WO2005001068A1

WO2005001068A1 - A BIFIDOBACTERIUM BREVE LMC520 STRAIN CONTAINING A PLASMID Pbc520, A METHOD FOR PREPARATION OF CONJUGATED FATTY ACIDS AND FERMENTED MILKS CONTAINING SUCH FATTY ACIDS USING THE SAME STRAIN, AND USE OF A PLASMID pBC520

Info

Publication number: WO2005001068A1
Application number: PCT/KR2004/001571
Authority: WO
Inventors: Chil-Surk Yoon; Soo-Hyun Chung; Kwang-Won Hong; In-Hwan Kim; Jae-Hong Jeong
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-06-28
Filing date: 2004-06-28
Publication date: 2005-01-06
Anticipated expiration: 2005-12-28
Also published as: KR100515850B1; KR20050001981A

Abstract

Disclosed is a novel Bifidobacterium breve strain LMC520 carrying a cryptic plasmid having an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and convert with high efficiency the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol containing the produced fatty acid. In addition, the present invention discloses the use of the cryptic plasmid, a method of producing a fatty acid with a conjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid by culturing the B. breve LMC520 under anaerobic or aerobic conditions, and a method of preparing a fermented milk containing the fatty acid or the acylglycerol.

Description

DESCRIPTION

A B±f±d bacter±υm breve LMC520 STRAIN CONTAINING A PLASMID pBC520, A METHOD FOR PREPARATION OF CONJUGATED FATTY ACIDS AND FERMENTED MILKS CONTAINING SUCH FATTY ACIDS USING THE SAME STRAIN, AND USE OF A PLASMID pBC520

Technical Field The present invention relates to a novel Bifidobacterium breve LMC520 carrying a cryptic plasmid having an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and convert with high efficiency the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol containing the produced fatty acid. In addition, the present invention relates to a method of producing a fatty acid with a conjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid by culturing the B. breve - LMC520 under anaerobic or aerobic conditions, and a method of preparing fermented milk containing the fatty acid or the acylglycerol .

Background Art Conjugated linoleic acids (hereinafter, referred to simply as "CLA") are a representative group of fatty acids with conjugated double bonds. CLA is known to have a range of physiological activities. These include beneficial effects on preventing and alleviating atherosclerosis, reducing blood levels of cholesterol, LDL-cholesterol and triglyceride (TG) , reducing body fat accretion, and treating diabetes. In particular, studies with experimental animals revealed that CLA effectively inhibits the incidence of cancer by the exposure to carcinogens . in the large intestine, the stomach, the breast and the skin [Ha, Y. L., et al., Cancer Research 50:1097-1101 (1990); Ip, C, et al., Lipids. 51:6118-6124 (1991); Belury, M.A. Nutr. Rev. 53:83-89 (1995)]. In addition, CLA intake in humans resulted in a reduction in body fat accretion [Blankson, H., et al., J. of Nutrition. 130:2943-2948 (2000)]. Linoleic acid is an 18 carbon fatty acid with two double bonds in cis-9 and cis-12 configuration. CLA represents a mixture of positional and geometric isomers of linoleic acid with conjugated double bonds in either cis or trans configuration. The double bonds of CLA may be in the cis and/or trans configurations in the positions of 8,10; 9,11; 10,12; 11,13; and 12,14. Among the CLA isomers, cis-9, trans-ll octadecadienoic acid and trar-s-10, cis-12 octadecadienoic acid are known to have nutritional and physiological effects. CLA is naturally contained in foods at trace levels, and, in particular, present in fermented milk products and meat from ruminant animals [Fogerty, A.C., et al., Nutrition

Reports International. 38:937-944 (1988); Lin, T.Y. et al., Scientfic Agriculture. 45:284-295 (1997)]. Among CLA isomers naturally present in foods, especially dairy products derived from ruminant animals, cis- 9, tra.ns-11 octadecadienoic acid represents 85-95% of the total CLA. In contrast, products produced by monogastric animals, for example, chicken eggs, chicken meat, pork, quail eggs and duck meat, rarely contain CLA, or contain CLA in trace amounts less than one-tenth of CLA levels in milk and beef. This is because, in ruminant animals, CLA is produced as intermediates in the hydrogenation of a dietary unsaturated fatty acid, linoleic acid, by rumen bacteria, such as Butyrivibrio fibrisolvens, but monogastric animals do not have the bacteria and digestive organs serving this function. Although ruminant animals produce CLA, since meat, milk and fermented milk products, such as yogurts, derived from ruminants typically contain CLA in trace amounts (0.55 to 9.12 mg/g of fat), it is unrealistic to expect the excellent physiological benefits of CLA from the intake of the traditional dairy products . Experimental studies with animals demonstrate that CLA effectively reduces the number of tumor cells in breast cancer when CLA intake is higher than 0.1% [Ip, C, et al., Cancer Research 54:1212-1215 (1994)]. Therefore, to expect the anticarcinogenic properties of CLA in humans, in addition to the conventional meat and milk products, dietary supplements with high contents of CLA need to be ingested. Currently, in America, Europe and other countries, chemically synthesized CLA is marketed as health food, which can be prepared by isomerization of linoleic acid under alkaline conditions using catalysts [American Oil Chemists' Society Official Method Cd 7-58, pages 1-11, American Oil Chemists' Society, Champaign, III. (1973)]. However, the CLA synthesized by the alkaline isomerization method is disadvantageous because it is chemically synthesized and contains residual materials derived from chemical compounds used in its synthesis and high levels of oxidative products of fat. In this regard, there is an increasing interest in the production of natural CLA, such as CLA present in meat, milk and fermented milk products. After natural CLA was known to be generated as intermediates in the hydrogenation of linoleic acid by the obligate anaerobic bacterium Butyrivibrio fibrisolvens derived from ruminants, interest in .the production of natural

CLA by bacteria increased. However, the employment of CLA- producing anaerobic bacteria is problematic in that the bacteria are difficult to culture in vitro since the bacteria require stringent anaerobic conditions for growth. The problems further include that produced CLA is often converted to other compounds, and some of the bacteria are pathogenic. Thus, these bacteria are currently not utilized in industrial fields associated with the production of natural CLA. U.S. Pat. Nos. 5,856,149 and 6,060,304 employ a Lactobacillus strain, Lactobacillus reuteri PYR8 (ATCC55739) . However, this Lactobacillus strain has limited industrial

^■ applications for the following reasons: it requires a stringent anaerobic environment for growth, utilizes linoleic acid only in a free fatty acid form as a substrate for CLA production, and has an optimal growth pH of higher than 7.5, which is not suitable for commercial production of fermented milk products (pH 4.2-4.5), such as yogurts. In addition, methods of preparing fermented milks •using lactic acid bacteria are disclosed in Korean Pat. No. 10-0337377 and Korean Pat. Laid-open Publication No. 2001- 0089858. Final fermented milk products prepared using Lactobacillus reuteri ATCC 55739 in ^'the Korean patent and Bifidobacterium longum ATCC 15707 in the Korean Laid-open Publication contained CLA of 160-400 ppm and 3.6-8.5 mg/g of fat, respectively. These CLA contents are only about 1.5 to 2.7-fold higher than the CLA contents of 4.1-14.8 mg/100 ml (or 2.3-5.4 mg/g of fat) of currently available yogurts. These increase rates in CLA contents are considered to be very small with regard to commercial production of yogurts exerting the beneficial physiological effects of CLA. This is because the CLA-producing lactic acid bacteria used in the above-mentioned patent and laid-open publication convert linoleic acid as a CLA precursor to CLA with very low efficiency. In addition, Lin et al. reported that lactic acid bacteria with a CLA-producing capacity convert linoleic acid to CLA with about 10% efficiency [T.Y. Lin, et al., Food Chemistry 67:1-5 (1999)]. A recent report by M. Coakley et al. reveled that Bifidobacterium breve and B. dentium have a high efficiency in CLA production, and that B. breve converts linoleic acid to CLA with 65% efficiency [M. Coakley, et al., J. of Applied Microbiology 94:138-145 (2003)]. However, since the B. breve is not a microorganism directly isolated from the human intestine, it is problematic in terms of causing toxicity in the human body. In addition, Korean Laid-open Publication No. 10-

