CLA by microorganisms in the bowels, only the cis-9 and trans-11 are specifically produced, unlike the chemical synthesis in which cis-9 and trans-11, and cis-12 and trans-10 are produced in a ratio of about 50%. Though a small amount of cis-9 and trans-l 1 has been found in the meat and milk from ruminant animals, cis-9 and trans-11 are detected only in a small amount in dairy products using skim milk. Even the Kimchii which is fermented using various lactic acid bacteria is known not to contain cis-9 and trans-11.

Therefore, Korean Patent Application No. 10-2001-0047292 disclosed a method for adding pure cis-9 and trans-11 type CLA which is synthesized by a microorganism of Lactobacillus genus to food. However, only an infinitesimal amount of CLA was found in an actual product.

Also, in order a CLA producing microorganism to be used as an effective ingredient in food or medicaments, it should survive at a high level similar to that upon production while the product is distributed and also should maintain a high viability and activity in the stomach and bowels after intake into a body of an animal including human beings. In addition, it should be excellent in resistance to antibiotics to hold a predominant position in the competition with harmful bacteria in the bowels, including superbacteria.

However, it was found that a number of strains, among microorganisms which are used as an effective ingredient in commercially available products, cannot survive during storage or passage through the stomach after intake and have weak resistance to antibiotics.

Therefore, it is still desired to develop a strain that shows a high viability and activity in animal bodies as well as excellent CLA productivity and itself can be directly added to food and medicaments as an effective ingredient.

Disclosure of Invention Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide novel strains which can produce conjugated linoleic acid, have excellent resistance to acids and antibiotics and can indirectly produce CLA when added to food and medicaments.

It is another object of the present invention to provide a capsule formulation comprising the strain and CLA which can be used to produce functional fermented food, dairy food and medicaments.

In accordance with the present invention, the above and other objects can be accomplished by the provision of novel strains capable of producing CLA.

The strains are characterized in that they are isolated from feces of infants and they can convert LA to CLA.

The strains include Bifìdobacterium breve CBG-C2, Bifidobacterium pseudocartesaulatum CBG-C4 and Enterococcus faeciunri CBG-C5.

The strains can be isolated as follow: Strains are isolated from feces of Korean infants and randomly selected 300 colonies are cultured in a medium with LA as a substrate. The medium is extracted with hexane and the extracts were measured for their absorbance to screen strains which have excellent CLA productivity.

The strains screened by the above procedures were once again examined whether they can produce CLA from LA, by subjecting fatty acids produced by the strains to gas chromatography (GC).

The strains thus isolated were three types, designated CBG-C2, CBG-C4 and CBG-C5, respectively and were deposited at Korean Agricultural Culture Collection

in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001, KACC 91003 and KACC 91002, respectively, on April 3,2002.

Also, CBG-C2 (KCTC 10462BP) and CBG-C4 (KCTC 10208BP) were deposited at the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology on March 25,2002 and April 10,2003, respectively.

The strains obtained by the above-described method are excellent in CLA productivity and have strong resistance to acids such as stomach acid and bile, and antibiotics.

CLA produced by microorganisms according to the present invention comprises only the stereostructures of cis-9 and trans-11, which are isomers having various physiological activities, as conventionally known to the art. Fatty acids and acyglycerol containing these structures can be widely used for development of dairy products from various animals, dairy products from vegetable materials, fermented food and functional health food, probiotics and the like.

Therefore, the strains according to the present invention can be effectively used in biosynthesis of isomers of CLA by biological methods such as immobilization.

The strains according to the present invention can be added to food and medicaments as not only a live strain but also a killed strain. This is because the strains according to the present invention can release CLA to a culture fluid or reaction fluid while accumulating a large amount of CLA in the strains.

Therefore, according to the present invention, the strain which are cultured in a medium containing LA may be added as an effective ingredient to various compositions such as food and medicaments so that CLA is indirectly produced and it is thus possible to promote development of functional products containing CLA in

a large amount by resolve the conventional problems in that CLA cannot be directly added to food and medicaments.

Also, the present invention provides a food or pharmaceutical composition containing the strain.

The compositions according to the present invention contain at least one selected from Bifidobacteriu7n breve CBG-C2, Bificlobacterium pseudocartenulatum CBG-C4 and Enterococcusfaeciunt CBG-C5 as an effective ingredient.

