WO2003087344A1 - New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the preparation methods thereof - Google Patents

New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the preparation methods thereof Download PDF

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Publication number
WO2003087344A1
WO2003087344A1 PCT/KR2003/000742 KR0300742W WO03087344A1 WO 2003087344 A1 WO2003087344 A1 WO 2003087344A1 KR 0300742 W KR0300742 W KR 0300742W WO 03087344 A1 WO03087344 A1 WO 03087344A1
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Prior art keywords
cla
strain
strains
cbg
present
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PCT/KR2003/000742
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French (fr)
Inventor
So-Mi Kim
Deok-Kun Oh
Dae-Heoun Baek
Hong-Sig Sin
Si-Ho Park
Yu-Jin Lee
Soo-Jong Um
Young-Soy Rho
Jong-Sup Park
Dong-Myong Kim
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Chebigen Inc.
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Priority to JP2003584288A priority Critical patent/JP4108613B2/en
Priority to AU2003221135A priority patent/AU2003221135A1/en
Publication of WO2003087344A1 publication Critical patent/WO2003087344A1/en

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    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04B—TRANSMISSION
    • H04B3/00—Line transmission systems
    • H04B3/54—Systems for transmission via power distribution lines
    • H04B3/56—Circuits for coupling, blocking, or by-passing of signals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
    • 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
    • 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 THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A23C11/106—Addition of, or treatment with, microorganisms
    • 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 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.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q20/00—Payment architectures, schemes or protocols
    • G06Q20/04—Payment circuits
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q20/00—Payment architectures, schemes or protocols
    • G06Q20/08—Payment architectures
    • G06Q20/20—Point-of-sale [POS] network systems
    • G06Q20/202—Interconnection or interaction of plural electronic cash registers [ECR] or to host computer, e.g. network details, transfer of information from host to ECR or from ECR to ECR
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q20/00—Payment architectures, schemes or protocols
    • G06Q20/30—Payment architectures, schemes or protocols characterised by the use of specific devices or networks
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q20/00—Payment architectures, schemes or protocols
    • G06Q20/30—Payment architectures, schemes or protocols characterised by the use of specific devices or networks
    • G06Q20/34—Payment architectures, schemes or protocols characterised by the use of specific devices or networks using cards, e.g. integrated circuit [IC] cards or magnetic cards
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/535—Pseudocatenulatum
    • 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
    • C12R2001/13—Brevibacterium
    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04B—TRANSMISSION
    • H04B2203/00—Indexing scheme relating to line transmission systems
    • H04B2203/54—Aspects of powerline communications not already covered by H04B3/54 and its subgroups
    • H04B2203/5429—Applications for powerline communications

Definitions

  • the present invention relates to novel strains capable of producing conjugated linoleic acid (hereinafter referred to as CLA).
  • CLA which is a conjugated isomer of linoleic acid (hereinafter referred to as
  • LA an essential fatty acid
  • an essential fatty acid is a natural fatty acid found in a small amount in milk or muscle of ruminants.
  • CLA has conjugated double bonds at positions cis-9 and trans-11 or at positions trans-10 and cis-12, in intra and trans configuration. Particularly, by a conjugated double bond at cis-9 and trans-11 positions, physiological activities useful to human bodies are expressed.
  • CLA is known to show reduction in development of the artery sclerosis
  • CLA may be usefully used as an effective ingredient of functional food and medicaments.
  • CLA is mainly contained in animal food, particularly at a large amount in ruminant animals. It is shown that beef contains 2.9 ⁇ 4.3 mg CLA/fat, lamb meat contains 5.6 mg CLA/fat and marine products contain as little as 0.3-0.6 mg CLA/fat. For dairy products, milk contains 5.5 mg CLA/fat and cheese contains 3 ⁇ 7 rag CLA/fat. With respect to human beings' daily CLA intake, it is presumed that oriental people who principally keep a vegetable diet intake 0.1 g of CLA per day and western people who eat more meat intake 0.4 g of CLA per day.
  • the conventional methods include methods for chemically synthesizing CLA from LA, such as urea addition, molecular distillation, HPLC, etc., and there are several isolated microorganism able to convert LA into CLA.
  • the chemical synthesis methods have problems, such that they require expensive equipments or too much time is taken for processes.
  • conventional chemical synthesis methods produce a kind of CLA along with various kinds of isomers, it is very inefficient to perform production of a kind of CLA by such chemical synthesis methods. Therefore, the most efficient method for producing CLA is to produce CLA by a microorganism, followed by isolation.
  • Representative microorganisms capable of producing CLA includes microorganisms in the bowels such as Lacto bacillus, Propionibacterium, Butyrivibrio fibrisolvens etc. and are usefully used as an effective ingredient in functional food or medicaments such as probiotics and feed which are fed to animals in many countries.
  • Korean Patent Application No. 10-2001-0047292 disclosed a method for adding pure cis-9 and trans-11 type CLA which is synthesized by a microorganism of Lactobacillus genus to food. However, only an infinitesimal amount of CLA was found in an actual product.
  • a CLA producing microorganism in order to be used as an effective ingredient in food or medicaments, it should survive at a high level similar to that upon production while the product is distributed and also should maintain a high viability and activity in the stomach and bowels after intake into a body of an animal including human beings. In addition, it should be excellent in resistance to antibiotics to hold a predominant position in the competition with harmful bacteria in the bowels, including superbacteria.
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide novel strains which can produce conjugated linoleic acid, have excellent resistance to acids and antibiotics and can indirectly produce CLA when added to food and medicaments.
  • the above and other objects can be accomplished by the provision of novel strains capable of producing CLA.
  • the strains are characterized in that they are isolated from feces of infants and they can convert LA to CLA.
  • the strains include Bifidobacterium breve CBG-C2, Bifidobacterium pseudocartenulatum CBG-C4 and Enterococcus faecium CBG-C5.
  • strains can be isolated as follow:
  • Strains are isolated from feces of Korean infants and randomly selected 300 colonies are cultured in a medium with LA as a substrate. The medium is extracted with hexane and the extracts were measured for their absorbance to screen strains which have excellent CLA productivity.
  • the strains screened by the above procedures were once again examined whether they can produce CLA from LA, by subjecting fatty acids produced by the strains to gas chromatography (GC).
  • GC gas chromatography
  • the strains thus isolated were three types, designated CBG-C2, CBG-C4 and CBG-C5, respectively and were deposited at Korean Agricultural Culture Collection in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001, KACC 91003 and KACC 91002, respectively, on April 3, 2002. Also, CBG-C2 (KCTC 10462BP) and CBG-C4 (KCTC 10208BP) were deposited at the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology on March 25, 2002 and April 10, 2003, respectively.
  • the strains obtained by the above-described method are excellent in CLA productivity and have strong resistance to acids such as stomach acid and bile, and antibiotics.
  • CLA produced by microorganisms according to the present invention comprises only the stereostructures of cis-9 and trans-11, which are isomers having various physiological activities, as conventionally known to the art.
  • Fatty acids and acyglycerol containing these structures can be widely used for development of dairy products from various animals, dairy products from vegetable materials, fermented food and functional health food, probiotics and the like. Therefore, the strains according to the present invention can be effectively used in biosynthesis of isomers of CLA by biological methods such as immobilization.