2003-0002688 applied by the present inventors discloses a novel microorganism with a maximum 95% CLA production efficiency, Bifidobacterium breve LMC7, which was deposited at the Korean Collection for Type Cultures (KCTC) in the Korean Research Institute of Bioscience and Biotechnology (KRIBB) under an accession number KCTC 1017 BP. Bifidobacteria are predominant in the intestinal tracts of breast-fed infants. In Korea, to obtain the physiological effects of bifidobacteria, fermented milk products, such as yogurts, are supplemented with bifidobacteria. However, Streptococcus thermophilus and Lactobacillus acidophilus play a major role in yogurt fermentation. These two bacteria ferment lactose into lactic acid while growing together in milk, thereby lowering a natural pH of yogurts to about 4.2 to 4.5. The yogurts prepared in this way have nutritional and physiological benefits, as well as excellent quality features according to measures of fermented milk products, such as soft tastes and flavor. Typically, S. thermophilus is good at fermenting milk to soft products but poor at producing lactic acid. In contrast, the Lactobacillus strain has a potent ability to produce lactic acid, and thus, lowers a pH value of fermented products to about 4.2 and provides unique flavor to yogurts . Thus, on industrial scales, yogurts are prepared for 6 to 10 hours by starter culture using a mixture of a Lactobacillus strain, a bifidobacterial strain and S. thermophilus at a proper ratio. In case of the Bifidobacterium breve LMC7 (KCTC 1017 BP) disclosed in the Korean Laid-open Publication No. 10- 2003-0002688 applied by the present inventors, yogurt preparation was achieved using a mono-culture of the bacterium. However, . there are problems in yogurt preparation with the B. breve LMC7, as follows. Fermentation is performed for 24 to 48 hours, which is a longer than the typical yogurt fermentation time. When natural CLA-containing yogurts are to be prepared, the use of a CLA precursor (linoleic acid or monolinolein) in an amount of higher than 0.1% results in a large reduction in the CLA production efficiency of the B. breve LMC7 and incomplete fermentation. In this case, yogurts with 0.1% or higher CLA contents cannot be produced within the typical yogurt fermentation time. In addition, when continuously cultured by sub-culturing, the B. breve LMC7 becomes reduced in CLA synthesis efficiency, and this decrease in CLA synthesis is difficult to restore. Therefore, the present inventors made efforts to find novel bifidobacteria which are capable of shortening the long yogurt fermentation time to the general yogurt fermentation time, that is, within 6 to 10 hours, converting a substrate for CLA production to CLA with a high efficiency within the typical yogurt fermentation time, and maintaining its CLA synthesis efficiency during sub-culturing. Leading to the present invention, the above-mentioned efforts to overcome the limitations encountered in the prior art, made by the present inventors, resulted in successful isolation of a novel Bifidobacterium breve strain from Korean people. The novel -B...breve strain efficiently converts a CLA precursor to a compound having a conjugated double bond structure, is capable of rapidly performing fermentation for preparation of fermented milk products even when used in starter culture with a CLA precursor in a mixed form with Lactobacillus acidophilus and Streptococcus thermophilus, which are conventionally used as starter bacteria in industrial yogurt production, and possesses a cryptic plasmid serving an essential role in CLA synthesis.

Disclosure Technical Solution It is therefore an object of the present invention to provide a novel Bifidobacterium sp. strain carrying a cryptic plasmid which has an ability to use as a substrate a fatty acid with unconjugated double bonds at its carbon chain or an acylglycerol containing the fatty acid, and convert with high efficiency the substrate to a fatty acid with conjugated double bonds at its carbon chain or an acylglycerol containing the produced fatty acid. It is another object of the present invention to provide a method of producing a fatty acid with a conjugated double bond structure or an acylglycerol containing the fatty acid by using the novel bacterial strain and the plasmid. - It is a further object of the present invention to provide a method of preparing a fermented milk product with improved functionality by culturing the novel bacterial strain alone or in a mixed form with Lactobacillus acidophilus and Streptococcus thermophilus . It is yet another object of the present invention to provide the use of the plasmid according to the present invention, which has an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and converts with high efficiency the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol -containing the produced fatty acid, in transformation of a bacterium not carrying the plasmid and other bacteria not having CLA production ability to possess an ability to produce CLA.

Description of Drawings FIG. 1 shows results of gas chromatography for CLA and other fatty acids, which are produced by the B. breve LMC520 of the present invention grown in a MRS medium containing linoleic acid; FIG. 2 shows results of electrophoresis analysis of PCR products obtained by PCR performed using a PCR mixture containing a template DNA isolated from the B. breve LMC520 of the present invention and a pair of primers for analysis of 16S rRNA of-the B. breve LMC520 (M: Hinc-III-digested- λ DNA molecular size marker; Pbi, BiADO, BiANG, BiBIF, BiBRE, BiCATg and BiLONg: primer sets for amplification of 16S rRNA) ; FIG. 3 is a photograph showing results of electrophoresis for the size and digested patterns with the restriction enzymes of plasmid DNA isolated from the present Bifidobacterium sp. strain (lane C: undigested pBC520 plasmid DNA as a control; lanes 1 to 9: linearized pBC520 plasmid DNA by digestion respectively with Xhol, Sail, C-Zal, Hindlll, EcoRI , Pstl, BamHI , SacII and Accl ; M:

Hindlll-digested λ DNA molecular size marker) ; FIG. 4 shows electrophoresis results showing that a pBC520 plasmid is substantially removed from the B. breve LMC520 of the present invention when plasmid replication is inhibited by curing, wherein upper bands represent bacterial chromosomes, and lower bands represent the pBC520 plamsid DNA (M: ff±ndlll-digested λ DNA molecular size marker; 1: intact B. breve LMC520, 2: cured B. breve LMC520-1; 3: another cured B. breve LMC520-2) ; FIG. 5 shows a nucleotide sequence of a pBC520 plasmid DNA of the B. breve LMC520 of the present invention; FIG. 6 is a graph showing changes in viable cell number when the B. breve LMC520 of the present invention is cultured in a complex medium containing fat milk and nonfat milk powder; FIG. 7 is a graph showing changes in levels of fatty acids with conjugated double bonds when the B. breve LMC520 of the present invention is cultured in a complex medium containing fat milk and non-fat milk powder; FIG. 8 is a graph showing changes in CLA production ability of the B. breve LMC520 of the present invention when the LMC520 strain is sub-cultured; FIG. 9 is a graph showing contents of fatty acids with conjugated double bonds in a milk medium according to 5 a substrate added when the B. breve LMC520 of the present invention is cultured in the milk medium (LA: linoleic acid; ML: monolinolein; DL: d-ilinolein; 50% MG: 50% monoglyceride-containing safflower oil; 90% MG: 90% monoglyceride-containing safflower oil) ; and 10 FIG. 10 is a graph showing contents of fatty acids with conjugated double bonds in yogurts upon fermentation of the B. breve LMC520 of the present invention alone and in a mixed form with other lactic acid bacteria (B: yogurt prepared by single fermentation with the B. breve LMC520;

15 A: yogurt prepared by single fermentation with L. acidophilus; T: yogurt prepared by single fermentation with S. thermophilus; AT: yogurt prepared by co-fermentation with S. thermophilus and L. acidophilus; BT: yogurt prepared by co-fermentation with the B. breve LMC520 and S.

20 thermophilus; AB: yogurt prepared by co-fermentation with •^•» the B. breve LMC520 and L. acidophilus; ABT: yogurt prepared by co-fermentation with the B. breve LMC520, S. thermophilus and L. acidophilus) .