The composition may comprise an additional effective ingredient such as an organic acid to enhance CLA production rate and improve growth stability of strain.

The organic acid in the composition according to the present invention may be CLA which is chemically synthesized or is isolated and purified from microorganisms.

Particularly, the composition according to the present invention may comprise any one selected from the strains according to the present invention and CLA at the same time.

The composition according to the present invention may further contain various adjuvant components in addition to the effective ingredient, when needed.

The food composition according to the present invention may comprise vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6 and vitamin B 12, folic acid, vitamin C, vitamin D3, vitamin E and the like, minerals such as copper, calcium, iron, magnesium, potassium, zinc and the like, or lactic acid bacteria and the like.

Also, as one of the food composition according to the present invention, a heath drink composition may comprise an additional component such as a flavor or natural carbohydrates, like other drinks. The flavor includes natural sweetening agents such as thaumatin and stevia extract and synthetic sweetening agents such as

saccharin and aspartame. The natural carbohydrate includes monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, polysaccharides such as dextrin, cyclodextrin and the like, and sucrose alcohols such as xylitol, sorbitol, erythritol and the like.

The usage or intake of the pharmaceutical composition according to the present invention is preferably 60 to 130 uM (Cancer Epidemiol. Biol. Prev. 2000.

9: 689-696) and the usage or intake of the food composition according to the present invention is preferably 3.4 to 6 g/day (J. Nutr. 2000.130 : 2943-2948), though it may be adjusted, as needed. The composition is stably absorbed into a body regardless of the intake. The usage or intake can be adjusted according to various factors including the type and amount of an effective ingredient and other components contained in the composition, formulation type and age, body weight, general health condition, sex and diet of a patient, administration time, administration route and release rate of the composition, treatment duration, simultaneously used drugs.

Upon administration to a human body, the composition would not show side effects, as compared to conventional food and medicaments containing chemically synthesized CLA.

The composition may be formulated by combining at least one pharmaceutically acceptable or edible carrier with the effective ingredient.

The pharmaceutically acceptable or edible carrier which can be used in the present invention includes a saline solution, sterile water, Ringer's solution, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof and may be combined with a conventional additive such as an antioxidant, a buffering agent, a bacteriostatic agent and the like, when needed. Also, the composition can be formulated into injections such as aqueous solutions, suspensions,

emulsions, pills, capsules, granules or tablets by further adding a diluting agent, a dispersing agent, a surfactant, a binder and a lubricant. Further, the composition may be preferably formulated by a proper method known to the art, or a method described in Remington's Pharmaceutical Science (the latest), Mack Publishing Company, Easton PA, according to diseases or components.

The composition according to the present invention can be formulated in the form of granules, powders, coated tablets, tablets, capsules, liquids and solutions, extracts, suppositories, syrups, juices, suspensions, emulsions, release-sustained formulation of an active compound and the like.

According to the present invention, there is provided a capsule formulation of the composition.

The capsule formulation comprises a coating material and a core material enclosed in the coating material.

As the core material of the capsule formulation comprises preferably both any one selected from the strains according to the present invention and CLA.

As the coating material enclosing a core material in the capsule, water- soluble polysaccharides which are excellent in absorption, dispersion and adhesion are usable.

The water-soluble polysaccharides which can be used in the present invention is at least one selected from the group consisting of starch, agar, carageenan, alginic acid, sodium alginate, polymethacrylate, wheat protein, soy protein, cellulose derivatives such as methylcellulose, hydroxylpropylcellulose, hydroxyl-propylmethylcellulose and the like; gums such as xanthan gum, arabic gum, locust bean gum, guar gum, tamarind gum, tara gum, karaya gum, tragacanth gum, ghatti gum and the like ; and gellan, xanthan, pectin (LM, HM type, dextran, glucan,

glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, gelatin and casein.

In the preparation of the capsule, the amount of the coating material can be adjusted according to the final use and purpose and is preferably 1 to 80 % by weight based on the weight of the core.

In addition to the water-soluble polysaccharides, the capsule may further comprise substances which are commonly used in a coating material to improve release control effect or to increase solubility.

Also, the capsule may further comprise an emulsifying agent, a protective agent and plasticizer, as needed.