  • the strains according to the present invention can be added to food and medicaments as not only a live strain but also a killed strain. This is because the strains according to the present invention can release CLA to a culture fluid or reaction fluid while accumulating a large amount of CLA in the strains.
  • the strain which are cultured in a medium containing LA may be added as an effective ingredient to various compositions such as food and medicaments so that CLA is indirectly produced and it is thus possible to promote development of functional products containing CLA in a large amount by resolve the conventional problems in that CLA cannot be directly added to food and medicaments.
  • the present invention provides a food or pharmaceutical composition containing the strain.
  • the compositions according to the present invention contain at least one selected from Bifidobacterium breve CBG-C2, Bifidobacterium pseudocartenulatum CBG-C4 and Enterococcus faecium CBG-C5 as an effective ingredient.
  • the composition may comprise an additional effective ingredient such as an organic acid to enhance CLA production rate and improve growth stability of strain.
  • an organic acid in the composition according to the present invention may be CLA which is chemically synthesized or is isolated and purified from microorganisms.
  • composition according to the present invention may comprise any one selected from the strains according to the present invention and CLA at the same time.
  • the composition according to the present invention may further contain various adjuvant components in addition to the effective ingredient, when needed.
  • the food composition according to the present invention may comprise vitamins such as vitamin A, vitamin Bl, vitamin B2, vitamin B3, vitamin B6 and vitamin B12, folic acid, vitamin C, vitamin D3, vitamin E and the like, minerals such as copper, calcium, iron, magnesium, potassium, zinc and the like, or lactic acid bacteria and the like.
  • a heath drink composition may comprise an additional component such as a flavor or natural carbohydrates, like other drinks.
  • the flavor includes natural sweetening agents such as thaumatin and stevia extract and synthetic sweetening agents such as saccharin and aspartame.
  • the natural carbohydrate includes monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, polysaccharides such as dextrin, cyclodextrin and the like, and sucrose alcohols such as xylitol, sorbitol, erythritol and the like.
  • the usage or intake of the pharmaceutical composition according to the present invention is preferably 60 to 130 uM (Cancer Epidemiol. Biol. Prev. 2000. 9:689-696) and the usage or intake of the food composition according to the present invention is preferably 3.4 to 6 g/day (J. Nutr. 2000. 130:2943-2948), though it may be adjusted, as needed.
  • the composition is stably absorbed into a body regardless of the intake.
  • the usage or intake can be adjusted according to various factors including the type and amount of an effective ingredient and other components contained in the composition, formulation type and age, body weight, general health condition, sex and diet of a patient, administration time, administration route and release rate of the composition, treatment duration, simultaneously used drugs.
  • the composition Upon administration to a human body, the composition would not show side effects, as compared to conventional food and medicaments containing chemically synthesized CLA.
  • the composition may be formulated by combining at least one pharmaceutically acceptable or edible carrier with the effective ingredient.
  • the pharmaceutically acceptable or edible carrier which can be used in the present invention includes a saline solution, sterile water, Ringer's solution, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof and may be combined with a conventional additive such as an antioxidant, a buffering agent, a bacteriostatic agent and the like, when needed.
  • the composition can be formulated into injections such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets by further adding a diluting agent, a dispersing agent, a surfactant, a binder and a lubricant.
  • the composition may be preferably formulated by a proper method known to the art, or a method described in Remington's Pharmaceutical Science (the latest), Mack Publishing Company, Easton PA, according to diseases or components.
  • composition according to the present invention can be formulated in the form of granules, powders, coated tablets, tablets, capsules, liquids and solutions, extracts, suppositories, syrups, juices, suspensions, emulsions, release-sustained formulation of an active compound and the like.
  • a capsule formulation of the composition According to the present invention, there is provided a capsule formulation of the composition.
  • the capsule formulation comprises a coating material and a core material enclosed in the coating material.
  • the core material of the capsule formulation comprises preferably both any one selected from the strains according to the present invention and CLA.
  • water- soluble polysaccharides which are excellent in absorption, dispersion and adhesion are usable.
  • the water-soluble polysaccharides which can be used in the present invention is at least one selected from the group consisting of starch, agar, carageenan, alginic acid, sodium alginate, polymethacrylate, wheat protein, soy protein, cellulose derivatives such as methylcellulose, hydroxylpropylcellulose, hydroxyl-propylmethylcellulose and the like; gums such as xanthan gum, arabic gum, locust bean gum, guar gum, tamarind gum, tara gum, karaya gum, tragacanth gum, ghatti gum and the like; and gellan, xanthan, pectin (LM, HM type, dextran, glucan, glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, gelatin and casein.
  • starch agar, carageenan, alginic acid, sodium alginate, polymethacrylate, wheat protein, soy protein, cellulose derivatives
  • the amount of the coating material can be adjusted according to the final use and purpose and is preferably 1 to 80 % by weight based on the weight of the core.
  • the capsule may further comprise substances which are commonly used in a coating material to improve release control effect or to increase solubility.
  • the capsule may further comprise an emulsifying agent, a protective agent and plasticizer, as needed.
  • the capsule formulation may be produced by using one of methods which are commonly used for encapsulation.
  • a method for preparing the capsule according to the present invention is performed by a typical encapsulation method, which comprises a process for emulsification, in which strains which are obtained by culturing the strain according to the present invention, an organic acid and a coating material for capsule are dispersed in a solvent with emulsion stability, a process for production of capsule membrane, in which the emulsified dispersion is stirred to form capsule membrane, and a process for curing, in which a curing agent and a reagent are added to cure the capsule membrane.
  • the capsule formulation thus obtained can protect the strain from outer circumstances by antioxidation and thus, can be stored stably at a low temperature for a long period of time.
  • fatty acids such as CLA are unstable to heat, enzymes, acids, alkalis, microorganisms and the like.
  • microencapsulation by a proper coating material may improve storage stability and convenience for internal use.
  • the capsule formulation maintains a live strain number at a uniform level in the bowels under acid conditions by regular release control.
  • the capsule fonnulation can increase specific gravity and dispersibility in the aqueous phase of the body by microencapsulation to further enhance the bioavailability, thereby improving applicability to preserved food, milk and drinks, and dairy products.
  • the capsule formulation allows the strains according to the present invention to grow stably in the bowels.
  • the strains which have been grown in the bowels can continuously produce CLA, whereby it is possible to provide inhibition of development of the artery sclerosis, reduction of body fat, improvement of immunity, anticancer effect, growth promoting effect and the like.
  • the strains according to the present invention hold dominant species in the bowels, thereby acting as probiotics to inhibit growth of harmful microorganisms in the bowels. Therefore, it is possible to continuously activate and improve the bowel function.
  • composition comprising the capsule formulation includes, for example, dairy products (milk, soy milk, processed milk), fermented milk (liquid type yogurt, curd type yogurt), fermented food (Kimchii, soy and bean paste), animal feed, health supplementary food and the like.
  • Food compositions containing the strains according to the present invention as an effective ingredient include animal feed, fermented food such as various kinds of Kimchii, soy and bean pastes and fermented dairy products such as yogurt and cheese and can be effectively expected to prevent cancers, to enhance immunity and to reduce body fat.
  • compositions containing the strains according to the present invention as effective ingredients can be used in the prevention and treatment of diseases inhibited by CLA, such as cancers, artery sclerosis, diabetes and obesity.
