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WO2005001068A1 - Souche de bifidobacterium breve lmc520 contenant un plasmide pbc520, procede de preparation d'acides gras conjugues et de laits fermentes contenant de tels acides gras au moyen de ladite souche, utilisation d'un plasmide pbc520 - Google Patents

Souche de bifidobacterium breve lmc520 contenant un plasmide pbc520, procede de preparation d'acides gras conjugues et de laits fermentes contenant de tels acides gras au moyen de ladite souche, utilisation d'un plasmide pbc520 Download PDF

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WO2005001068A1
WO2005001068A1 PCT/KR2004/001571 KR2004001571W WO2005001068A1 WO 2005001068 A1 WO2005001068 A1 WO 2005001068A1 KR 2004001571 W KR2004001571 W KR 2004001571W WO 2005001068 A1 WO2005001068 A1 WO 2005001068A1
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fatty acid
cla
substrate
double bond
plasmid
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Chil-Surk Yoon
Soo-Hyun Chung
Kwang-Won Hong
In-Hwan Kim
Jae-Hong Jeong
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1315—Non-milk proteins or fats; Seeds, pulses, cereals or soja; Fatty acids, phospholipids, mono- or diglycerides or derivatives therefrom; Egg products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • 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.
  • 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 .
  • CLA Conjugated linoleic acids
  • 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.
  • 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
  • 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.
  • 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 .
  • 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.
  • CLA natural CLA
  • CLA present in meat, milk and fermented milk products 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
  • 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.
  • S. thermophilus is good at fermenting milk to soft products but poor at producing lactic acid.
  • 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 .
  • 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.
  • Bifidobacterium breve LMC7 KCTC 1017 BP
  • yogurt preparation was achieved using a mono-culture of the bacterium.
  • Fermentation is performed for 24 to 48 hours, which is a longer than the typical yogurt fermentation time.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ DNA molecular size marker; 1: intact B. breve LMC520, 2: cured B. breve LMC520-1; 3: another cured B. breve LMC520-2) ; FIG.
  • 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. 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;
  • 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.
  • thermophilus 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) .
  • Plasmid 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 .
  • 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.
  • 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.
  • the cryptic plasmids are not essential for the general growth of bacteria, and provide particular specificity to bacteria containing the cryptic plasmids .
  • 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.
  • curing as used herein, has the following meaning. Plasmids are generally stable in cells, but are unstable under some unfavorable conditions .
  • Agents causing curing include plasmid replication interrupters, such as acridine orange, acriflavin and ethidium bromide, and DNA synthesis inhibitors, such as mitomycin C.
  • plasmid replication interrupters such as acridine orange, acriflavin and ethidium bromide
  • DNA synthesis inhibitors such as mitomycin C.
  • 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.
  • curing is caused by exposing bacteria to very poor environment in which normal bacterial growth is impossible.
  • lactic acid bacteria refers 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.
  • Lactobacillus examples 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 milks are greatly classified into liquid forms and concentrated forms according to the content of milk solids non-fat.
  • the fermented milks are categorized .into the liquid fermented milk.
  • the fermented milks are categorized into the concentrated fermented milk.
  • 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.
  • lacic acid refers to a fatty acid molecule that is composed of 18 carbons having two double bonds in cis configuration at positions 9 and 12.
  • conjugated linoleic acid 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.
  • transformation refers to a method of introducing a gene having a specific genetic property into a host cell.
  • 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.
  • U.S. Pat. No. 5,683,909 discloses a method of constructing a shuttle vector for expression of exogenous genes in Streptococcus sp.
  • 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.
  • strain containing the cryptic plasmid 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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 IO 8 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.
  • the novel bacterium is excellent with respect to the applicability to milk products .
  • 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.
  • 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.
  • a pBC520 plasmid of the present invention may be provided as a plasmid for transformation of other lactic acid bacteria, bifidobacteria, and the like.
  • 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.
  • 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.
  • 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.
  • the milk as a raw material used in the preparation of the fermented milk preferably contains a total fat content of 4% or lower.
  • 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 IO 8 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.
  • 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.
  • 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 .
  • 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 .
  • 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.
  • the peak at 13.4 min corresponds to ethylesters of cis-9, trans-11 octadecadienoic acid.
  • the peak at 8.7 min corresponds to an ethylester of heptadecanoic acid used as an internal standard
  • the peak at 10.5 min corresponds to an ethylester of oleic acid in the MRS medium.
  • the final bacterial isolate of the present invention was evaluated for carbohydrate fermentation using an API 50 CHL test kit (API, France) .
  • API 50 CHL test kit API, France
  • 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.
  • Bifidobacterium breve ATCC 15700 a standard, known bacterial strain
  • Table 2 TABLE 2 Comparison of Bifidobacterium breve strains LMC 520 and ATCC 15700 for carbohydrate fermentation properties
  • a predetermined -amount of the cells was resuspended in 450 ⁇ l of a DNA extraction solution (250 ⁇ l of an extraction buffer (100 mM tris-HCl, 40 mM EDTA, pH 9.0), 50 ⁇ l of 10% SDS, 150 ⁇ l 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.
  • a DNA extraction solution 250 ⁇ l of an extraction buffer (100 mM tris-HCl, 40 mM EDTA, pH 9.0), 50 ⁇ l of 10% SDS, 150 ⁇ l of benzylchloride
  • 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
  • PCR was carried out using the isolated DNA from the Bifidobacterium breve LMC520 as a template.
  • 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.
  • EtBr ethidium bromide
  • 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 .
  • 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.
  • a sucrose solution 25% sucrose, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0, 20 mg/ml of lysozy e
  • 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 ⁇ l 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.
  • 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.
  • the undigested plasmid DNA isolated from the Bifidobacterium breve LMC520 was shown as three bands (lane C of FIG. 3) .
  • the plasmid DNA was digested with restriction enzymes, other background bands disappeared.
  • 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".
  • 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.
  • 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 ⁇ g/ml of ethidium bromide at 37°C for 24 hrs, and subsequently cultured twice more under the same conditions .
  • 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.
  • 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 ⁇ g/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.
  • 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.
  • 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) .
  • ORFs Open Reading Frames
  • 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) .
  • the membrane protein had a 100% homology
  • the mobilization protein had a 71% homology
  • the replication protein had ⁇ a 93% homology, to a corresponding protein of the pKJ50 plasmid.
  • 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.
  • the pKJ50 plasmid does not have a Stul site,-, whereas the pBC520 plasmid has a Stul site, resulting in different restriction mapping.
  • EXAMPLE 1 Growth properties of the -5. -breve LMC520
  • 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.
  • 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.
  • 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-
  • 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.
  • 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.
  • 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 .
  • MRS Mert Laboratories, Detroit, MI, USA
  • the measured CLA levels during the sub-culturing of the B. breve LMC520 are given in FIG. 8.
  • strain of the present invention produced -CLA in an amount of 705 ⁇ g/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 ⁇ g/ml to 715 ⁇ g/ml.
  • the B. breve LMC520 was not found to be reduced in its CLA production ability even during sub- culturing of more than thirty rounds .
  • 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 .
  • 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.
  • 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%.
  • 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.
  • the present strain showed a higher CLA production capacity when using monolinolein than the case of using linoleic acid as the substrate.
  • monolinolein is advantageous in producing CLA in high levels, and produced CLA-containing monolinolein is well absorbed by the body.
  • 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.
  • the B. breve LMC520 produced CLA of 24.5 and 38.6 mg/100 ml, respectively.
  • 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.
  • 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.
  • 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
  • EXAMPLE 5 CLA production by starter culture using a mixture of the B. breve LMC520, Streptococcus thermophilus and Lactobacillus acidophilus .
  • B. breve LMC520 Streptococcus thermophilus
  • 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.
  • 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.
  • Example 3 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 . .
  • 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.
  • 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 (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.
  • 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.
  • 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.
  • 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.
  • This low pH of yogurts prepared by the co-fermentation is expected to have beneficial effects on taste and stability for storage.
  • 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.
  • CLA contents in the yogurts were increased by about 12% in comparison with the case of being maturated for 12 hrs.
  • 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)].
  • 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%.
  • 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.
  • 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.
  • 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 .
  • breve LMC7 strain has another problem of having reduced CLA production capacity when sub-cultured.
  • 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.
  • 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.
  • the pBC520 plasmid has various industrial applications .
  • 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.
  • the present strain can improve functionality of fermented products in the body.
  • 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.
  • 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.
  • 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 .