The capsule formulation may be produced by using one of methods which are commonly used for encapsulation. For example, a method for preparing the capsule according to the present invention is performed by a typical encapsulation method, which comprises a process for emulsification, in which strains which are obtained by culturing the strain according to the present invention, an organic acid and a coating material for capsule are dispersed in a solvent with emulsion stability, a process for production of capsule membrane, in which the emulsified dispersion is stirred to form capsule membrane, and a process for curing, in which a curing agent and a reagent are added to cure the capsule membrane.

The capsule formulation thus obtained can protect the strain from outer circumstances by antioxidation and thus, can be stored stably at a low temperature for a long period of time.

In general, fatty acids such as CLA are unstable to heat, enzymes, acids, alkalis, microorganisms and the like. However, microencapsulation by a proper coating material may improve storage stability and convenience for internal use.

The capsule formulation maintains a live strain number at a uniform level in the bowels under acid conditions by regular release control.

Also, the capsule formulation can increase specific gravity and dispersibility in the aqueous phase of the body by microencapsulation to further enhance the bioavailability, thereby improving applicability to preserved food, milk and drinks, and dairy products.

Therefore, the capsule formulation allows the strains according to the present invention to grow stably in the bowels. The strains which have been grown in the bowels can continuously produce CLA, whereby it is possible to provide inhibition of development of the artery sclerosis, reduction of body fat, improvement of immunity, anticancer effect, growth promoting effect and the like. Also, the strains according to the present invention hold dominant species in the bowels, thereby acting as probiotics to inhibit growth of harmful microorganisms in the bowels.

Therefore, it is possible to continuously activate and improve the bowel function.

The composition comprising the capsule formulation includes, for example, dairy products (milk, soy milk, processed milk), fermented milk (liquid type yogurt, curd type yogurt), fermented food (Kimchii, soy and bean paste), animal feed, health supplementary food and the like.

Food compositions containing the strains according to the present invention as an effective ingredient include animal feed, fermented food such as various kinds of Kimchii, soy and bean pastes and fermented dairy products such as yogurt and cheese and can be effectively expected to prevent cancers, to enhance immunity and to reduce body fat.

Pharmaceutical compositions containing the strains according to the present invention as effective ingredients can be used in the prevention and treatment of

diseases inhibited by CLA, such as cancers, artery sclerosis, diabetes and obesity.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 is a view showing the result of a HPLC assay to confirm the element composition of fatty acids produced by the strains according to the present invention; Fig. 2 is a view showing the growth conditions of the strains according to the present invention in a medium with linoleic acid (LA) added, the CLA production and the change in pH of the medium; Fig. 3 is a view showing the growth conditions of the strains according to the present invention in a medium without linoleic acid (LA) and the CLA production; Fig. 4 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium with linoleic acid (LA) added; Fig. 5 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium without linoleic acid (LA); and Fig. 6 is a view showing the result of measuring the growth level (a) and CLA production (b) of the strains according to the present invention which have been cultured in media, to which glucose, fructose, lactose and sucrose, respectively, were added as a carbon source.

Examples Now, the present invention will be explained in further detail by the following examples. However, the present invention is not limited thereto.

Example 1: Isolation, identification and characterization of strain 1) Isolation and identification of inventive strains Feces of infants were collected to isolate the strains according to the present invention.

In order to isolate microorganisms which can produce CLA, 10 types of feces were taken from infants of breast feeding, weaning feeding and combined feeding and were inoculated into the MRS medium with sterilized liquid paraffin embedded therein. The medium was diluted with sterilized physiological saline (0.5%, w/v) so that about 30 to 150 colonies could be formed, plated on the MRS (Man Rogosa Sharpe) medium with 0.05% of L-cysteine, and incubated in an anaerobic tank along with a Gas pack (MGC, mitsubishi) at 37 C for 72 hours. At this time, LA was well emulsified in Tween 80 (0.5 %, w/v), filtered through a cotton filter and added to the MRS medium. In order to establish the anaerobic conditions during cultivation, the sterilized culture vessel was filled with the MRS medium without any room.

About 300 colonies were randomly selected from the cultured medium.

