WO2019112217A1 - Lactobacillus reuteri oh0335 strain providing high yield of reuterin from glycerol and use thereof - Google Patents

Lactobacillus reuteri oh0335 strain providing high yield of reuterin from glycerol and use thereof Download PDF

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WO2019112217A1
WO2019112217A1 PCT/KR2018/014545 KR2018014545W WO2019112217A1 WO 2019112217 A1 WO2019112217 A1 WO 2019112217A1 KR 2018014545 W KR2018014545 W KR 2018014545W WO 2019112217 A1 WO2019112217 A1 WO 2019112217A1
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strain
present
lactobacillus
glycerol
culture
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French (fr)
Korean (ko)
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김철호
오백록
박두상
서정우
허선연
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한국생명공학연구원
주식회사 엑티브온
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • 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
    • 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
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • 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
    • A23V2400/11—Lactobacillus
    • 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
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to Lactobacillus luteriol OH 0335 strain, which highly produces ruterin from glycerol, and its use.
  • microorganisms are one of the causes of corruption in foods, feeds, and cosmetics.
  • toxic and carcinogenic mycotoxins produced by microorganisms are harmful to health due to various adverse effects at the time of ingestion (Schaefer et al., Microbiology 2010, 156: 1589-1599).
  • corruption of food, feed, cosmetics, etc. has enormous economic impact.
  • add a biological or chemical preservative to prevent this decay.
  • chemical preservatives safety issues such as body scaling are constantly emerging and have a negative effect on the human body depending on the type and amount of the substance.
  • income levels have improved, there has been a growing demand for biocompatible biological preservatives along with a health-oriented tendency and avoiding the use of chemical preservatives.
  • Lactobacillus luteri is known to produce 3-hydroxypropionaldehyde (3-HPA), a natural antimicrobial substance called ruterin.
  • Rutherin has a broad spectrum of antimicrobial spectrum because it has antimicrobial activity against yeast, fungi as well as pathogenic bacteria (Doleyres et al., A ppl Microbiol Biotechnol 2005, 68: 467-474). Rutherin has been actively pursuing research into the food industry and medical treatment.
  • Korean Patent No. 1483012 discloses a method for producing 3-hydroxypropionic acid in high yield using recombinant Escherichia coli
  • Korean Patent No. 1505172 discloses a method for producing 3-hydroxypropionic acid Microbes and a method for producing 3-hydroxypropionic acid using the same, "but, as in the present invention, the lactobacillus luterian OH 0335 strain producing high levels of ruterin from glycerol and its use has never been disclosed.
  • the present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to newly isolate Lactobacillus luterian OH 0335 strain (KCTC13362BP), a GRAS strain high in production of ruterin called 3-hydroxypropionaldehyde (3-HPA) And the strain was able to produce about 300 mM rutherin from 540 mM glycerol. This enables the production of very high levels of rutherin from glycerol, thus completing the present invention.
  • Lactobacillus luterian OH 0335 strain KCTC13362BP
  • 3-HPA 3-hydroxypropionaldehyde
  • the present invention provides a lactobacillus ( lactobacillus) reuteri ) OH0335 strain (KCTC13362BP).
  • the present invention also provides a microorganism preparation for producing rutherin containing the above strain or a culture solution thereof as an active ingredient.
  • the present invention provides an antimicrobial composition comprising the strain or a culture solution thereof as an active ingredient.
  • the present invention also provides an antimicrobial feed composition comprising the strain or a culture thereof.
  • the present invention also provides a dressing composition comprising the strain or a culture thereof.
  • the present invention also provides a method for producing rutherin comprising the step of culturing the strain.
  • the Lactobacillus luteriol OH 0335 strain of the present invention was confirmed to have an excellent ability to convert glycerol to ruterin. Therefore, the lactic acid producing strain Lactobacillus luterian OH 0335 according to the present invention is a kind of lactic acid bacterium which is a generically Recognized As Safe (GRAS), and when the waste glycerol is used for the production of ruterin, Unlike strains, it is very useful in related industries because it can provide advantages such as safety, environmental aspect, and recycling of resources.
  • GRAS generically Recognized As Safe
  • FIG. 1 is a diagram showing a taxonomic diagram of a novel Lactobacillus luteriol OH 0335 strain isolated in the present invention.
  • FIG. 2 is a graph showing the production ability of 1,3-propanediol in the novel Lactobacillus luteriol OH 0335 strain isolated from the present invention and the production ability of rutherin using the reactivation cell.