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Abstract

La présente invention concerne une nouvelle souche de Bifidobacterium breve LMC520 comprenant un plasmide cryptique qui a la capacité d'utiliser, en tant que substrat, un acide gras ayant une structure à double liaison non conjuguée au niveau de sa chaîne carbone ou un acylglycérol contenant l'acide gras, puis de convertir avec une grande efficacité, le substrat en un acide gras ayant une structure à liaison double conjuguée ou un acylglycérol contenant l'acide gras produit. La présente invention concerne également l'utilisation du plasmide cryptique; un procédé de production d'un acide gras ayant une structure à liaison double conjuguée au niveau de sa chaîne carbone ou un acylglycérol contenant l'acide gras, et ce, par la mise en culture de B. breve LMC520 dans des conditions anaérobie ou aérobie; et un procédé de préparation d'un lait fermenté contenant l'acide gras ou l'acylglycérol.
PCT/KR2004/001571 2003-06-28 2004-06-28 Souche de bifidobacterium breve lmc520 contenant un plasmide pbc520, procede de preparation d'acides gras conjugues et de laits fermentes contenant de tels acides gras au moyen de ladite souche, utilisation d'un plasmide pbc520 Ceased WO2005001068A1 (fr)

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CN113170820A (zh) * 2021-05-20 2021-07-27 浙江李子园食品股份有限公司 一种含共轭亚油酸和共轭亚麻酸的发酵乳及其制备方法

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KR101589465B1 (ko) * 2015-05-21 2016-02-01 주식회사 쎌바이오텍 성장 촉진을 위한 비피도박테리움 브레베 cbt br3 균주 및 이를 포함하는 성장촉진용 기능성 식품 조성물

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CN110878273A (zh) * 2019-12-30 2020-03-13 江南大学 一株短双歧杆菌及其在制备共轭脂肪酸中的应用
CN113170820A (zh) * 2021-05-20 2021-07-27 浙江李子园食品股份有限公司 一种含共轭亚油酸和共轭亚麻酸的发酵乳及其制备方法
CN113170820B (zh) * 2021-05-20 2022-06-17 浙江李子园食品股份有限公司 一种含共轭亚油酸和共轭亚麻酸的发酵乳及其制备方法

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