Each was passage cultured two times in the MRS medium and inoculated in a 20 in, test tube at a level of 1%, followed by cultivation for 48 hours. The culture fluid was extracted with hexane and the extract was measured for absorbance at 233 nm to determine the amount of produced CLA, which was then corrected by the amount of strain, that is, the absorbance at 600 nm to compare the relative amount of produced

CLA.

The measurement of the live strain number was performed by plate-culturing in the MRS agar medium by 10 times dilution, placing the medium in an anaerobic tank (Difco, USA) with a gas pack for 48 hours and thereafter, counting the number of produced colonies.

Thus, three strains which were excellent in the CLA productivity were selected and designated CBG-C2, CBG-C4 and CBG-C5, respectively. The strains were identified for their genus and species by analysis of their 16S rRNA sequence.

As a result, it was proved that the CBG-C2 strain belongs to Bifidobacteriurn breve, the CBG-C4 strain belongs to Bifidobacteriuna pseudocartenulatum and the CBG-C5 strain belongs to Ente7 0coccus faecium.

The strains were deposited at Korean Agricultural Culture Collection in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001 (CBG-C2), KACC 91003 (CBG-C4) and KACC 91002 (CBG-C5), respectively, on April 3,2002. Also, CBG-C2 (KCTC 10462BP) and CBG- C4 (KCTC 10208BP) were deposited at the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology on March 25, 2002 and April 10,2003, respectively.

2) Confirmation of composition of inventive strains In order to confirm that the strains according to the present invention can produce CLA by examining the composition of fatty acids produced by the strains, the following GC was performed.

Each strain was cultured in a medium containing LA (500 ßg/lDt) for 48 hours and centrifuged.

The strain was suspended in a medium or distilled water, mixed with isopropyl alcohol in a volume of twice the volume of the strain and the vigorously stirred. Then, a 1.5 volume of hexane was added and thoroughly mixed for 3 minutes by shaking.

The resulting mixture was centrifuged at 3000 rpm for 5 minutes and the supernatant was measured for absorbance at 233 nm. The extracted fatty acids were methylesterified according to the method described in American Oil Chemists's Society: Official Method and Recommended Practoces pf AOCS, 4th. ed. (1989) to form a sample for the GC analysis.

The conditions of GC were as follows: the GC DS-6200 (DONAM) with FID attached was used, the colurnn was HP-FFAP capillary column (30mx0. 25mm, thickness 0. 25film), the oven temperature was 210 C, the injector temperature was 250C and the detector temperature was 270 C. The carrier gas was helium and eluted at a flow rate of 1 mt/min, and the split ratio was 50 : 1. The peak areas were determined using an integrating meter (Model 3390A, Hewlett-packard, USA) equipped to the apparatus. The identification of CLA was performed through comparison with the retention time of a reference material and the content of CLA was determined by the ratio of the area of CLA to the area of a reference material.

The results are shown in Fig. 1.

As shown in Fig. 1, all the strains showed LA peaks and CLA peaks.

Therefore, it was noted that the strains according to the present invention could produce CLA using LA as a substrate.

3) Determination of optimal condition for CLA production by inventive strains

In order to determine the optimal conditions for CLA production by the strains according to the present invention, the strains were cultured under two conditions, in which one is in a medium with LA and the other is in a medium without LA, and characterized for their growth.

Firstly, each strain which had been activated by pre-cultivation was inoculated at 1% into 20 of the MRS medium containing LA (500 gg/i-n)-Every hour, the medium was collected and measured for the strain level, amount of CLA and pH. The results are shown in Fig. 2.

As shown in Fig. 2, the strains showed increase in the amount of produced CLA when entering to the log phase and produced the highest amount of CLA just prior to the stationary phase. The pHs of the media were about 6.5 at the initial stage and drops as the time went on, whereby it was about 4.2 at about 50 hours.

Meanwhile, each strain which had been activated by pre-cultivation was inoculated at 1% into the MRS medium without LA, dispensed in a 20 mE test tube and cultured. During cultivation, a growth curve was made and from 18 hours after beginning the cultivation, every 6 to 12 hours, the pH of the medium was measured.

The strain was collected and suspended with a Tris-HCl buffer solution in a volume of 10 times the volume of the strain and subjected to an enzyme reaction to examine the change in the factor of CLA isomerase according to the change of the growth curve. Here, LA was added to the enzyme reaction at a concentration of 100 ug/in.

The results are shown in Fig. 3.