  • the present invention relates to a lactobacillus reuteri ) OH0335 strain (KCTC13362BP).
  • the Lactobacillus luterian OH0335 strain (KCTC13362BP) of the present invention was confirmed to be able to highly produce ruterin at about 300 mM from 540 mM glycerol. Therefore, the strain of the present invention can produce ruterin at 280 mM or more, preferably 280 to 320 mM, more preferably 290 to 310 mM, still more preferably 295 to 305 mM.
  • the above strain was deposited on October 13, 2017 with the deposit number KCTC13362BP by the Korea Research Institute of Bioscience and Biotechnology (KCTC).
  • the present invention also provides a microorganism preparation for producing rutherin containing a strain or a culture solution thereof as an active ingredient.
  • the strain may be a lactobacillus reuteri strain, preferably, but not limited to, Lactobacillus lutea OH 0335 strain (KCTC13362BP).
  • the present invention provides an antimicrobial composition comprising the strain or a culture solution thereof as an active ingredient.
  • antimicrobial is meant the ability to reduce, prevent, inhibit, or eliminate the growth or survival of the bacteria at any concentration.
  • the Lactobacillus luterian OH0335 strain (KCTC13362BP) of the present invention produces a natural antimicrobial substance 3-hydroxypropionaldehyde called ruterin as a GRAS strain, and ruterin has antibacterial activity against yeast and mold as well as pathogenic bacteria
  • the antimicrobial spectrum has very wide characteristics.
  • the composition may be in the form of a food, a food additive, a feed or a feed additive, and the food may be selected from the group consisting of dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, But are not limited to, be selected from the group consisting of drinks, meats, sausages, breads, chocolates, candies, snacks, confections, pizza, ramen, gums, ice cream, soups, beverages, alcoholic beverages and vitamins .
  • dairy products milk, soy milk, processed milk
  • fermented milk liquid yogurt
  • the food of the present invention may contain a functional food.
  • various auxiliary ingredients may be added as necessary.
  • vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.
  • health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages.
  • the flavoring agent include natural sweetening agents such as tau martin and stevia extract, and synthetic sweetening agents such as saccharine and aspartame.
  • natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • the strain or its culture obtained in the step of culturing the strain of the present invention is used as a food additive
  • the strain or the culture thereof may be added as it is, or may be used together with other food or food ingredients, .
  • the amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).
  • the present invention also provides an antimicrobial feed composition comprising the strain or a culture thereof.
  • the feed additive of the present invention is added to the base feed at a certain ratio.
  • the basic diet may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, and mineral premix.
  • the vitamin premix can be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin
  • the mineral premix can be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium.
  • feeds may include, but are not limited to, livestock feed, broiler feed, cattle feed, and the like.
  • the present invention also provides a dressing composition comprising the strain or a culture thereof.
  • compositions of the present invention may be prepared according to conventional methods for preparing dressing compositions and may generally be in lyophilized or encapsulated form or in a culture suspension or in the form of a dry powder.
  • one or more pharmaceutically acceptable conventional carriers or one or more additives to the effective amount of the above-mentioned Lactobacillus lutea OH0335 strain (KCTC13362BP) or a culture thereof as a main component, Can be manufactured.
  • the carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives.
  • the additives include one or more of flavorings, vitamins and antioxidants Can be used.
  • the carrier and the additive may be any pharmaceutically acceptable ones.
  • the diluent include lactose monohydrate, trehalose, corn starch, soybean oil, Microcrystalline cellulose or mannitol is preferred and the lubricant is preferably magnesium stearate or talc and the binder may be polyvinylpyrrolidone (PVP) or polyvinylpyrrolidone And hydroxypropylcellulose (HPC).
  • the disintegrant may be selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone
  • the sweetener is selected from white sugar, fructose, sorbitol or aspartame.
  • the stabilizer examples include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta-cyclodextrin) white bee's wax or xanthan gum and the preservative is selected from the group consisting of methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate).
  • the strain culture obtained by inoculating and culturing the strain culture obtained by inoculating and culturing the Lactobacillus luteriol OH 0335 strain (KCTC13362BP) with a microorganism having a probiotic activity is administered to an animal as a probiotic activity- Or may be administered to an animal together with food, medicines, animal drugs or lactic acid bacteria, and is preferably used as a feed additive or supplementary feed for livestock, and the nutrient rich in colostrum and useful physiological activity It is possible to increase the rate of growth of swine and eliminate the occurrence of diarrhea in pigs.