Best Mode

25 The terms as used herein will be defined in brief, as follows . The term "plasmid", as used herein, has the meaning common in the art, 'that is, refers to a circular non- chromosomal element present in bacteria. Plasmid preparation, digestion and ligation of plasmid DNA, plasmid transformation, and the like may be achieved by methods well known to those skilled in the art. -The methods are described, for example, in a guidebook, ^Molecular Cloning: A Laboratory Manual, Second Edition' [Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989) ] . The term "cryptic plasmid", as used herein, refers to an extrachromosomal element that is usually smaller and is maintained at higher copy numbers in a single cell, than general plasmids . However, although the cryptic plasmids are usually small, they are not always smaller than the general plasmids. The general plasmids range from several to tens of killobases in length. In contrast, the cryptic plasmids are several killobases long. Also, the cryptic plasmids are not always present at higher copy numbers than the general plasmids. However, the general plasmids are present at several to tens of copy numbejrs, whereas the cryptic plasmids may be present at tens to hundreds of copy numbers. These small plasmids have been described in E. coli, Shigella sonnei, Salmonella enteritidis, Salmonella enterica, Neisseria ghonorrhoeae, Staphylococcus aureus, Lactobacilli, and the like. Typically, the cryptic plasmids are not essential for the general growth of bacteria, and provide particular specificity to bacteria containing the cryptic plasmids . In the present invention, it is to be understood that the term "cryptic plasmid" means a novel pBC520 plasmid according to the present invention, which has an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and converts the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol containing the produced fatty acid. The term "curing", as used herein, has the following meaning. Plasmids are generally stable in cells, but are unstable under some unfavorable conditions . Under unfavorable conditions, plasmids disappear in bacterial hosts during the bacterial growth, and this phenomenon is expressed as "curing". Agents causing curing include plasmid replication interrupters, such as acridine orange, acriflavin and ethidium bromide, and DNA synthesis inhibitors, such as mitomycin C. In the present invention, ethidium bromide is used to cause curing of a novel cryptic plasimd of the present invention by interrupting plasmid replication in order to evaluate CLA production ability of the cryptic plasmid of the present invention. In addition to the method employing plasmid replication interrupting agents, curing is caused by exposing bacteria to very poor environment in which normal bacterial growth is impossible. When bacteria are exposed to environments making even chromosomal replication difficult, they are devoted entirely to replicating chromosomes, and thus, plasmid replication does not occur. Eventually, plasmids are removed during sub-culturing. It is well known in the art that these plasmid removals are used as a tool to evaluate the functions of the plasmids removed from bacteria. The term "lactic acid bacteria", as used herein, refer to bacteria that decompose glucose or lactose to lactic acid or acetic acid. Lactic acid bacteria commonly used in the preparation of fermented milk products include the genus Lactobacillus, the genus Streptococcus and the genus Bifidobacterium. Examples of the genus Lactobacillus include L. bulgaricus, L. casei and L. acidophilus. Examples of the genus Streptococcus include S. thermophilus. The genus Bifidobacterium is evolutionally closer to the genus Actinomycetes, but is treated as lactic acid bacteria because it produces lactic acid and has beneficial effects on the body. Lactic acid bacteria inhabit the intestinal tract of various animals, and, in the gastrointestinal tract, protect mucous membranes, improve abnormal fermentation in the intestine, stimulate calcium absorption by the body, and the like. By virtue of these beneficial physiological effects, lactic acid bacteria are used as medicines, for example, for treating intestinal disorders, and feed additives . Lactic acid bacteria are characterized by the following properties: lactose metabolizing ability to convert lactose in milk into lactic acid; protein degradation ability to degrade milk proteins to peptides, absorb the peptides and degrade ^■ the absorbed peptides to amino acids; food preservation ability by production of lactic acid and acetic acid; and ability to produce antimicrobial agents including hydroperoxide, diacetyl and bacteriocin. The term "fermented milk", as used herein, has a general meaning and refers to milk obtained by fermenting raw milk or milk products by lactic acid bacteria, yeast, and the like. The fermented milk varies depending on types and raw materials thereof, solid contents, microorganism types, and production places. In Korea, the fermented milks are greatly classified into liquid forms and concentrated forms according to the content of milk solids non-fat. When the content of non-fat milk solids is higher than 3.0%, the fermented milks are categorized .into the liquid fermented milk. When the content of non-fat milk solids is higher than 8.0%, the fermented milks are categorized into the concentrated fermented milk. In addition, the concentrated fermented milk products are sub-grouped into plain yogurts containing fruit pieces and typically eaten with a spoon and drink yogurts containing fruit juice and thus being drinkable. The term "linoleic acid", as used herein, refers to a fatty acid molecule that is composed of 18 carbons having two double bonds in cis configuration at positions 9 and 12. The term "conjugated linoleic acid (CLA)", as used herein, is a general term for positional and geometric isomers of linoleic acid with conjugated double bonds in cis and trans configurations in the positions 9 and 11, and 10 and 12. The term "transformation", as used herein, refers to a method of introducing a gene having a specific genetic property into a host cell. In the present invention, the term "transformation" is intended to mean a method in which the cryptic plasmid of the present invention is manipulated by genetic recombination so that it becomes replicable and expressible in lactic acid bacteria, such as Lactobacillus or Streptococcus, and bifidobacteria, and is introduced into such a bacterium to provide CLA production ability thereto . A recombinant plasmid used in the transformation method according to the present invention may be prepared by methods known in the art. A plasmid genetically manipulated to express an exogenous gene comprises a replication origin for replication in bacteria to essentially express the exogenous gene therein, an operable promoter for normal expression of the exogenous gene in the bacteria, a marker^' gene for identification gene expression in the bacteria, and the expressible exogenous gene to provide an improved fermentation property to the bacteria. This recombinant plasmid may be constructed as a shuttle vector to be massively replicated in E. coli and expressed in a target bacterium. For example, U.S. Pat. No. 5,683,909 discloses a method of constructing a shuttle vector for expression of exogenous genes in Streptococcus sp. strains and a transformation method using the shuttle vector, and this description is cited as a reference in the present invention. In addition, a number of recombinant shuttle vectors derived from Bifidobacterium and Lactobacillus are described in many publications. For example, Korean Pat. Laid-open Publication No. 2002-0069324 discloses a novel shuttle vector derived from Bifidobacterium and a transformation method using the same. U.S. Pat. No. 5,688,683 describes a shuttle vector derived from Lactobacillus and a transformation method using the same. The contents of the above-mentioned publications are referred just as references, but are not intended to be limiting. It will be apparent to those skilled in the art that a certain method, which is known in the art, of preparing a recombinant vector for expression in Bifidobacterium, Lactobacillus and Streptococcus bacteria can be used in the present invention for preparing a recombinant vector for expression of the present cryptic plasmid in the above-mentioned lactic acid bacteria. The present invention relates to a novel Bifidobacterium sp. strain carrying a cryptic plasmid which has an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and converts with high efficiency the substrate to have a conjugated double bond structure. The novel Bifidobacterium sp. strain is capable of rapidly performing fermentation in the presence of a substrate for CLA production in a mixed form with Lactobacillus acidophilus and Streptococcus thermophilus as starter bacteria. The novel Bifidobacterium sp. strain containing the cryptic plasmid according to the present invention utilizes at least one selected from among fatty acids including unconjugated double bonds, preferably, in at least the cis- 9, cis-12 configuration, or acylglycerols containing the fatty acids, as a substrate for production of a compound with a conjugated double bond structure. In a preferred aspect of the present invention, the fatty acid including unconjugated double bonds in the cis- 9, cis-12 configuration and the acylglycerol containing the fatty acid are linoleic acid and monolinolein, respectively. In addition, any strains of Lactobacillus acidophilus and Streptococcus thermophilus are used as starter bacteria with the novel Bifidobacterium sp. strain as long as their growth and acid productivity are not inhibited by a fatty acid including unconjugated double bonds or an acylglycerol containing the fatty acids to be added as a substrate. In addition, the present invention relates to a fermented milk which is produced by culturing a mixture of the novel Bifidobacterium sp. strain, Lactobacillus acidophilus and Streptococcus thermophilus and has a 0.1% or higher CLA content within at least nine hours, and a method of preparing the fermented milk. By the fermentation of the mixed bacteria, the substrate is converted to have a conjugated double bond structure including at least the cis-9, trans-11 configuration. A fatty acid having this structure and an acylglycerol containing this fatty acid may be used for various applications, for example, in milk products, foods for intestinal regulation of infants, probiotics, health functional foods, medicines, feed additives and cosmetic materials . In a preferred aspect of the present invention, the fatty acid having the conjugated double bond structure or the acylglycerol containing this fatty acid indicate a CLA produced using linoleic acid or monolinolein as a substrate or an acylglyceride containing the fatty acid. The novel Bifidobacterium sp. strain performing the above-mentioned function according to the present invention was selected by the present inventors by primarily selecting CLA-producing bacteria from about 500 Bifidobacterium sp. strains isolated from the feces of over 70 volunteers including Korean healthy infants, juveniles and adults, and subjecting the selected Bifidobacterium sp. strains to CLA production tests and culturing in a mixed form with Streptococcus thermophilus and Lactobacillus acidophilus. The finally selected Bifidobacterium sp. strain was identified to have different properties from the available conventional strains and known standard strains of the genus Bifidobacterium. The present inventors expressed the new isolate as "Bifidobacterium breve LMC520@/pBC520", and deposited the new isolate at an international depository authority, the Korean Collection for Type Cultures (KCTC) in the Korean Research Institute of Bioscience and Biotechnology (KRIBB) on March 28, 2003, under an accession number KCTC 10455 BP. The novel bacterial strain (KCTC 10455 BP) according to the present invention, unlike the conventional Bifidobacterium species, has a high growth of higher than IO⁸ cfu/ml even in linoleic acid-containing fat milk and non-fat milk media, and is rarely killed during storage or transport of fermented milk products prepared using the novel bacterial strain. Therefore, the novel bacterium is excellent with respect to the applicability to milk products . In addition, the present invention includes a method of producing a fatty acid containing a conjugated double bond structure in its carbon chain or an acylglycerol containing -the fatty acid by culturing the Bifidobacterium breve LMC520 alone or in a mixed form with Streptococcus thermophilus and Lactobacillus acidophilus. A substrate of the Bifidobacterium breve LMC520 includes at least one selected from among fatty acids including unconjugated double bonds, preferably, at least in the cis-9, cis-12 configuration, or acylglycerols containing the fatty acids . A product including the conjugated double bond structure is characterized by containing, at least, the cis-9, trans-11 configuration. More preferably, the fatty acid including unconjugated double bonds is linoleic acid, and the acylglycerol is monolinolein. Herein, the linoleic acid or monolinolein used as the substrate is preferably added to a medium in an amount of about 0.1% or higher with respect to the yield of a final product, but the present invention is not limited to the use of linoleic acid or monolinolein. To produce the fatty acid including a conjugated double bond structure or the acylglycerol containing the fatty acid, a pBC520 plasmid of the present invention may be provided as a plasmid for transformation of other lactic acid bacteria, bifidobacteria, and the like. Herein, the present pBC520 plasmid may be manipulated by recombinant DNA techniques to ensure its expression in a bacterial strain to be transformed therewith by a method known in the art. When the novel Bifidobacterium sp. strain is cultured alone or in a mixed form with Streptococcus thermophilus and Lactobacillus acidophilus in a medium containing fat milk or non-fat milk, a fermented milk may be produced, which contains a fatty acid having conjugated double bonds at least in the cis-9, trans-11 configuration in its carbon chain, or an acylglycerol including the fatty acid. Herein, in case of a fermented milk as described above, the fatty acid or the acylglycerol containing the fatty acid is preferably contained in the fermented milk at concentrations of 0.1% or higher to obtain a final product with satisfactory quality properties. Also, the milk as a raw material used in the preparation of the fermented milk preferably contains a total fat content of 4% or lower. Hereinafter, the novel Bifidobacterium sp. strain of ■ the present invention will be described in more detail with regard to the isolation method and microbial properties thereof. Isolation and selection of Bifidobacterium species strains The present inventors collected the feces from over 70 Korean healthy infants, juveniles and adults, diluted the feces with physiological saline to IO⁸ times, taking 0.1 ml from each of the dilutions, and isolating Bifidobacterium sp. strains using a bifidobacteria selection medium, TP medium. ^• The composition of the TP medium is given. in Table 1, below.