As shown in Fig. 3, it took more time to reach the stationary phase when culturing in the medium without LA than in the medium with LA. The final pH of the medium was about 4.2.

Therefore, it was noted that the strains according to the present invention

could produce the greatest amount of CLA just before they enter from the log phase to stationary phase.

Also, the results obtained from the case where the strains were cultured in media containing and the results obtained from the case where the strains were cultured in media without containing LA are useful to understand the growth stage of the strains showing the highest growth factor per unit strain in development of products by fermentation of microorganisms in the presence of a substrate and in indirect production CLA by adding the strains as an effective ingredient, respectively.

4) Confirmation of amount of CLA distributed in medium or inventive strain upon cultivation of inventive strains In order to examine amounts of CLA distributed in a medium and the strain when the strains according to the present invention were cultured, the following experiments were performed.

Firstly, each strain was inoculated into a medium containing LA and measured for the strain level and CLA production at 18,24, 36 and 48 hours. The results are shown in Fig. 4.

As shown in Fig. 4, at the early stage of cultivation, that is, 18 hours later, the ratio of an amount of CLA in a strain to an amount of CLA in a medium was 1: 1.85 for CBG-C2,1 : 2.1 for CBG-C4 and 1: 1.54 for CBG-C5. As the time went on, the amount of CLA in the medium increased. 48 hours later, the ratio was 1: 3.5 for CBG-C2,1 : 2.9 for CBG-C4 and 1: 2 for CBG-C5, as shown in Table 1.

<Table 1> Productivity CLA Distribution CLA (, Yg)/dry cell conversion conversion (%) (%) weight (mg) supernatant 213. 68 54. 7 78 pellet 59.97 22 supernatant 145.96 75 CBG-C4 38.8 pellet 48.20 25 supernatant 126.72 67 pellet 61. 70 33

Meanwhile, each strain was inoculated into a medium without LA and cultured for 18,24, 36 and 48 hours. After cultivation, the strain was collected mixed with a buffer solution containing LA. The mixture solution was left for 1 hour for reaction and measured for the distribution of produced CLA. The results are shown in Fig. 5.

As shown in Fig. 5, as the cultivation progressed, the amount of CLA increased. After 36 hours, the distribution of CLA produced by the strain for 1 hour was 1.3 : 1,1. 6: 1 and 0.6 : 1 in a ratio of strain to medium for CBG-C2, CBG-C4 and CBG-C5, respectively, as shown in Table 2.

<Table 2> Productivity CLA Distribution CLA (, ug)/dry cell conversion (%) (%) weight (mg) supernatant 63.20 43 CBG-C2 100 pellet 82.90 57 supernatant 49.21 39 CBG-C4 80 pellet 75.02 61 CBG-C5 supernatant 38. 6 52. 53 60 pellet 25. 37 40

Therefore, it was noted that the strains according to the present invention can secret the produced CLA to a medium while accumulating in their body.

Such properties of the strains are useful to be added as an effective ingredient to food and medicaments for indirect production of CLA.

5) Growth of inventive strains and CLA production according to the type of carbon source In order to examine the growth of the strains and their CLA production according to the type of a carbon source, glucose, the strains were cultured in media containing various carbon sources including fructose, lactose and sucrose and the growth level and CLA production were measured. The results are shown in Fig. 6.

As shown in Fig. 6a, when the medium contains LA, a carbon source which is the most suitable for each strain, is glucose for CBG-C2 and lactose and sucrose for CBG-C4, and when the medium does not contain LA, it is lactose for CBG-C2 and lactose and sucrose for CBG-C4.

Meanwhile, after cultivation in the medium with LA for 48 hours, CBG-C2 and CBG-C4 had the highest CLA production when using glucose as a carbon source while CBG-C5 was not affected by the carbon source.

6) Verification of resistance of inventive strains to pH, bile salt and stomach acid The pH resistance, bile salt resistance and antibiotic resistance of the strains according to the present invention were confirmed as follows.

In order to examine the use of the novel strains which had been isolated and

identified according to the present invention in application to products fermented by inoculating the CLA producing lactic acid bacteria or additives using CLA contained in the strains, growth features of the strains were compared.

1) Resistance to pH In order to confirm the pH resistance of the strains according to the present invention, the strains were cultured in a medium containing LA and a medium without containing LA and the change of each medium in pH was observed. The results are shown in Fig. 2 and Fig. 3.