  • Fig. 1 is a view showing the result of a HPLC assay to confirm the element composition of fatty acids produced by the strains according to the present invention
  • Fig. 2 is a view showing the growth conditions of the strains according to the present invention in a medium with linoleic acid (LA) added, the CLA production and the change in pH of the medium;
  • LA linoleic acid
  • Fig. 3 is a view showing the growth conditions of the strains according to the present invention in a medium without linoleic acid (LA) and the CLA production;
  • Fig. 4 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium with linoleic acid (LA) added;
  • LA linoleic acid
  • Fig. 5 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium without linoleic acid (LA); and
  • Fig. 6 is a view showing the result of measuring the growth level (a) and CLA production (b) of the strains according to the present invention which have been cultured in media, to which glucose, fructose, lactose and sucrose, respectively, were added as a carbon source. Examples
  • Example 1 Isolation, identification and characterization of strain
  • Feces of infants were collected to isolate the strains according to the present invention.
  • LA was well emulsified in Tween 80 (0.5 %, w/v), filtered through a cotton filter and added to the MRS medium.
  • Tween 80 0.5 %, w/v
  • the sterilized culture vessel was filled with the MRS medium without any room. About 300 colonies were randomly selected from the cultured medium.
  • the culture fluid was extracted with hexane and the extract was measured for absorbance at 233 nm to determine the amount of produced CLA, which was then con"ected by the amount of strain, that is, the absorbance at 600 nm to compare the relative amount of produced CLA.
  • the measurement of the live strain number was performed by plate- culturing in the MRS agar medium by 10 times dilution, placing the medium in an anaerobic tank (Difco, USA) with a gas pack for 48 hours and thereafter, counting the number of produced colonies.
  • CBG-C2 three strains which were excellent in the CLA productivity were selected and designated CBG-C2, CBG-C4 and CBG-C5, respectively.
  • the strains were identified for their genus and species by analysis of their 16S rRNA sequence.
  • the CBG-C2 strain belongs to Bifidobacterium breve
  • the CBG-C4 strain belongs to Bifidobacterium pseudocartenulatum
  • the CBG-C5 strain belongs to Enterococcus faecium.
  • CBG-C2 Korean Agricultural Culture Collection in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001 (CBG-C2), KACC 91003 (CBG-C4) and KACC 91002 (CBG-C5), respectively, on April 3, 2002.
  • CBG-C2 KCTC 10462BP
  • CBG- C4 KCTC 10208BP
  • Each strain was cultured in a medium containing LA (500 ⁇ glml) for 48 hours and centrifuged.
  • the strain was suspended in a medium or distilled water, mixed with isopropyl alcohol in a volume of twice the volume of the strain and the vigorously stirred. Then, a 1.5 volume of hexane was added and thoroughly mixed for 3 minutes by shaking. The resulting mixture was centrifuged at 3000 rpm for 5 minutes and the supernatant was measured for absorbance at 233 nm.
  • the extracted fatty acids were methylesterified according to the method described in American Oil Chemists's Society: Official Method and Recommended Practoces pf AOCS, 4th. ed.(1989) to form a sample for the GC analysis.
  • the conditions of GC were as follows: the GC DS-6200 (DONAM) with
  • the column was HP-FFAP capillary column (30m ⁇ 0.25mm, thickness 0.25/.H1), the oven temperature was 210 ° C , the injector temperature was 250 ° C and the detector temperature was 270 ° C .
  • the carrier gas was helium and eluted at a flow rate of 1 mi/min, and the split ratio was 50:1.
  • the peak areas were determined using an integrating meter (Model 3390A, Hewlett-packard, USA) equipped to the apparatus.
  • the identification of CLA was performed through comparison with the retention time of a reference material and the content of CLA was determined by the ratio of the area of CLA to the area of a reference material. The results are shown in Fig. 1. As shown in Fig. 1, all the strains showed LA peaks and CLA peaks.
  • strains according to the present invention could produce CLA using LA as a substrate.
  • the strains showed increase in the amount of produced CLA when entering to the log phase and produced the highest amount of CLA just prior to the stationary phase.
  • the pHs of the media were about 6.5 at the initial stage and drops as the time went on, whereby it was about 4.2 at about 50 hours.
  • each strain which had been activated by pre-cultivation was inoculated at 1% into the MRS medium without LA, dispensed in a 20 mi test tube and cultured. During cultivation, a growth curve was made and from 18 hours after beginning the cultivation, every 6 to 12 hours, the pH of the medium was measured.
  • the strain was collected and suspended with a Tris-HCl buffer solution in a volume of 10 times the volume of the strain and subjected to an enzyme reaction to examine the change in the factor of CLA isomerase according to the change of the growth curve.
  • LA was added to the enzyme reaction at a concentration of 10O ⁇ g/mi.
  • results obtained from the case where the strains were cultured in media containing and the results obtained from the case where the strains were cultured in media without containing LA are useful to understand the growth stage of the strains showing the highest growth factor per unit strain in development of products by fermentation of microorganisms in the presence of a substrate and in indirect production CLA by adding the strains as an effective ingredient, respectively.
  • each strain was inoculated into a medium containing LA and measured for the strain level and CLA production at 18, 24, 36 and 48 hours. The results are shown in Fig. 4.
  • each strain was inoculated into a medium without LA and cultured for 18, 24, 36 and 48 hours. After cultivation, the strain was collected mixed with a buffer solution containing LA. The mixture solution was left for 1 hour for reaction and measured for the distribution of produced CLA. The results are shown in Fig. 5.
  • strains according to the present invention can secret the produced CLA to a medium while accumulating in their body.
  • strains are useful to be added as an effective ingredient to food and medicaments for indirect production of CLA.
  • a carbon source which is the most suitable for each strain is glucose for CBG-C2 and lactose and sucrose for CBG-C4, and when the medium does not contain LA, it is lactose for CBG-C2 and lactose and sucrose for CBG-C4.
  • CBG-C2 and CBG-C4 had the highest CLA production when using glucose as a carbon source while CBG-C5 was not affected by the carbon source.
  • the pH resistance, bile salt resistance and antibiotic resistance of the strains according to the present invention were confirmed as follows. In order to examine the use of the novel strains which had been isolated and identified according to the present invention in application to products fermented by inoculating the CLA producing lactic acid bacteria or additives using CLA contained in the strains, growth features of the strains were compared.
  • the strains were cultured in a medium containing LA and a medium without containing LA and the change of each medium in pH was observed. The results are shown in Fig. 2 and Fig.3.
  • the strain according to the present invention could grow and propagate at pH in the range of 3.0 to 6.0 and particularly, the CBG-C4 strain showed the most excellent pH resistance.
  • strains according to the present invention can grow and propagate in a wide range from weak alkalinity to weak acidity, particularly in the stomach under acidic conditions resulting from the secretion of stomach acid.
  • the strains could satisfactorily grow and propagate to the bile concentration of 1000 ⁇ g/ i, whereas the control of Bacillus subtilis could no grow even at 100 ⁇ g/mt. Therefore, it was noted that the strain according to the present invention could stably grow and propagate in the gastrointestinal tract where the bile is secreted.
  • the strains which had been cultured overnight in a 20 mi test tube were diluted in the MRS medium to form a solid medium. Lactobacillus reuteri was used as control. Antibiotics used in this experiment were ampicillin, tetracycline, streptomycin, rifamycin and kanamycin, each of which was prepared at concentrations of 50 ⁇ g/mi, 1000 ⁇ g/mi and 10000 ⁇ g/ml.