TABLE 1 Composition of the TP medium

Plates were incubated at 37°C for 48 hours in an anaerobic jar (using a gas pack system) and an anaerobic incubator in which air therein is substituted with an anaerobic mixed gas (H₂:C0₂:N₂=5: 15: 80) . Primarily, over 500 bifidobacterial strains were isolated according to the above method, and evaluated for CLA-producing ability. To evaluate over 500 isolates for the CLA-producing ability, each isolate was inoculated in MRS (Difco Laboratories, Detroit, MI, USA) liquid media supplemented with 0.08% linoleic acid and 0.05% L-cystein-HCl, and incubated under anaerobic conditions at 37°C for 24 hours. Produced CLA was identified by gas chromatography. To isolate fatty acids from MRS culture fluids, each of the culture fluids was mixed with heptadecanoic acid as an internal standard and a chloroform/methanol (1 : 1) solution in a separatory funnel. After being vigorously mixed, the funnel was allowed to stand for one hour under gravity to separate fatty acids into a chloroform layer (a lower layer). The lower layer was mixed with a 0.88% KC1 solution. After vigorous mixing, only the chloroform layer (a lower layer) was collected. The solvent chloroform was evaporated using a rotary vacuum evaporator to recover the fatty acids . For analysis of fatty acid composition, the recovered fatty acids were put into a test tube, and mixed with 20 ml of 2% sulfuric acid in anhydrous ethyl alcohol. The test tube was airtight with a stopper, and incubated in a water bath at 80°C for one hour to allow ethyl- esterification of the fatty acids . The ethyl-esterified fatty acids were extracted with 2 ml of n-hexane, and contents of CLA and other fatty acids were measured using gas chromatography (Varian 3800, USA) . By the above procedure, the most excellent bacterial strain in ^■ ,an ability to convert linoleic acid to CLA was finally selected, which is the novel Bifidobacterium sp. strain {Bifidobacterium breve LMC520) of the present invention. Gas chromatograms for analysis of CLA levels produced by the Bifidobacterium breve LMC520 strain in a MRS culture fluid are given in FIG. 1. FIG. 1-A is a gas chromatogram of ethylesters of each fatty acid contained in the MRS medium, in which the peak at 11.7 min corresponds to ethylesters of the linoleic acid added to the medium. FIG. 1-B is a gas chromatogram of ethylesters of CLA and other fatty acids in a MRS culture fluid, produced by the Bifidobacterium breve LMC520 strain, in which the peak at 13.4 min corresponds to ethylesters of cis-9, trans-11 octadecadienoic acid. In the A and B of FIG. 1, the peak at 8.7 min corresponds to an ethylester of heptadecanoic acid used as an internal standard, and the peak at 10.5 min corresponds to an ethylester of oleic acid in the MRS medium. These results indicate that the linoleic acid added to the MRS medium is almost converted to CLA.

Properties of the isolated Bifidoba cteri υm sp. strain (1) Morphology and sugar fermentation ability The Bifidobacterium breve LMC520 of the present invention was incubated in a MRS solid medium supplemented with 0.05% L-cystein-HCl using an anaerobic incubator. The final bacterial isolate was identified to be Gram-positive and not form spores . When suspended in physiological saline and microscopically observed, the final bacterial isolate was found to have the morphology of Bacillus species that show morphologic pleomorphism. In addition, when a F6PPK (fructose-6-phosphate phosphoketolase) assay was carried out using a cultured- medium obtained after the isolate was activated by being inoculated in a TPY broth according to the Scardovi' s method [Scardovi, V. 1984. Genus Bifidobacterium. in Bergey's Manual of Systematic Bacteriology. 9th Ed. Vol. 2. pp.1418-1434. Williams & Wilkins Publishers. Baltimore, MD, USA] , F6PPK-positive results were obtained, indicating that the novel bacterial strain belongs to Bifidobacterium species. On the other hand, the final bacterial isolate of the present invention was evaluated for carbohydrate fermentation using an API 50 CHL test kit (API, France) . As a result, the final bacterial isolate showed differences in fermentation of melezitose and trehalose in comparison with the carbohydrate fermentation properties of a standard, known bacterial strain, Bifidobacterium breve ATCC 15700. These results indicate that the present bacterial strain is a novel Bifidobacterium breve strain (Table 2) . TABLE 2 Comparison of Bifidobacterium breve strains LMC 520 and ATCC 15700 for carbohydrate fermentation properties

(3) Genetic properties Genetic properties of the Bifidobacterium breve LMC520 of the present invention was investigated by 16S rRNA analysis and size and nucleotide sequence analysis of plamsmid DNA. First, for the 16S rRNA analysis, the B. breve LMC520 strain was grown in a MRS medium supplemented with 0.05% L- cystein-HCl for 24 hours. The cultured cells were collected by centrifugation. The collected cells were washed with

^•physiological saline. A predetermined -amount of the cells was resuspended in 450 μl of a DNA extraction solution (250 μl of an extraction buffer (100 mM tris-HCl, 40 mM EDTA, pH 9.0), 50 μl of 10% SDS, 150 μl of benzylchloride) , and incubated in a water bath at 50°C for 30 min. Then, DNA was precipitated by isopropanol, and the isolated DNA was used as a template in 16S rRNA analysis. 16S rRNA analysis for the Bifidobacterium genus was carried out using species-specific or group-specific primers, summarized in Table 3, below, and these primers were designed based on primers suggested by T. Matsuki et al. [Matsuki, T., et al., FEMS Microbiology Letters 167, 113-121 (1998)] and D. Roy et al. [Roy, D., et al . , FEMS Microbiology Letters 191, 17-24 (2000)]. With these

^•.primers, PCR was carried out using the isolated DNA from the Bifidobacterium breve LMC520 as a template.