As shown in Fig. 2 and Fig. 3, as the growth of the strains come into the stationary phase whether the medium contains LA or not, the pH of the medium was dramatically reduced. It was found that the final pH dropped to pH 4.2. Therefore, it was noted that typical fermentation by lactic acid bacteria occurs in the medium.

Based on the above results, various MRS media having pH in the range of 2 to 6 were prepared to confirm the growth of the strains according to the pH change of the medium. Each strain was inoculated into a media and cultured at 37 C for 48 hours while the medium was observed to know whether the strain was growing and propagating. The results are shown in Table 3.

<Table 3> pH2 pH3 pH4 pH5 pH6 CBG-C2-+ + + ++ CBG-C4-+ + ++ ++ CBG-C5-+ + + ++ (- : no propagation, +: a little propagation, ++: abundant propagation) As seen from Table 3, the strain according to the present invention could

grow and propagate at pH in the range of 3.0 to 6.0 and particularly, the CBG-C4 strain showed the most excellent pH resistance.

Therefore, it was noted that the strains according to the present invention can grow and propagate in a wide range from weak alkalinity to weak acidity, particularly in the stomach under acidic conditions resulting from the secretion of stomach acid.

2) Resistance to bile In order to confirm the bile resistance of the strains according to the present invention, various MRS media containing sodium deoxycholate, a component of the bile, at concentrations of 0,100, 300,500, 800 and 1000 µg/ml were prepared.

Each medium inoculated with the strains and cultured at 37°C for 48 hours while the growth of the strains was examined. Bacillus subtilis which does not show resistance to the bile was used as control. The results are shown in Table 4.

<Table 4> CGB-C2 CGB-C4 CGB-C5 Bacillus subtilis O, ug/mQ ++ ++ ++ ++ 100 g/m ++ ++ ++- 300 ug/mQ ++ ++ ++- 500 µg/ml ++ ++ ++- 800, ug/mQ ++ ++ ++- 1000 ug/in, + ++ +- (- : no propagation, +: a little propagation, ++: abundant propagation) As seen from Table 4, the strains could satisfactorily grow and propagate to the bile concentration of 1000 µg/ml, whereas the control of Bacillus subtilis could no grow even at 100 µg/ml.

Therefore, it was noted that the strain according to the present invention could stably grow and propagate in the gastrointestinal tract where the bile is secreted.

3) Resistance to antibiotics In order to confirm the resistance to antibiotics of the strains according to the present invention, the strains which had been cultured overnight in a 20 1111 test tube were diluted in the MRS medium to form a solid medium. Lactobacillus reuteri was used as control.

Antibiotics used in this experiment were ampicillin, tetracycline, streptomycin, rifamycin and kanamycin, each of which was prepared at concentrations of 50 µg/ml, 1000 µg/ml and 10000 µg/ml.

Each of the antibiotics at different concentrations was dropped on paper discs in an amount of 40, ut, followed by drying. Each disc was placed on the medium with the strains mixed and cultured at 37 C for 48 hours while examining the growth of the strains. The results are shown in Table 5.

<Table 5> Antibiotic Lactobacillu Antibiotics concentration CBG-C2 CBG-C4 CBG-C5 s reuteri (µg/ml) 50 Ampicillin 1000 10000 50--+ + Tetracycline 1000--+ 10000--+ 50 + + + + Streptomycin 1000 10000 50 - + - - Rifamycin 1000 - - - - 10000 - - - - 50 + + + + Kanamycin 1000 - + + - 10000

As seen from Table 5, the strains according to the present invention showed resistance to total 2 or 3 antibiotics. Particularly, CBG-C4 shows most excellent resistance to antibiotics at high levels, such as kanamycin at 1000 µg/ml and tetracycline at 10000 µg/ml.

Therefore, it was noted that the strains according to the present invention have resistance to various kinds of antibiotics at a wide range of concentration.

Example 2 Preparation of capsules and dairy products containing strains and CLA Capsules and dairy products containing the strains according to the present

invention were prepared as follows.

In order to prepare a coating material for microencapsulation of conjugated linoleic acid, a mixture comprising 4 types of vegetable polysaccharides, gellan, xanthan, starch and agar, in a concentration of 1 to 5% (w/v) was prepared.