  • the strains according to the present invention showed resistance to total 2 or 3 antibiotics.
  • CBG-C4 shows most excellent resistance to antibiotics at high levels, such as kanamycin at 1000 ⁇ g/mi and tetracycline at 10000 ⁇ g/mi.
  • strains according to the present invention have resistance to various kinds of antibiotics at a wide range of concentration.
  • Capsules and dairy products containing the strains according to the present invention were prepared as follows.
  • a coating material for microencapsulation of conjugated linoleic acid a mixture comprising 4 types of vegetable polysaccharides, gellan, xanthan, starch and agar, in a concentration of 1 to 5% (w/v) was prepared.
  • Sorbitan monostearate having a HLB (hydrophilic lipophilic balance) value of 4.7 as an emulsifying agent was added to the mixture, heated at 60 ° C so that it was completely dissolved, sterilized by heating and cooled down to 40 ° C to form an aqueous mixture solution.
  • sorbitan monostearate was treated at a concentration of 0.01 to 1% (w/v) and the coating material and the strain was mixed in a ratio of 7:3(w/w) and homogenized.
  • the homogenized strain suspension was treated with cooled water at 10 °C by spraying to form microcapsules.
  • the microcapsules were added in an amount of 1% (w/v) to prepare Kimchii which is one of the low-temperature fermented products.
  • the microcapsules were added in an amount of 4.2% (w/v) and for curd type yogurt and fermented soy milk, the microcapsules were added in an amount of 7.3% (w/v).
  • Each product was stored in a refrigerator kept at a low temperature of 4 ° C or 10 ° C .
  • the dairy products prepared as described above were subjected to an experiment to examine the preservation of microcapsules. As a result, it was shown that when stored in a refrigerator at 4 ° C and 10 ° C for 7 to 14 days, strains were reduced by about lO 1""2 strains/mi. Such reduction rate is very insignificant, judging from a common basis.
  • the capsules according to the present invention could stably maintain the number of strains even when stored for a long period of time and have excellent storage stability.
  • the strains according to the present invention can produce CLA from LA at a high efficiency and show outstanding resistance to stomach acids, bile salts, and antibiotics.
  • strains according to the present invention can accumulate CLA in their body after production and thus have effect to make CLA indirectly produced.
  • composition comprising the strain according to the present invention may be prepared in the form of a capsule comprising the strain according to the present invention in a coating material comprising a water-soluble polysaccharide and be usefully used in functional food and medicaments.
  • strains according to the present invention can be effectively used to biosynthesize not only CLA but also isomers of CLA in large quantities by a biological method such as immobilization.

Abstract

The present invention relates to novel strains capable of producing conjugated linoleic acid (CLA). The strains include Bifidobacterium breve CBG-C2, Bifidobacterium pseudocartenulatum CBG-C4 and Enterococcus faecium CBG-C5. The strains are excellent in producing CLA and are able to secret the produced CLA to a medium or to accumulate in the bodies thereof. Also, the strains show strong resistance to antibiotics and acids such as stomach acid or bile salt. A composition comprising the strain according tot he present invention is prepared in the form a capsule comprising the strain according to the present invention and CLA encapsulated in a coating material comprising water soluble polysaccharides and may be used in functional foods and medicaments.

Description

NEW STRAINS CAPABLE OF PRODUCING CONJUGATED LINOLEIC
ACID, CAPSULATED COMPOSITION COMPRISING THEM, AND THE
PREPARATION METHODS THEREOF
Technical Field
The present invention relates to novel strains capable of producing conjugated linoleic acid (hereinafter referred to as CLA).
Background Art CLA, which is a conjugated isomer of linoleic acid (hereinafter referred to as
LA), an essential fatty acid, is a natural fatty acid found in a small amount in milk or muscle of ruminants.
CLA has conjugated double bonds at positions cis-9 and trans-11 or at positions trans-10 and cis-12, in intra and trans configuration. Particularly, by a conjugated double bond at cis-9 and trans-11 positions, physiological activities useful to human bodies are expressed.
CLA is known to show reduction in development of the artery sclerosis
(Artery. 1997. 22:266-277), enhancement of immune system (J. Nut. 1999. 129:32-
38), anticancer effect (Anticancer research. 1997. 17:969-973), growth promotion (J. Nut. 2000. 130:2981-2989) and excellent effects of treating some diseases including diabetes and to inhibit the obesity through reduction of body fat (Am. J. Physiol.
1998. 275:R667-R672). Owing to such properties, CLA may be usefully used as an effective ingredient of functional food and medicaments.
CLA is mainly contained in animal food, particularly at a large amount in ruminant animals. It is shown that beef contains 2.9~4.3 mg CLA/fat, lamb meat contains 5.6 mg CLA/fat and marine products contain as little as 0.3-0.6 mg CLA/fat. For dairy products, milk contains 5.5 mg CLA/fat and cheese contains 3~7 rag CLA/fat. With respect to human beings' daily CLA intake, it is presumed that oriental people who principally keep a vegetable diet intake 0.1 g of CLA per day and western people who eat more meat intake 0.4 g of CLA per day.
So far, various methods have been proposed to mass-produce CLA. The conventional methods include methods for chemically synthesizing CLA from LA, such as urea addition, molecular distillation, HPLC, etc., and there are several isolated microorganism able to convert LA into CLA. However, the chemical synthesis methods have problems, such that they require expensive equipments or too much time is taken for processes. Also, since conventional chemical synthesis methods produce a kind of CLA along with various kinds of isomers, it is very inefficient to perform production of a kind of CLA by such chemical synthesis methods. Therefore, the most efficient method for producing CLA is to produce CLA by a microorganism, followed by isolation. Representative microorganisms capable of producing CLA includes microorganisms in the bowels such as Lacto bacillus, Propionibacterium, Butyrivibrio fibrisolvens etc. and are usefully used as an effective ingredient in functional food or medicaments such as probiotics and feed which are fed to animals in many countries.
However, the direct addition of CLA as an effective ingredient to food or medicaments is not allowed in Korea. Therefore, in order to employ CLA as an effective ingredient in food and medicaments, a method comprising directly adding a strain of a microorganism capable of producing CLA should be used, rather than a method comprising directly adding CLA isolated from a microorganism. As known up to now, in the synthesis of CLA by microorganisms in the bowels, only the cis-9 and trans-11 are specifically produced, unlike the chemical synthesis in which cis-9 and trans-11, and cis-12 and trans- 10 are produced in a ratio of about 50%. Though a small amount of cis-9 and trans-1 1 has been found in the meat and milk from ruminant animals, cis-9 and trans-11 are detected only in a small amount in dairy products using skim milk. Even the Kimchii which is fermented using various lactic acid bacteria is known not to contain cis-9 and trans-11. Therefore, Korean Patent Application No. 10-2001-0047292 disclosed a method for adding pure cis-9 and trans-11 type CLA which is synthesized by a microorganism of Lactobacillus genus to food. However, only an infinitesimal amount of CLA was found in an actual product.
Also, in order a CLA producing microorganism to be used as an effective ingredient in food or medicaments, it should survive at a high level similar to that upon production while the product is distributed and also should maintain a high viability and activity in the stomach and bowels after intake into a body of an animal including human beings. In addition, it should be excellent in resistance to antibiotics to hold a predominant position in the competition with harmful bacteria in the bowels, including superbacteria.