TABLE 3 Primers used in 16S rRNA analysis for the Bifidobacterium genus

PCR products obtained using PCR mixtures were separated on a 1% agarose gel by electrophoresis. The separated" PCR products were stained with ethidium bromide (EtBr) and visualized by UV illumination to investigate size thereof. The results are given in FIG. 2. As apparent from the electrophoresis result of FIG. 2, among the PCR samples amplified using the template DNA isolated from the Bifidobacterium breve LMC520 and several pairs of the primers, DNA bands were found only in the cases using a pair of common primers Pbi of the Bifidobacterium genus and a pair of B. breve-specific primers BiBre, indicating that the fragments were selectively amplified by the two pairs of primers, respectively. The amplified fragments were, as expected, respectively 914 bp and 288 bp in size. Therefore, the present Bifidobacterium breve LMC520 was also identified to belong to Bifidobacterium species by the 16S rRNA analysis. On the other hand, the present Bifidobacterium breve

LMC520 has another genetic property of having a cryptic plasmid. The cryptic plasmid was isolated from the present bacterial strain, and its size was analyzed, as follows. The Bifidobacterium breve LMC520 was grown in a MRS medium supplemented with 0.05% L-cystein-HCl for 12 hrs, and the cultured medium was centrifuged at 10,000 rpm for 10 min. After the supernatant was discarded, the cell pellet was washed with TES (30 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 8.0). Centrifugation was carried out under the same conditions . After the supernatant was discarded, the pellet was suspended in 6 ml of a sucrose solution (25% sucrose, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0, 20 mg/ml of lysozy e) . After incubation at 37°C for one hour, the cell suspension was mixed with 12 ml of an alkali SDS solution (3% SDS, 0.2 M NaOH) . After being incubated at room temperature for 10 min, the resulting cell lysate was mixed with 9 ral of 3 M sodium acetate (pH 4.8) and centrifuged at 10,000 rpm for 15 min. The supernatant was transferred to a new centrifuge bottle, mixed with an equal volume of isopropanol, and centrifuged under the same conditions. The DNA pellet was dried, and mixed with 10 ml of sterile distilled water and a 0.2 volume of 10 M ammonium acetate and then an equal volume of phenol/chloroform (1:1, v/v) . After centrifugation, the pellet was washed with 70% ethanol, dried and resuspended in 300 μl of TER (TE + 0.1 mg/ml RNase A) . The isolated plasmid DNA was stored at -20°C until its use in the following experiment. The isolated plasmid DNA was evaluated for its size by electrophoresis on a 1% agarose gel. In this plasmid size analysis, various restriction enzymes including Xhol, Sail, Clal, Hindlll, EcoRI, Pstl, BamHL, SacII and Accl were used according to the protocols provided by their manufacturer (Takara,- Japan) . The size and digested patterns with the restriction enzymes of the plasmid DNA isolated from the Bifidobacterium breve LMC520 according to the above procedure are given in FIG. 3. When electrophoresed on a 1% agarose gel, the undigested plasmid DNA isolated from the Bifidobacterium breve LMC520 was shown as three bands (lane C of FIG. 3) . When the plasmid DNA was digested with restriction enzymes, other background bands disappeared. In particular, SacII digestion made a single cut in the plasmid DNA and resulted in a single band, indicating that the present bacterial strain has a single plasmid type (lane 8 of FIG. 3) . The plasmid DNA was found to be about 5 kb in size, and was expressed as "pBC520". (3) The effect of pBC520 on CLA productivity of the Bifidobacterium breve LMC520 The effect of pBC520 on CLA productivity of the

Bifidobacterium breve LMC520 of the present invention was investigated by removing the plasmid DNA, that is, pBC520 from the B. breve LMC520 by curing and evaluating CLA productivity of the resulting bacterial strain. Primarily, curing of the pBC520 plasmid from the Bifidobacterium breve LMC520 was achieved as follows. The B. breve LMC520 was cultured in a 0.05% L-cystein-HCl- containing MRS broth supplemented with 500 μg/ml of ethidium bromide at 37°C for 24 hrs, and subsequently cultured twice more under the same conditions . After colonies were formed on a 0.05% L-cystein-HCl-containing MRS plate, single colonies were picked and grown in a 0.05% L- cystein-HCl-containing MRS broth. Plasmid DNA was isolated from 'the cultured cells according to the same method as described above, and subjected to agarose gel electrophoresis. The results are given in FIG..4. As shown in FIG. 4, the plasmid DNA about 5 kb in size was removed from the Bifidobacterium breve LMC520. The pBC520 plasmid-lacking bacterial strains were compared with the parent Bifidobacterium breve LMC520 with respect to CLA production ability. Two pBC520 plasmid- lacking strains and the parent strain were individually grown in MRS (Difco Laboratories, Detroit, MI, USA) media supplemented with 0.08% linoleic acid (represented "LA" in Table 4, below) and 0.05% L-cystein-HCl-containing 500 μg/ml of ethidium bromide at 37°C for 24 hrs under anaerobic conditions . Produced CLA was analyzed by gas chromatography. The parent strain was found to convert most of the linoleic acid used as a substrate to CLA, and thus, the linoleic acid rarely remained in the culture fluid. In contrast, the two pBC520 plasmid-lacking strains were found to convert almost none of the linoleic acid to CLA, and thus, most of the added linoleic acid remained in the culture fluids (Table 4, below) . From the above results, it can be seen that the pBC520 plasmid is necessary for the conversion of linoleic acid to CLA by using B. breve LMC 520 of this invention.

TABLE 4 Conversion ability of linoleic acid to CLA by the parent B. breve LMC520 and the plasmid-lacking strains

(4) Nucleotide sequence analysis of the pBC520 plasmid DNA of the Bifidobacterium breve LMC520 Since the plasmid DNA pBC520 was shown to play an essential role in the CLA production of the Bifidobacterium breve LMC520 of the present invention, the present inventors performed nucleotide sequence analysis of the pBC520 plasmid according to the following method. Since the pBC520 plasmid of the present invention was shown to be linearized to a size of about 5 kb by digestion with SacII in the above experiment, its linearized form by SacII digestion was ligated with pBluescript II KS (-) digested with the same restriction enzyme by T4 DNA ligase. E. coli NM522 was transformed with the resulting plasmid. Plasmid DNA was isolated and purified according to an alkali lysis method well known in the art, and was subjected to nucleotide sequence analysis. Nucleotide sequence was analyzed using a sequence analyzer (SEQ 4x4 personal sequencing system) and a sequence analysis kit (Termo Sequenase Cy5.5 Dye termination Cycle Sequencing kit) . Analysis of the resulting sequence was performed using the BLAST program of the NCBI (National Center for Biotechnology Information) . •^•> The obtained nucleotide sequence and its BLAST search results are given in FIG. 5. The pBC520 plasmid was found to have a 62% GC content, a size of 4,962 bp and three major ORFs (Open Reading Frames) . Among the ORFs, the first one is 501 bp in length that corresponds to nucleotides 474 to 974 of the nucleotide sequence, and encodes a 17.3-kDa membrane protein (pi 4.55) of 167 amino acids. The second ORF is 1,179 bp in length that corresponds to • nucleotides 1,044 to 2,222 of the nucleotide sequence, and encodes a 44.6-kDa mobilization protein (pi 9.42) of 393 amino acids. The third ORF is 909 bp in length that corresponds to nucleotides 3,614 to 4,522 of the nucleotide sequence, and- encodes a 34.2-kDa replication protein (pi 8.47) of 303 amino acids . Amino acid sequences of the proteins encoded by the pBC520 plasmid were compared with those of proteins encoded by a pKJ50 plasmid from Bifidobacterium longum [known as not having the ability of CLA conversion) . As a result, the membrane protein had a 100% homology, the mobilization protein had a 71% homology, and the replication protein had ■a 93% homology, to a corresponding protein of the pKJ50 plasmid. Thus, the pBC520 plasmid is very similar to the pKJ50 plasmid of B. longum in size and nucleotide sequence, but showed different restriction enzyme digestion patterns due to partially different nucleotide sequences. In particular, the pKJ50 plasmid does not have a Stul site,-, whereas the pBC520 plasmid has a Stul site, resulting in different restriction mapping.