Sorbitan monostearate having a HLB (hydrophilic lipophilic balance) value of 4.7 as an emulsifying agent was added to the mixture, heated at 60 C so that it was completely dissolved, sterilized by heating and cooled down to 40 C to form an aqueous mixture solution. At this time, sorbitan monostearate was treated at a concentration of 0. 01 to 1% (w/v) and the coating material and the strain was mixed in a ratio of 7 : 3 (w/w) and homogenized.

The homogenized strain suspension was treated with cooled water at 10 C by spraying to form microcapsules.

The microcapsules were added in an amount of 1% (w/v) to prepare Kimchii which is one of the low-temperature fermented products. In preparation of various dairy products, for milk, soy milk, liquid type yogurt, the microcapsules were added in an amount of 4.2% (w/v) and for curd type yogurt and fermented soy rnilk, the microcapsules were added in an amount of 7.3% (w/v). Each product was stored in a refrigerator kept at a low temperature of 4 C or 10 C.

The dairy products prepared as described above were subjected to an experiment to examine the preservation of microcapsules. As a result, it was shown that when stored in a refrigerator at 4 C and 10 C for 7 to 14 days, strains were reduced by about 10l~2 strains/in. Such reduction rate is very insignificant, judging from a common basis.

Therefore, it was noted that the capsules according to the present invention could stably maintain the number of strains even when stored for a long period of

time and have excellent storage stability.

Industrial Applicability The strains according to the present invention can produce CLA from LA at a high efficiency and show outstanding resistance to stomach acids, bile salts, and antibiotics.

Also, the strains according to the present invention can accumulate CLA in their body after production and thus have effect to make CLA indirectly produced.

Therefore, a composition comprising the strain according to the present invention may be prepared in the form of a capsule comprising the strain according to the present invention in a coating material comprising a water-soluble polysaccharide and be usefully used in functional food and medicaments.

Further, the strains according to the present invention can be effectively used to biosynthesize not only CLA but also isomers of CLA in large quantities by a biological method such as immobilization.

PCT Original (for SUBMISSION) - printed on 12. 04.2003 12:11 : 37 PM 0-1 Form-PCT/RO/134 (EASY) indications Relating to Deposited Microorganism (s) or Other Biological Material (PCT Rule 13bis) 01-1 Prepared using PCT-EASY Version 2. 92 (updated 01. 01. 2003) 0-2 International Application No. 0-3 Applicants or agent's file reference 03PP030 1 The indications made below relate to the deposited microorganism (s) or other biological material referred to in the description on : 1-1 page 12 1-2 line 1-3 Identification of Deposit- 1-3-1 Name of depositary institution Korean Collection for Type Cultur es 1-3-2 Address of depositary institution 52, Oun-dong, Yusong-Ku, Taejon 3 O 5-3 3 3, Republic of Korea 1-3-3 Date of deposit 10 April 2003 (10. 04. 2003) 1-3-4 Accession Number KCTC 10462BP 14 Additional Indications NONE 1-s Designated States for Which all designated States Indications are Made 1-6 Separate Furnishing of Indications NONE These indications will be submitted to the Intemational Bureau later 2 The indications made below relate to the deposited microorganism (s) or other biological materiat referred to in the description on : 12 21 line 12 2-2 line : L2 2-3 Identification of Deposit 2-3-1 Name of depositary institution Korean Collection for Type Cultures 2-3-. 2 Address of depositary institution 52, Oun-dong, Yusong-K-u, Taejon. 3 05-333 Republic of Korea 2-3-3 Dateofdepos ! t 10 April 2003 (10. 04. 2003) 2-3-4 Accession Number KCTC 1020 8BP 2-4 Additional Indications NONE 2=s-Designated States tor Which all designated States Indications are Made 2-6 Separate Furnishing of Indications NONE These indications will be submitted to the International Bureau later PCT Original (for SUBMISSION) - printed on 12.04. 2003 12 : 11 : 37 PM FOR RECEIVING OFFICE USE ONLY 0-4 This form was received with the international application : (yes or no) 0-4-1 Authorised officer *" __ FOR INTERNATIONAL BUREAU USE ONLY 0-5 This form was received by the international Bureau on : 0-5-1 Authorized officer