However, it was found that a number of strains, among microorganisms which are used as an effective ingredient in commercially available products, cannot survive during storage or passage through the stomach after intake and have weak resistance to antibiotics.
Therefore, it is still desired to develop a strain that shows a high viability and activity in animal bodies as well as excellent CLA productivity and itself can be directly added to food and medicaments as an effective ingredient. Disclosure of Invention
Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide novel strains which can produce conjugated linoleic acid, have excellent resistance to acids and antibiotics and can indirectly produce CLA when added to food and medicaments.
It is another object of the present invention to provide a capsule formulation comprising the strain and CLA which can be used to produce functional fermented food, dairy food and medicaments. In accordance with the present invention, the above and other objects can be accomplished by the provision of novel strains capable of producing CLA.
The strains are characterized in that they are isolated from feces of infants and they can convert LA to CLA.
The strains include Bifidobacterium breve CBG-C2, Bifidobacterium pseudocartenulatum CBG-C4 and Enterococcus faecium CBG-C5.
The strains can be isolated as follow:
Strains are isolated from feces of Korean infants and randomly selected 300 colonies are cultured in a medium with LA as a substrate. The medium is extracted with hexane and the extracts were measured for their absorbance to screen strains which have excellent CLA productivity.
The strains screened by the above procedures were once again examined whether they can produce CLA from LA, by subjecting fatty acids produced by the strains to gas chromatography (GC).
The strains thus isolated were three types, designated CBG-C2, CBG-C4 and CBG-C5, respectively and were deposited at Korean Agricultural Culture Collection in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001, KACC 91003 and KACC 91002, respectively, on April 3, 2002. Also, CBG-C2 (KCTC 10462BP) and CBG-C4 (KCTC 10208BP) were deposited at the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology on March 25, 2002 and April 10, 2003, respectively. The strains obtained by the above-described method are excellent in CLA productivity and have strong resistance to acids such as stomach acid and bile, and antibiotics.
CLA produced by microorganisms according to the present invention comprises only the stereostructures of cis-9 and trans-11, which are isomers having various physiological activities, as conventionally known to the art. Fatty acids and acyglycerol containing these structures can be widely used for development of dairy products from various animals, dairy products from vegetable materials, fermented food and functional health food, probiotics and the like. Therefore, the strains according to the present invention can be effectively used in biosynthesis of isomers of CLA by biological methods such as immobilization.
The strains according to the present invention can be added to food and medicaments as not only a live strain but also a killed strain. This is because the strains according to the present invention can release CLA to a culture fluid or reaction fluid while accumulating a large amount of CLA in the strains.
Therefore, according to the present invention, the strain which are cultured in a medium containing LA may be added as an effective ingredient to various compositions such as food and medicaments so that CLA is indirectly produced and it is thus possible to promote development of functional products containing CLA in a large amount by resolve the conventional problems in that CLA cannot be directly added to food and medicaments.
Also, the present invention provides a food or pharmaceutical composition containing the strain. The compositions according to the present invention contain at least one selected from Bifidobacterium breve CBG-C2, Bifidobacterium pseudocartenulatum CBG-C4 and Enterococcus faecium CBG-C5 as an effective ingredient.
The composition may comprise an additional effective ingredient such as an organic acid to enhance CLA production rate and improve growth stability of strain. The organic acid in the composition according to the present invention may be CLA which is chemically synthesized or is isolated and purified from microorganisms.
Particularly, the composition according to the present invention may comprise any one selected from the strains according to the present invention and CLA at the same time. The composition according to the present invention may further contain various adjuvant components in addition to the effective ingredient, when needed.
The food composition according to the present invention may comprise vitamins such as vitamin A, vitamin Bl, vitamin B2, vitamin B3, vitamin B6 and vitamin B12, folic acid, vitamin C, vitamin D3, vitamin E and the like, minerals such as copper, calcium, iron, magnesium, potassium, zinc and the like, or lactic acid bacteria and the like.
Also, as one of the food composition according to the present invention, a heath drink composition may comprise an additional component such as a flavor or natural carbohydrates, like other drinks. The flavor includes natural sweetening agents such as thaumatin and stevia extract and synthetic sweetening agents such as saccharin and aspartame. The natural carbohydrate includes monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, polysaccharides such as dextrin, cyclodextrin and the like, and sucrose alcohols such as xylitol, sorbitol, erythritol and the like. The usage or intake of the pharmaceutical composition according to the present invention is preferably 60 to 130 uM (Cancer Epidemiol. Biol. Prev. 2000. 9:689-696) and the usage or intake of the food composition according to the present invention is preferably 3.4 to 6 g/day (J. Nutr. 2000. 130:2943-2948), though it may be adjusted, as needed. The composition is stably absorbed into a body regardless of the intake. The usage or intake can be adjusted according to various factors including the type and amount of an effective ingredient and other components contained in the composition, formulation type and age, body weight, general health condition, sex and diet of a patient, administration time, administration route and release rate of the composition, treatment duration, simultaneously used drugs. Upon administration to a human body, the composition would not show side effects, as compared to conventional food and medicaments containing chemically synthesized CLA.
The composition may be formulated by combining at least one pharmaceutically acceptable or edible carrier with the effective ingredient. The pharmaceutically acceptable or edible carrier which can be used in the present invention includes a saline solution, sterile water, Ringer's solution, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof and may be combined with a conventional additive such as an antioxidant, a buffering agent, a bacteriostatic agent and the like, when needed. Also, the composition can be formulated into injections such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets by further adding a diluting agent, a dispersing agent, a surfactant, a binder and a lubricant. Further, the composition may be preferably formulated by a proper method known to the art, or a method described in Remington's Pharmaceutical Science (the latest), Mack Publishing Company, Easton PA, according to diseases or components.
The composition according to the present invention can be formulated in the form of granules, powders, coated tablets, tablets, capsules, liquids and solutions, extracts, suppositories, syrups, juices, suspensions, emulsions, release-sustained formulation of an active compound and the like. According to the present invention, there is provided a capsule formulation of the composition.
The capsule formulation comprises a coating material and a core material enclosed in the coating material.
As the core material of the capsule formulation comprises preferably both any one selected from the strains according to the present invention and CLA.
As the coating material enclosing a core material in the capsule, water- soluble polysaccharides which are excellent in absorption, dispersion and adhesion are usable.
The water-soluble polysaccharides which can be used in the present invention is at least one selected from the group consisting of starch, agar, carageenan, alginic acid, sodium alginate, polymethacrylate, wheat protein, soy protein, cellulose derivatives such as methylcellulose, hydroxylpropylcellulose, hydroxyl-propylmethylcellulose and the like; gums such as xanthan gum, arabic gum, locust bean gum, guar gum, tamarind gum, tara gum, karaya gum, tragacanth gum, ghatti gum and the like; and gellan, xanthan, pectin (LM, HM type, dextran, glucan, glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, gelatin and casein.
In the preparation of the capsule, the amount of the coating material can be adjusted according to the final use and purpose and is preferably 1 to 80 % by weight based on the weight of the core.
In addition to the water-soluble polysaccharides, the capsule may further comprise substances which are commonly used in a coating material to improve release control effect or to increase solubility.