Mode for Invention Hereinafter, the above-noted properties and CLA production ability of the Bifidobacterium breve LMC520 will be confirmed in the following examples. Also, the usefulness of the present B. breve LMC520 will be verified by fermentation experiments with the B. breve LMC520 alone or in a mixed form with Streptococcus thermophilus and Lactobacillus acidophilus. The following examples are set forth to illustrate, but are not to be construed as the limit of the present invention.

Characterization of the B. breve LMC520

EXAMPLE 1: Growth properties of the -5. -breve LMC520

To evaluate the Bifidobacterium breve LMC520 of the present invention for potential to be used in the preparation of fermented milk products, its growth properties were investigated. A medium was prepared by adding 3% non-fat milk powder and 1% sucrose to milk (2% fat) and supplemented with 0.05% linoleic acid. The present strain was inoculated in the medium and cultured at 37°C for 48 hrs. Changes in viable cell number and pH and CLA content of the medium were estimated at regular intervals of time. In addition, after the culture was completed, the culture fluid was stored at 4°C for a predetermined period for evaluation for changes in viable cell number, pH and CLA content. The culture of the present B. breve LMC520 reached over IO⁹ higher viable cells per ml within 24 hrs. Also, the viable cell number of higher than IO⁹ cells/ml was maintained after storage at 4°C for 3 days. These results indicate that the B. breve LMC520 has high potential for use in the preparation of fermented milk products (FIG. 6) . -pH of the medium decreased according to the culture time. At 48 hrs, the medium had a pH of 4.96, and this pH value was maintained even after the storage at 4°C for 3 days (FIG. 6) . CLA production by the fermentation of the present strain was increased during 18 hrs of the culture, and, thereafter, maintained at a constant level. After 6 and 18 hours of the fermentation, CLA was synthesized in amounts of 187 μg/ml and 289 μg/ml, respectively. The final CLA amount was maintained even after storage (FIG. 7) . As apparent from the above results, the present B. breve LMC520 is a fermentative microorganism having a high capacity in the initial CLA production and has high potential to be used in the production of high content CLA-

•vcontaining fermented milk products. ^•.

EXAMPLE 2: CLA production ability of the B. breve LMC520 during sub-culturing

The B. breve LMC520 of the present invention possesses a pBC520 plasmid that plays an essential role in the conversion of linoleic acid to CLA. When the pBC520 plasmid is stably maintained in bacterial cells during sub- culturing, the CLA production ability is expressed in subsequently sub-cultured bacterial cells. Therefore, in this example, the B. breve LMC520 was evaluated to determine ^• whether the CLA production ability is maintained during sub-culturing. The B. breve LMC520 was grown in a MRS medium supplemented with 0.05% L-cystein-HCl and 0.1% agar at 37°C for 24 hrs and stored at 4°C, and sub-cultured using the same medium once per week. During sub-culturing, CLA produced by the B. breve LMC520 was measured after the B. breve LMC520 was cultured in a MRS (Difco Laboratories, Detroit, MI, USA) broth supplemented with 0.08% linoleic acid and 0.05% L-cystein-HCl at 37°C for 24 hrs under anaerobic conditions . The measured CLA levels during the sub-culturing of the B. breve LMC520 are given in FIG. 8. During the sub-culturing period, the Bifidobacterium sp. strain of the present invention produced -CLA in an amount of 705 μg/ml upon the first sub-culturing, and, during the 2nd to 32nd sub-culturing, CLA production were maintained in very stable levels in a range of 672 μg/ml to 715 μg/ml. Thus, the B. breve LMC520 was not found to be reduced in its CLA production ability even during sub- culturing of more than thirty rounds . These results indicate that the cryptic plasmid DNA carried by the present strain is stably transferred to subsequent generations of the present strain during sub-culturing, resulting in maintenance of the CLA synthesis ability of the present strain in subsequent generations.

EXAMPLE 3: CLA production ability of the B. breve LMC520' according to substrate types

The B. breve LMC520 of the present invention was evaluated for its applicability in the preparation of fermented milk products by investigating CLA production ability thereof according to substrate types . As substrates for CLA production, linoleic acid, monolinolein, dilinolein, 50% monoglyceride-containing safflower oil, and 90% monoglyceride-containing safflower oil were used. Each of the substrates was added to milk in an amount of 0.05%. The milk was pasteurized, inoculated with the present strain, and incubated at 37°C for 18 hrs. Then, CLA contents in the milk were measured. Herein, among the substrates, monolinolein means to contain monoglycerides of higher than 99% and have a fatty acid composition including linoleic acid of higher than 99%. 50% and 90% monoglyceride-containing safflower oils are prepared by synthesizing safflower oil with monoglyceride contents of 50% or higher and 90% or higher, respectively, using raw safflower oil with a 100%- triglyceride structure, have the identical fatty acid composition to the raw safflower oil, and thus, contain linoleic acid at levels of higher than 70%. The Bifidobacterium sp. strain of the present invention produced CLA in an -amount of 28.2 mg/100 ml when using linoleic acid as a substrate convertible to CLA, and 40.6 mg/100 ml when using monolinolein. Thus, the present strain showed a higher CLA production capacity when using monolinolein than the case of using linoleic acid as the substrate. In addition, because of having good emulsifying capacity, monolinolein is advantageous in producing CLA in high levels, and produced CLA-containing monolinolein is well absorbed by the body. On the other hand, the use of dilinolein as the substrate resulted in production of CLA of 8.5 mg/100 ml, indicating that the present B. breve LMC520 is poor in converting dilinolein to CLA. When using 50% and 90% monoglyceride-containing safflower oils as the substrate, the B. breve LMC520 produced CLA of 24.5 and 38.6 mg/100 ml, respectively. These results demonstrate that safflower oil monoglycerides have a high industrial value as a substrate convertible to CLA (FIG. 9) . Since synthesized CLA levels were not greatly reduced upon the use of monoglyceride-containing safflower oils prepared from raw safflower oils in comparison with the case of using monolinolein as the substrate, monoglycerides derived from other edible oils, for example, soybean oil, corn oil, cottonseed oil and sunflower oil, which contain linoleic acid at levels of higher than 50%, have a potential to be used as a substrate convertible to CLA.

- EXAMPLE 4 : CLA levels according to the amount of a substrate added in yogurt production using the B. breve LMC520

The B. breve LMC520 of the present invention was evaluated for how much it increases CLA contents in currently commercially available yogurts when directly applied in the preparation of the yogurts. Also, substrate amounts required for preparing fermented milk having a 0.1% or higher CLA content within at least 9 hours after culturing were investigated. 90% monoglyceride-containing safflower oil was added to a medium as a substrate for CLA production at various concentrations of 0.05% to 0.5%, and raw milk for yogurt v preparation was prepared by modulating the fat content of 3.5% fat-milk to 2% using a cream separator. According to the composition listed in Table 5, below, materials were mixed, homogenized and pasteurized. The B. breve strains LMC520 and LMC7 were individually activated by being grown in a MRS broth supplemented with 0.05% L-cystein-HCl for 18 hrs, and then inoculated in each medium.