  • the present invention also provides a method for producing rutherin comprising the step of culturing the strain.
  • the method of producing the rutherin of the present invention may further comprise the step of recovering rutherin from the culture broth of the strain.
  • the step of recovering the ruthenin from the culture broth of the strain can be carried out by conventional separation techniques such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction and the like, They can be used in combination to separate them.
  • the method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and is not limited to a specific method.
  • Pig small intestine and duodenum samples were collected from a pig slaughterhouse in Iksan, Jeollabuk-do, and 1 g of finely crushed samples were suspended in 10 mL of physiological saline, diluted to 1 ⁇ 10 -4 times, and cultured in MRS solid medium containing bromocresol purple 5 g / L of yeast extract, 20 g / L dextrose, 1 g / L of polysorbate 80, 2 g / L of ammonium citrate, 5 g / L of sodium acetate, 10 g / L of protease peptone NO.3, 10 g / L of beef extract, 100 ⁇ l each was plated on each of the wells at a temperature of 37 ° C, and the plate was immersed in a Bactron Anaerobic Chamber (Bruker Daltonik, Billerica, CA, USA) for 24 hrs.
  • MRS solid medium containing bromocresol purple 5 g / L of yeast extract,
  • the colonies were re-inoculated on the MRS medium containing bromocresol purple and purified by MALDI Biotyper (Bruker Daltonik, Billerica, MA USA), 81 strains of Lactobacillus luterium It was isolated.
  • 3 mL of MRS medium (15 mL falcon tube) was pre-cultured for 24 hours in an anaerobic condition using a Bactron Anaerobic Chamber at 37 ° C, and then cultured in 50 mL of lactic acid culture medium (10 g / L Protease Peptone NO.3, 10 g / L beef L of yeast extract, 20 g / L dextrose, 1 g / L polysorbate 80, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 g / L dipotassium sulfate) (250 mL round-bottomed flask) was incubated in an anaerobic condition for 24 hours at 37 ° C in an initial inoculation OD of 0.5 using a Bactron Anaerobic Chamber.
  • lactic acid culture medium (10 g / L Protease
  • the culture was centrifuged at 13,000 rpm for 5 minutes, and the cells were recovered and washed twice with 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in a 200 mM glycerol solution, allowed to stand in a 30 ° C incubator, and sampled at intervals of 1 hour. The reaction samples were centrifuged at 13,000 rpm for 5 minutes and then used for analysis. Quantitative analysis of ruterin used acrolein as a standard.
  • 16S rRNA gene sequence was analyzed for molecular biology identification of Lactobacillus luterian OH0335 strain, which has the highest production of ruterin.
  • the chromosomal DNA was isolated from a single colony using a genomic DNA extraction kit (Invitrogen, Germany), and the primer 5'-AGAGTTTGATCMTGGCTCAG-3 '(27f, SEQ ID NO: 2) for amplifying the Lactobacillus 16S rRNA gene and 5' 16S rRNA gene DNA was amplified by PCR using -TACGGYTACCTTGTTACGACTT-3 '(1492 r, SEQ ID NO: 3).
  • PCR amplification was carried out by preparing a PCR reaction solution (50 ⁇ l) containing Ex Taq polymerase (Takara) (2.5 U), polymerase buffer, dNTP mixture (1 mM each), 1 ml of each primer (100 pmol) and 500 ng of template DNA , And gene amplification (Takara, Japan) for 30 times at 96 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 2 minutes.
  • the PCR reaction solution was electrophoresed on 1% agarose gel, and E. coli EPI300 was transformed with pGEM-TEasy vector (Promega, USA) after confirming amplification of the DNA fragment of the expected size.
  • the plasmid DNA was extracted from the transformed recombinant E. coli (Qiagen, USA) and the restriction enzyme Eco RI was treated to confirm that the DNA fragment of the desired size was cloned and the nucleotide sequence was determined (SEQ ID NO: 1). Based on the result of homology analysis of the nucleotide sequence, the isolated lactic acid bacterium was named Lactobacillus reuteri OH 0335.