Also, the capsule may further comprise an emulsifying agent, a protective agent and plasticizer, as needed.
The capsule formulation may be produced by using one of methods which are commonly used for encapsulation. For example, a method for preparing the capsule according to the present invention is performed by a typical encapsulation method, which comprises a process for emulsification, in which strains which are obtained by culturing the strain according to the present invention, an organic acid and a coating material for capsule are dispersed in a solvent with emulsion stability, a process for production of capsule membrane, in which the emulsified dispersion is stirred to form capsule membrane, and a process for curing, in which a curing agent and a reagent are added to cure the capsule membrane. The capsule formulation thus obtained can protect the strain from outer circumstances by antioxidation and thus, can be stored stably at a low temperature for a long period of time.
In general, fatty acids such as CLA are unstable to heat, enzymes, acids, alkalis, microorganisms and the like. However, microencapsulation by a proper coating material may improve storage stability and convenience for internal use. The capsule formulation maintains a live strain number at a uniform level in the bowels under acid conditions by regular release control.
Also, the capsule fonnulation can increase specific gravity and dispersibility in the aqueous phase of the body by microencapsulation to further enhance the bioavailability, thereby improving applicability to preserved food, milk and drinks, and dairy products.
Therefore, the capsule formulation allows the strains according to the present invention to grow stably in the bowels. The strains which have been grown in the bowels can continuously produce CLA, whereby it is possible to provide inhibition of development of the artery sclerosis, reduction of body fat, improvement of immunity, anticancer effect, growth promoting effect and the like. Also, the strains according to the present invention hold dominant species in the bowels, thereby acting as probiotics to inhibit growth of harmful microorganisms in the bowels. Therefore, it is possible to continuously activate and improve the bowel function. The composition comprising the capsule formulation includes, for example, dairy products (milk, soy milk, processed milk), fermented milk (liquid type yogurt, curd type yogurt), fermented food (Kimchii, soy and bean paste), animal feed, health supplementary food and the like.
Food compositions containing the strains according to the present invention as an effective ingredient include animal feed, fermented food such as various kinds of Kimchii, soy and bean pastes and fermented dairy products such as yogurt and cheese and can be effectively expected to prevent cancers, to enhance immunity and to reduce body fat.
Pharmaceutical compositions containing the strains according to the present invention as effective ingredients can be used in the prevention and treatment of diseases inhibited by CLA, such as cancers, artery sclerosis, diabetes and obesity.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Fig. 1 is a view showing the result of a HPLC assay to confirm the element composition of fatty acids produced by the strains according to the present invention; Fig. 2 is a view showing the growth conditions of the strains according to the present invention in a medium with linoleic acid (LA) added, the CLA production and the change in pH of the medium;
Fig. 3 is a view showing the growth conditions of the strains according to the present invention in a medium without linoleic acid (LA) and the CLA production; Fig. 4 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium with linoleic acid (LA) added;
Fig. 5 is a view for comparison of the amounts of CLA distributed in the medium and the strains, respectively, when the strains according to the present invention are cultured in a medium without linoleic acid (LA); and
Fig. 6 is a view showing the result of measuring the growth level (a) and CLA production (b) of the strains according to the present invention which have been cultured in media, to which glucose, fructose, lactose and sucrose, respectively, were added as a carbon source. Examples
Now, the present invention will be explained in further detail by the following examples. However, the present invention is not limited thereto.
Example 1 : Isolation, identification and characterization of strain
1) Isolation and identification of inventive strains
Feces of infants were collected to isolate the strains according to the present invention.
In order to isolate microorganisms which can produce CLA, 10 types of feces were taken from infants of breast feeding, weaning feeding and combined feeding and were inoculated into the MRS medium with sterilized liquid paraffin embedded therein. The medium was diluted with sterilized physiological saline (0.5%, w/v) so that about 30 to 150 colonies could be formed, plated on the MRS(Man Rogosa Sharpe) medium with 0.05% of L-cysteine, and incubated in an anaerobic tank along with a Gas pack (MGC, mitsubishi) at 37 °C for 72 hours. At this time, LA was well emulsified in Tween 80 (0.5 %, w/v), filtered through a cotton filter and added to the MRS medium. In order to establish the anaerobic conditions during cultivation, the sterilized culture vessel was filled with the MRS medium without any room. About 300 colonies were randomly selected from the cultured medium.
Each was passage cultured two times in the MRS medium and inoculated in a 20 mi test tube at a level of 1%, followed by cultivation for 48 hours. The culture fluid was extracted with hexane and the extract was measured for absorbance at 233 nm to determine the amount of produced CLA, which was then con"ected by the amount of strain, that is, the absorbance at 600 nm to compare the relative amount of produced CLA.
The measurement of the live strain number was performed by plate- culturing in the MRS agar medium by 10 times dilution, placing the medium in an anaerobic tank (Difco, USA) with a gas pack for 48 hours and thereafter, counting the number of produced colonies.
Thus, three strains which were excellent in the CLA productivity were selected and designated CBG-C2, CBG-C4 and CBG-C5, respectively. The strains were identified for their genus and species by analysis of their 16S rRNA sequence.
As a result, it was proved that the CBG-C2 strain belongs to Bifidobacterium breve, the CBG-C4 strain belongs to Bifidobacterium pseudocartenulatum and the CBG-C5 strain belongs to Enterococcus faecium.
The strains were deposited at Korean Agricultural Culture Collection in the National Institute of Agricultural Biotechnology under Accession numbers KACC 91001 (CBG-C2), KACC 91003 (CBG-C4) and KACC 91002 (CBG-C5), respectively, on April 3, 2002. Also, CBG-C2 (KCTC 10462BP) and CBG- C4(KCTC 10208BP) were deposited at the Korean Collection for Type Cultures (KCTC) in Korea Research Institute of Bioscience and Biotechnology on March 25, 2002 and April 10, 2003, respectively.
2) Confirmation of composition of inventive strains
In order to confirm that the strains according to the present invention can produce CLA by examining the composition of fatty acids produced by the strains, the following GC was perfonned.
Each strain was cultured in a medium containing LA (500 βglml) for 48 hours and centrifuged. The strain was suspended in a medium or distilled water, mixed with isopropyl alcohol in a volume of twice the volume of the strain and the vigorously stirred. Then, a 1.5 volume of hexane was added and thoroughly mixed for 3 minutes by shaking. The resulting mixture was centrifuged at 3000 rpm for 5 minutes and the supernatant was measured for absorbance at 233 nm. The extracted fatty acids were methylesterified according to the method described in American Oil Chemists's Society: Official Method and Recommended Practoces pf AOCS, 4th. ed.(1989) to form a sample for the GC analysis. The conditions of GC were as follows: the GC DS-6200 (DONAM) with
FID attached was used, the column was HP-FFAP capillary column (30mχ0.25mm, thickness 0.25/.H1), the oven temperature was 210°C , the injector temperature was 250 °C and the detector temperature was 270 °C . The carrier gas was helium and eluted at a flow rate of 1 mi/min, and the split ratio was 50:1. The peak areas were determined using an integrating meter (Model 3390A, Hewlett-packard, USA) equipped to the apparatus. The identification of CLA was performed through comparison with the retention time of a reference material and the content of CLA was determined by the ratio of the area of CLA to the area of a reference material. The results are shown in Fig. 1. As shown in Fig. 1, all the strains showed LA peaks and CLA peaks.