TABLE 5 Amounts of raw materials for yogurt preparation

A substrate was added in various concentrations listed in Table 5, and yogurts were prepared by fermentation with B. breve strains LMC520 and LMC7 at 37°C for 9 hrs. CLA contents and pH of the yogurts are given in

Table 6, below. When yogurts were prepared by fermentation with the B. breve" LMC520, CLA contents in the yogurts' increased along with the concentrations of the substrate . The highest conversion rate of the substrate to CLA was found in the case of using the substrate in a 0.1% concentration. However, at substrate concentrations of higher than 0.1%, the conversion rate of the substrate to CLA decreased inversely with the substrate concentrations. In detail, when the substrate was added at concentrations of 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, its conversion rates to CLA were found to be 42.4%, 60.2%, 39.1%, 39.1%, 37.1% and 33.3%, respectively. According to the results of this example, when the substrate was added at concentrations of 0.3%, 0.4% and 0.5%, fermented yogurts had CLA contents of 117.4, 148.5 and 166.7 mg/100 ml, respectively. Also, when yogurts were prepared by 9-hour fermentations with the B. breve LMC7, CLA contents in the yogurts increased along with the concentrations of the substrate. However, at 0.2% and higher concentrations of the substrate, little increase in CLA contents was found, and, also, no remarkable drop in pH of yogurts was found. These results indicate that, at a 0.2% or higher substrate concentration, the B. breve LMC7 should be cultured for a longer period to increase its growth and CLA production levels. According to the results of this example, unlike the fermentation by the B. breve LMC520, when yogurt fermentation was carried out by the B. breve LMC7, a 0.1% or higher CLA content was not achieved by the 9-hour fermentation even at substrate concentrations of 0.3%, 0.4% and 0.5%. Also, fermentation was found to be incomplete. These results indicate that, unlike the B. breve LMC7, the present B. breve LMC520 can be used in a fermentation of 6 to 10 hrs, applied for the preparation of. commercially available yogurts, for preparing yogurts with an at least 0.1% or higher CLA content.

TABLE 6 CLA contents and pH of yogurts prepared by a 9-hour fermentation according to substrate concentrations

EXAMPLE 5: CLA production by starter culture using a mixture of the B. breve LMC520, Streptococcus thermophilus and Lactobacillus acidophilus . With respect to rapid fermentation rates of yogurts, and quality including texture, taste and flavor and functionality in the body of final fermented products, industrial yogurts are produced using a mixture of three starter bacteria consisting of Lactobacillus, Streptococcus thermophilus and Bifidobacterium. In this regard, the present B. breve LMC520 was evaluated for whether being industrially applicable in a mixed form with other bacterial types for preparing yogurts containing natural CLA. Thus, two types of bacteria were required, which are capable of being grown with the present novel strain, are not inhibited in growth even in the presence of linoleic acid or monolinolein as a substrate for preparation of yogurts with high CLA contents, and have excellent acid production capacity. Streptococcus thermophilus and Lactobacillus acidophilus strains satisfying these requirements were selected from commercially available lactic acid bacteria and lactic acid bacteria isolated from available fermented milk products and preserved in Department of Food & Nutrition, College of Health Sciences, Korea University, Korea. Then, the novel bacterial strain of the present invention was co-cultured with the selected S. thermophilus and L. acidophilus strains. The resulting yogurts were evaluated for CLA contents . 90% monoglyceride-containing safflower oil was added to a medium as a substrate for CLA production at a concentration of 0.3%, and raw milk for yogurt preparation and other materials were prepared according to the same composition as in Example 3. The B. breve LMC520 was activated by being grown in MRS broth supplemented with 0.05% L-cystein-HCl for 18 hrs. The S. thermophilus and L. acidophilus strains were activated by being grown in M17 medium and MRS medium, respectively, for 18 hrs. The activated strains were inoculated in a medium for yogurt preparation alone or in a mixed form, and fermentation was carried out for 9 hrs . . After the single fermentation or co-fermentation at 37°C for 9 hrs, CLA contents were measured in each yogurt, and the results are given in FIG. 10. CLA contents were found to be 116.2 mg/100 ml in a yogurt (B) prepared by single fermentation with the B. breve LMC520, 6.4 mg/100 ml in a yogurt (T) prepared by single fermentation with the S. thermophilus strain, and 6.2 mg/100 ml in a yogurt (A) prepared by single fermentation with the L. acidophilus strain. These results indicate that the S. thermophilus and L. acidophilus strains do not convert monolinolein added as a substrate to CLA. This conclusion was supported by a co-fermentation with the S. thermophilus and L. acidophilus strains, in which a resulting yogurt (AT) was found to have a CLA content of 6.5 mg/100 ml. In contrast, when prepared by a co-fermentation with the B. breve LMC520 and the S. thermophilus strain or the L. acidophilus strain, or a co- fermentation with the three bacterial strains, yogurts displayed high CLA contents. That is, a yogurt (BT) ' prepared by a co-fermentation with the B. breve LMC520 and the S. thermophilus strain contained CLA of 118.4 mg/100 ml. A yogurt (AB) prepared by a co-fermentation with the B. breve LMC520, the L. acidophilus strain contained CLA of 112.3 mg/100 ml. A yogurt (ABT) prepared by a co- fermentation of the B. breve LMC520 with the S. thermophilus and L. acidophilus strains contained CLA of 115.7 mg/100 ml. These results indicate that the B. breve LMC520 of the present invention has high CLA production capacity even in co-culture with S. thermophilus and L. acidophilus, which are typically used as starter bacteria in industrial yogurt production. EXAMPLE 6: CLA contents according to the concentration of a substrate in yogurts prepared by a co-culture

Yogurt fermentation was carried out using a mixture of the B. breve LMC520, S. thermophilus and L. acidophilus for 9 hrs . CLA levels in yogurts according to the added amounts of a substrate were measured. Also, the yogurts were evaluated for changes in CLA contents after a 12-hour maturation, and after a 5-day storage at 4°C when packaged into bottles. 90% monoglyceride-containing safflower oil was added to a medium as a substrate for CLA production at concentrations from 0.1% to 0.5%. Raw milk for yogurt preparation was prepared by modulating the fat content of 3.5% fat-milk to 2% using a cream separator. Other materials were prepared according to the same composition as in Example 3. The B. breve LMC520 was activated by being grown in MRS broth supplemented with 0.05% L-cystein-HCl for 18 hrs. The S. thermophilus and L. acidophilus strains were activated by being grown in M17 medium and MRS medium, respectively, for 18 hrs. The activated strains were inoculated to the medium for yogurt preparation. Yogurt fermentations were carried out using a mixture of the activated bacterial strains with various concentrations of the substrate at 37°C for 9 hrs . CLA contents and pH of produced yogurts were measured. Also, the yogurts were evaluated for changes in CLA contents and pH after a 12-hour maturation and after a 5-day storage at 4°C. The results are given in Table 7, below.

TABLE 7 CLA contents and pH of yogurts prepared using a starter mixture according to substrate concentrations

Upon yogurt fermentation using a mixture of the three bacteria, CLA contents in yogurts, similar to the single fermentation of the B. breve LMC520 in Example 3, increased along with the concentrations of the added substrate, and CLA levels against each substrate concentration were similar to those in Example 3. However, each yogurt was found to have a very ^'low pH of from 4.27 to 4.46 in comparison with the yogurts prepared by single fermentations with the B. breve LMC520, which ranged from 5.05 to 5.12 in pH. This is because the S. thermophilus and L. acidophilus strains used in this co-culture produced more lactic acid in the yogurt co-fermentation than the single fermentation by the B. breve LMC520. This low pH of yogurts prepared by the co-fermentation is expected to have beneficial effects on taste and stability for storage. After each of the fermented yogurts was maturated at 4°C for 12 hrs, the mature yogurts were evaluated for CLA contents. CLA contents in the mature yogurts were found to increase by 15.9% to 25.0% in comparison with the yogurts immediately after fermentation. After storage for 5 days, CLA contents in the yogurts were increased by about 12% in comparison with the case of being maturated for 12 hrs. On the other hand, experimental studies with animal models revealed that a diet containing 0.1% or higher CLA effectively inhibits cancer cells (breast cancer) [Ip, C, et al., Cancer Research 54:1212-1215 (1994)]. According to the results of this example, the yogurts prepared by co- fermentation using the substrate in various concentrations of 0.3%, 0.4% and 0.5% were found to, after being maturated, contain CLA of 145.8, 188 and 199.8 mg/100 ml, respectively, and, after storage for 5 days, 164.8, 209.6 and 222.8 mg/100 ml, respectively. Therefore, to prepare yogurts containing 0.1% or higher -.CLA, a substrate is proper to be added at a concentration ranging from 0.3% to 0.5%.

As described in the above Examples, the B. breve LMC520 of the present invention converted linoleic acid or monolinolein to CLA in similar levels to the Bifidobacterium breve LMC7 disclosed in Korean Laid-open Publication No. 10-2003-0002688 applied by the present inventors. However, the present B. breve LMC520 was superior to the B. breve LMC7 in practical applications, as follows . The present strain is capable of being used as a starter -in a mixed form with other lactic acid bacteria, S. thermophilus and L. acidophilus. In particular, the B. breve LMC7 required 24 to 48 hrs for fermentation, but the present strain greatly shortened the fermentation time to 9 to 12 hrs . The B. breve LMC7 strain has another problem of having reduced CLA production capacity when sub-cultured. However, the present LMC520 strain was found to be not reduced in its CLA production capacity even during over thirty rounds of sub-culturing, and the cryptic plasmid DNA carried by the present LMC520 strain is also stably transferred to subsequent generations and thus attributes for the present LMC520 strain to maintain the ability to synthesize CLA during sub-culturing. In addition, since the pBC520 -.plasmid carried by the present LMC52.Q strain is present as an extrachromosomal element, it is a gene capable of being introduced into CLA production capacity- lacking lactic acid bacteria, bifidobacteria, and the like. Therefore, the pBC520 plasmid has various industrial applications . In addition, since the present LMC520 strain survives in fermented products for a sufficient period, fermented products can be enriched with the present bifidobacterial strain as well as the conventional lactic acid bacteria. Thus, the present strain can improve functionality of fermented products in the body. In particular, the present LMC520 strain uses monolinolein as a substrate for CLA production so that the use of monolinolein is beneficial in producing high levels of CLA. Also, produced CLA-containing monolinolein is well absorbed by the body. Further, because it was isolated from the human feces, the B. breve LMC520 and the plasmid DNA of the present invention can be used without concern about pathogenicity for various applications, for example,- in fermented milk products, foods for intestinal regulation of infants, CLA-enriched bifidus milk products, probiotics, health functional foods, feed additives, medicines, and cosmetic materials . Industrial Applicability The B. breve LMC520 according to the present invention has an excellent ability to convert linoleic acid or monolinolein to CLA. Also, when used in a mixed form with Lactobacillus acidophilus and Streptococcus thermophilus, the B. breve LMC520 is suitable for the production of fermented milk products containing high levels of CLA for a period required for general fermented milk production. Further, the B. breve LMC520 or the pBC520 plasmid responsible for the CLA production ability of the B. breve LMC520, which is isolated from humans, can be used without concern about pathogenicity for various applications in food and medicine fields .

-NDIC-fflONSR-----A -NGTOD--POSI^ OROTHERBIOI-JOGICALMATORIAL (PCT Rule 13b--s)

A The ind-cat-ons made below relate to the deposited -mm-Qrganism or other biological ma-eπal refeired to in the description on pass 21 . line 14-25

RlDE-ST-FI -AΗONOFDEPOSrr Further deposits are on an additional sheetO Name of depositary lnst-tut-on

Korean Collection forType Cultures Address of deposit-try wstMiai^mcIudmgpostalco amlccnmtry) #52, Oun-dong, Yusong-ku, Taejon 305-806, Republic ofKoiea Date of deposit AccessionNumber 28/03/2003 KCTC10455BP

C ADOmONALi iCAΗaNSfaiεlb ti≠a Thisin-b-mation is continued on an addit-onal sheet D

DDESIGNAT--D STATES FOR WHICH INDICAΗONS ARE m^inii-dxtarscrey xfb cides^tied i&s)

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For receiving Office use only For -nte-nat-onal Bureau use only

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Claims

1. A Bifidobacterium breve, maintained in a high viable cell number of higher than IO⁹ cells/ml within 24 hours of culture and maintained in a high viable cell number of higher than 10^s cells/ml after ^•storage for 3 days after fermentation; having the property of carbohydrate fermentation of a standard known bacterial strain, Bifidobacterium breve ATCC 15700 and an ability to ferment melezitose and trehalose; and containing a pBC520 plasmid that has an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and convert the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol containing the produced fatty acid, and is stably maintained during sub-culturing.

2. The Bifidobacterium breve according to claim 1, wherein the Bifidobacterium breve is Bifidobacterium breveLMC520 (Accession number KCTC 10455 BP) ..

3. A pBC520 plasmid having an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid and convert the substrate to a fatty acid with a conjugated double bond structure or an acylglycerol containing the fatty acid, and having a nucleotide sequence of SEQ ID NO: 1.

4. The Bifidobacterium breve according to claim 2, wherein the substrate provided to be converted to form the conjugated double bond structure is one selected from fatty acids with unconjugated double bonds at least in a cis-9 , cis-12 configuration, acylglycerols containing the fatty acids and a mixture thereof.

5. The pBC520 plasmid according to claim 3, wherein the substrate provided to be converted to form the conjugated double bond structure is one selected from fatty acids with unconjugated double bonds at least in a cis-9, cis-12 configuration, acylglycerols containing the fatty acids and a mixture thereof.

6. The Bifidobacterium breve according to claim 2 or 4, wherein the conjugated double bond structure includes at least the. cis-9, trans-11 configuration as a .structural feature .

7. The pBC520 plasmid according to claim 3 or 5, wherein the conjugated double bond structure includes at least the cis-9, trans-11 configuration as a structural feature .

8. The Bifidobacterium breve according to claim 4, wherein the substrate is linoleic acid, monolinolein, dilinolein or a mixture thereof, wherein the monolinolein is a synthetic substrate that contain monoglycerides of 50% or more and have- a fatty acid composition including linoleic acid of 50% or more.

9. The Bifidobacterium breve according to any one of claims 2, 4 and 6, wherein the fatty acid with the conjugated double bond structure is conjugated linoleic acid (CLA) .

10. The pBC520 plasmid according to any one of claims 3, 5 and 7, wherein the fatty acid with the conjugated double bond structure is conjugated linoleic acid (CLA) .

11. A method of producing a fatty acid with a conjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid in a conjugated linoleic acid (CLA) production ability-lacking bacterium, in which: the CLA production ability-lacking bacterium is transformed with a pBC520 plasmid having an ability to use as a substrate a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid, and convert the substrate to the fatty acid with the conjugated double bond structure or the acylglycerol containing the produced fatty acid.

12. The method according to claim 11, wherein the substrate is one selected from fatty acids with unconjugated double bonds at least in a cis-9, cis-12 configuration, acylglycerols containing the fatty acids or a mixture thereof.

13. The method according to claim 11, wherein the fatty acid with the conjugated double bond structure or the acylglycerol containing the fatty acid includes at least a cis-9, trans-11 configuration as a structural feature.

14. The method according to claim 11, wherein the substrate is linoleic acid, monolinolein, dilinolein or a mixture thereof, wherein the monolinolein is a synthetic substrate that contain monoglycerides of 50% or more and have a fatty acid composition including linoleic acid of 50% or more.

15. The method according to claim 11, wherein the fatty acid with the conjugated double bond structure is conjugated linoleic acid (CLA) .

16. The method according to any one of claims 11, 12 and 14, wherein the substrate is added to a medium at a concentration of 0.05% or higher.

17. A method of producing a fermented milk comprising ^• a fatty acid with a conjugated double bond structure at its carbon chain thereof at least in a cis-9, trans-11 configuration or an acylglycerol containing the fatty acid, in which: a Bifidobacterium breve LMC520 containing a pBC520 plasmid is cultured in a culture medium containing fat milk or non-fat milk, and a fatty acid with an unconjugated double bond structure at its carbon chain or an acylglycerol containing the fatty acid as a substrate for production of the fatty acid with the conjugated double bond structure or the acylglycerol containing the fatty acid.

18. The method according to claim 17, wherein the ^•> Bifidobacterium breve LMC520 is cultured along with Lactobacillus acidophilus and Streptococcus thermophilus, which are not inhibited in growth by the substrate added for conjugated linoleic acid (CLA) production.

19. The method according to claim 17 or 18, wherein the fatty acid with the conjugated double bond structure at the carbon chain thereof at least in the cis-9, tmas-11 configuration or the acylglycerol containing the fatty acid is contained in the fermented milk at a concentration of 0.1% or higher.

20. The method according to claims 17 and 18, wherein the fermented milk is prepared by culturing at least for six hours.