Therefore, it was noted that the strains according to the present invention could produce CLA using LA as a substrate.
3) Determination of optimal condition for CLA production by inventive strains In order to determine the optimal conditions for CLA production by the strains according to the present invention, the strains were cultured under two conditions, in which one is in a medium with LA and the other is in a medium without LA, and characterized for their growth. Firstly, each strain which had been activated by pre-cultivation was inoculated at 1% into 2D of the MRS medium containing LA (500 g/m£). Every hour, the medium was collected and measured for the strain level, amount of CLA and pH. The results are shown in Fig. 2.
As shown in Fig. 2, the strains showed increase in the amount of produced CLA when entering to the log phase and produced the highest amount of CLA just prior to the stationary phase. The pHs of the media were about 6.5 at the initial stage and drops as the time went on, whereby it was about 4.2 at about 50 hours.
Meanwhile, each strain which had been activated by pre-cultivation was inoculated at 1% into the MRS medium without LA, dispensed in a 20 mi test tube and cultured. During cultivation, a growth curve was made and from 18 hours after beginning the cultivation, every 6 to 12 hours, the pH of the medium was measured.
The strain was collected and suspended with a Tris-HCl buffer solution in a volume of 10 times the volume of the strain and subjected to an enzyme reaction to examine the change in the factor of CLA isomerase according to the change of the growth curve. Here, LA was added to the enzyme reaction at a concentration of 10O μg/mi.
The results are shown in Fig. 3.
As shown in Fig. 3, it took more time to reach the stationary phase when culturing in the medium without LA than in the medium with LA. The final pH of the medium was about 4.2. Therefore, it was noted that the strains according to the present invention could produce the greatest amount of CLA just before they enter from the log phase to stationary phase.
Also, the results obtained from the case where the strains were cultured in media containing and the results obtained from the case where the strains were cultured in media without containing LA are useful to understand the growth stage of the strains showing the highest growth factor per unit strain in development of products by fermentation of microorganisms in the presence of a substrate and in indirect production CLA by adding the strains as an effective ingredient, respectively.
4) Confirmation of amount of CLA distributed in medium or inventive strain upon cultivation of inventive strains
In order to examine amounts of CLA distributed in a medium and the strain when the strains according to the present invention were cultured, the following experiments were performed. Firstly, each strain was inoculated into a medium containing LA and measured for the strain level and CLA production at 18, 24, 36 and 48 hours. The results are shown in Fig. 4.
As shown in Fig. 4, at the early stage of cultivation, that is, 18 hours later, the ratio of an amount of CLA in a strain to an amount of CLA in a medium was 1 :1.85 for CBG-C2, 1 :2.1 for CBG-C4 and 1 : 1.54 for CBG-C5. As the time went on, the amount of CLA in the medium increased. 48 hours later, the ratio was 1 :3.5 for CBG-C2, 1:2.9 for CBG-C4 and 1 :2 for CBG-C5, as shown in Table 1. <Table 1>
Figure imgf000019_0001
Meanwhile, each strain was inoculated into a medium without LA and cultured for 18, 24, 36 and 48 hours. After cultivation, the strain was collected mixed with a buffer solution containing LA. The mixture solution was left for 1 hour for reaction and measured for the distribution of produced CLA. The results are shown in Fig. 5.
As shown in Fig. 5, as the cultivation progressed, the amount of CLA increased. After 36 hours, the distribution of CLA produced by the strain for 1 hour was 1.3:1, 1.6:1 and 0.6:1 in a ratio of strain to medium for CBG-C2, CBG-C4 and CBG-C5, respectively, as shown in Table 2. <Table 2>
Figure imgf000019_0002
Therefore, it was noted that the strains according to the present invention can secret the produced CLA to a medium while accumulating in their body.
Such properties of the strains are useful to be added as an effective ingredient to food and medicaments for indirect production of CLA.
5) Growth of inventive strains and CLA production according to the type of carbon source
In order to examine the growth of the strains and their CLA production according to the type of a carbon source, glucose, the strains were cultured in media containing various carbon sources including fructose, lactose and sucrose and the growth level and CLA production were measured. The results are shown in Fig. 6. •
As shown in Fig. 6a, when the medium contains LA, a carbon source which is the most suitable for each strain, is glucose for CBG-C2 and lactose and sucrose for CBG-C4, and when the medium does not contain LA, it is lactose for CBG-C2 and lactose and sucrose for CBG-C4.
Meanwhile, after cultivation in the medium with LA for 48 hours, CBG-C2 and CBG-C4 had the highest CLA production when using glucose as a carbon source while CBG-C5 was not affected by the carbon source.
6) Verification of resistance of inventive strains to pH, bile salt and stomach acid
The pH resistance, bile salt resistance and antibiotic resistance of the strains according to the present invention were confirmed as follows. In order to examine the use of the novel strains which had been isolated and identified according to the present invention in application to products fermented by inoculating the CLA producing lactic acid bacteria or additives using CLA contained in the strains, growth features of the strains were compared.
1) Resistance to pH
In order to confirm the pH resistance of the strains according to the present invention, the strains were cultured in a medium containing LA and a medium without containing LA and the change of each medium in pH was observed. The results are shown in Fig. 2 and Fig.3.
As shown in Fig. 2 and Fig. 3, as the growth of the strains come into the stationary phase whether the medium contains LA or not, the pH of the medium was dramatically reduced. It was found that the final pH dropped to pH 4.2. Therefore, it was noted that typical fermentation by lactic acid bacteria occurs in the medium.
Based on the above results, various MRS media having pH in the range of 2 to 6 were prepared to confirm the growth of the strains according to the pH change of the medium. Each strain was inoculated into a media and cultured at 37°C for 48 hours while the medium was observed to know whether the strain was growing and propagating. The results are shown in Table 3. <Table 3>
Figure imgf000021_0001
(-: no propagation, +: a little propagation, ++: abundant propagation)
As seen from Table 3, the strain according to the present invention could grow and propagate at pH in the range of 3.0 to 6.0 and particularly, the CBG-C4 strain showed the most excellent pH resistance.
Therefore, it was noted that the strains according to the present invention can grow and propagate in a wide range from weak alkalinity to weak acidity, particularly in the stomach under acidic conditions resulting from the secretion of stomach acid.
2) Resistance to bile
In order to confirm the bile resistance of the strains according to the present invention, various MRS media containing sodium deoxycholate, a component of the bile, at concentrations of 0, 100, 300, 500, 800 and 1000 μg/mi were prepared. Each medium inoculated with the strains and cultured at 37 °C for 48 hours while the growth of the strains was examined. Bacillus subtilis which does not show resistance to the bile was used as control. The results are shown in Table 4. <Table 4>
Figure imgf000022_0001
(-: no propagation, +; a little propagation, ++: abundant propagation)
As seen from Table 4, the strains could satisfactorily grow and propagate to the bile concentration of 1000 βg/ i, whereas the control of Bacillus subtilis could no grow even at 100 μg/mt. Therefore, it was noted that the strain according to the present invention could stably grow and propagate in the gastrointestinal tract where the bile is secreted.
3) Resistance to antibiotics
In order to confirm the resistance to antibiotics of the strains according to the present invention, the strains which had been cultured overnight in a 20 mi test tube were diluted in the MRS medium to form a solid medium. Lactobacillus reuteri was used as control. Antibiotics used in this experiment were ampicillin, tetracycline, streptomycin, rifamycin and kanamycin, each of which was prepared at concentrations of 50 μg/mi, 1000 βg/mi and 10000 μg/ml.
Each of the antibiotics at different concentrations was dropped on paper discs in an amount of 40 μl, followed by drying. Each disc was placed on the medium with the strains mixed and cultured at 37°C for 48 hours while examining the growth of the strains. The results are shown in Table 5.
<Table 5>
Figure imgf000024_0001
As seen from Table 5, the strains according to the present invention showed resistance to total 2 or 3 antibiotics. Particularly, CBG-C4 shows most excellent resistance to antibiotics at high levels, such as kanamycin at 1000 μg/mi and tetracycline at 10000 μg/mi.
Therefore, it was noted that the strains according to the present invention have resistance to various kinds of antibiotics at a wide range of concentration.
Example 2 Preparation of capsules and dairy products containing strains and
CLA
Capsules and dairy products containing the strains according to the present invention were prepared as follows.
In order to prepare a coating material for microencapsulation of conjugated linoleic acid, a mixture comprising 4 types of vegetable polysaccharides, gellan, xanthan, starch and agar, in a concentration of 1 to 5% (w/v) was prepared. Sorbitan monostearate having a HLB (hydrophilic lipophilic balance) value of 4.7 as an emulsifying agent was added to the mixture, heated at 60 °C so that it was completely dissolved, sterilized by heating and cooled down to 40 °C to form an aqueous mixture solution. At this time, sorbitan monostearate was treated at a concentration of 0.01 to 1% (w/v) and the coating material and the strain was mixed in a ratio of 7:3(w/w) and homogenized.
The homogenized strain suspension was treated with cooled water at 10 °C by spraying to form microcapsules.
The microcapsules were added in an amount of 1% (w/v) to prepare Kimchii which is one of the low-temperature fermented products. In preparation of various dairy products, for milk, soy milk, liquid type yogurt, the microcapsules were added in an amount of 4.2% (w/v) and for curd type yogurt and fermented soy milk, the microcapsules were added in an amount of 7.3% (w/v). Each product was stored in a refrigerator kept at a low temperature of 4 °C or 10 °C .
The dairy products prepared as described above were subjected to an experiment to examine the preservation of microcapsules. As a result, it was shown that when stored in a refrigerator at 4°C and 10°C for 7 to 14 days, strains were reduced by about lO1""2 strains/mi. Such reduction rate is very insignificant, judging from a common basis.
Therefore, it was noted that the capsules according to the present invention could stably maintain the number of strains even when stored for a long period of time and have excellent storage stability.
Industrial Applicability
The strains according to the present invention can produce CLA from LA at a high efficiency and show outstanding resistance to stomach acids, bile salts, and antibiotics.
Also, the strains according to the present invention can accumulate CLA in their body after production and thus have effect to make CLA indirectly produced.
Therefore, a composition comprising the strain according to the present invention may be prepared in the form of a capsule comprising the strain according to the present invention in a coating material comprising a water-soluble polysaccharide and be usefully used in functional food and medicaments.
Further, the strains according to the present invention can be effectively used to biosynthesize not only CLA but also isomers of CLA in large quantities by a biological method such as immobilization.
Original (for SUBMISSION)- prinledcn 12.04.2003 1211:37 PM -1 Form -PCT/RO/134 (EASY) Indications Relating to Deposited Microorgaπism(s) or Other Biological Material (PCT Rule 13bis) -1-1 Prepared using PCT-EASY Version 2.92 (updated 01.01.2003) -2 International Application No.
-3 Applicant's or agent's file reference 03PPO3O
The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: -1 page 12 ? line 11 -3 Identification of Deposit -3-1 Name of depositary institution Korean Collection for Type Cultur -es -3? Address of depositary institution 52, Oun-dong, Yusong-Ku, Taejon 3 05-333,
Republic of Korea -3-3 Date of deposit 10 April 2003 (10.04.2003) -34 Accession Number KCTC 10462BP
Additional Indications NONE -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE
These indications will be submitted to the International Bureau later
The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: -1 page 12 -2 line 12 -3 Identification of Deposit -3-1 Name of depositary institution Korean Collection for Type Cultures -32 Address of depositary institution 52, Oun-dong, Yusong-Ku, Taejon. 3 05-333
Republic of Korea -3-3 Date of deposit 10 April 2003 (10.04.2003) -3-4 Accession Number KCTC 10208BP -4 Additional Indications NONE -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE
These indications will bo submitted to the International Bureau later Original (for SUBMISSION) - printed on 12.04.2003 12:11 :37 PM FOR RECEIVING OFFICE USE ONLY -4 This form was received with the international application: (yes or no) -4-1 Authorized officer
FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the international Bureau on: -5-1 Authorized officer

Claims

What is claimed is:
1. A novel strain capable of converting LA (linoleic acid) to CLA (conjugated linoleic acid).
2. The strain according to claim 1, which is Bifidobacterium breve CBG-C2.
3. The strain according to claim 2, wherein the strain is deposited under accession number KACC 91001 .
4. The strain according to claim 2, wherein the strain is deposited under accession number KCTC 10462BP .
5. The strain according to claim 1, which is Bifidobacterium pseudocartenulatum CBG-C4.
6. The strain according to claim 5, wherein the strain is deposited under accession number KACC 91003.
7. The strain according to claim 1, which is Enterococcus faecium CBG-C5.
8. The strain according to claim 8, wherein the strain is deposited under accession number KACC 91002.
9. The starain according to calim 7, wherein the strain is deposited under accession number KCTC 10208BP.
10. A composition for producing CLA comprising a strain claimed in any one of claims 1 to 9 as an effective ingredient.
11. The composition according to claim 10, which is for prevention or treatment of diseases suppressed by CLA such as cancers, arteriolo sclerosis, diabetes and obesity.
12. A capsule formulation comprising a strain claimed in any one of claims 1 to 9 and CLA as a core material and a water-soluble polysaccharide as a coating material.
13. A method for producing CLA from LA by using a strain claimed in any one of claims 1 to 9.
14. A method for producing CLA by adding a strain claimed in any one of claims 1 to 9 as an effective ingredient to food or medicaments.
PCT/KR2003/000742 2002-04-12 2003-04-12 New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the preparation methods thereof WO2003087344A1 (en)

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WO2006070891A1 (en) * 2004-12-28 2006-07-06 Meiji Seika Kaisha, Ltd. Novel strain conferring anti-disease properties to host and bacterial cell composition
EP1789531A1 (en) * 2004-08-16 2007-05-30 PL Bio Co., Ltd. Lactobacillus rhamnosus with body-fat reducing activity and the foods containing them
JP2008511312A (en) * 2004-09-02 2008-04-17 ピーエル バイオ カンパニー リミテッド Lactobacillus plantarum with reduced body fat and food containing it (LACOTABASILLUSPLANTARUMITWIBODY-FATREDUCINGAACTIVITYANDTHEFOODSCONTAININGTHEM)
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US8497114B2 (en) 2009-09-17 2013-07-30 Morinaga Milk Industry Co., Ltd. Anti-obesity agent, anti-obesity food or beverage, glucose tolerance-ameliorating agent, and food or beverage for amelioration of glucose tolerance
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