  • Example 2 New isolated lactic acid bacteria by fermentation Lactobacillus Luteri Of the strain OH0335 Ruterin production
  • 5 g / L yeast extract, 20 g / L dextrose, 1 g / L Polysorbate 80, and 10 g / L protein extract were added to the MRS medium (10 g / L Proteose Peptone NO.3, 10 g / L beef extract, L of ammonium citrate, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 2 g / L of dipotassium sulfate) and centrifuged at 13,000 rpm for 5 minutes. Was collected and washed twice with 50 mM sodium phosphate buffer (pH 7.5).
  • MRS medium 10 g / L Proteose Peptone NO.3, 10 g / L beef extract, L of ammonium citrate, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 0.05 g /
  • the washed cells were resuspended in a 200 mM glycerol solution, allowed to stand in a 30 ° C incubator, and sampled at intervals of 1 hour.
  • ruterin was produced at a cell concentration of 10 g DCW / L and 540 mM glycerol at 299.8 mM for 1 hour as shown in Table 2 below.
  • Example 3 Using a fermentor Oil price formula New isolated lactobacilli through culture Lactobacillus Luteri OH0335 The 1,3- Propanediol And Ruterin production
  • Lactobacillus luteriol OH 0335 strain was used to culture the MRS medium (20 g / L glycerol, 10 g / L protease peptone NO.3, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L deck L diatomite sulfate, 1 g / L polysorbate 80, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1,3-propanediol and by-products were investigated. As a result, as shown in Fig.
  • the cells cultured for 72 hours were centrifuged at 13,000 rpm for 5 minutes, collected, re-suspended in a 3 L MRS medium in a 5 L incubator, and reactivated for 4 to 6 hours.
  • the reactivation culture was centrifuged at 13,000 rpm for 5 minutes, and the cells were recovered and washed twice with 50 mM sodium phosphate buffer (pH 7.5).
  • the washed cells were resuspended in 540 mM glycerol solution at a cell concentration of 10 g DCW / L, subjected to a stationary reaction at 30 ° C in an incubator, and samples were collected at intervals of 1 hour.
  • Table 4 shows that ruterin was produced at 244.5 mM 2 hours after the reaction.

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Abstract

The OH0335 strain of Lactobacillus reuteri of the present invention is confirmed to have an extremely excellent capability of converting glycerol to reuterin. Consequently, the OH0335 strain of Lactobacillus reuteri providing a high yield of reuterin of the present invention is a type of lactobacillus which is generally recognized as safe (GRAS) to the human body, and thus, if used in the production of reuterin from waste glycerol, is safe, unlike conventional strains of GMO, while providing the advantage of being environmentally friendly and enabling recycling of resources, and thus is very useful to the relevant industries.

Description

글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리 OH0335 균주 및 이의 용도Lactobacillus luteriol OH 0335 strain producing high levels of rutherin from glycerol and uses thereof
본 발명은 글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리 OH0335 균주 및 이의 용도에 관한 것이다.The present invention relates to Lactobacillus luteriol OH 0335 strain, which highly produces ruterin from glycerol, and its use.
This application is financially supported by the 2017 Commercialization Project for Promising Technologies of the goverment of the Republic of Korea.This application is financially supported by the 2017 Commercialization Project for Promising Technologies of the Republic of Korea.
일반적으로 미생물은 식품, 사료, 화장품 등의 부패 원인 중의 하나이다. 특히, 미생물에 의해 생성된 독성 및 발암성 마이코톡신은 사람이 섭취시에는 여러 가지 부작용으로 인해 건강에 해롭다(Schaefer et al., Microbiology 2010, 156:1589-1599). 또한, 식품, 사료, 화장품 등의 부패는 막대한 경제적 영향을 미친다. 이러한 부패를 막기 위해, 생물학적 또는 화학적 보존제를 첨가한다. 그러나, 화학적 보존제의 경우 체내 축척 등 안전성에 관한 문제가 지속적으로 대두되고 있고 물질의 종류, 사용량에 따라 인체에 부정적 영향을 준다. 소득 수준이 향상되면서 건강지향적 성향과 함께 생체친화형 생물학적 보존제에 대한 요구가 높아지고 있고, 화학적 보존제의 사용을 피하고 있다.In general, microorganisms are one of the causes of corruption in foods, feeds, and cosmetics. In particular, toxic and carcinogenic mycotoxins produced by microorganisms are harmful to health due to various adverse effects at the time of ingestion (Schaefer et al., Microbiology 2010, 156: 1589-1599). In addition, corruption of food, feed, cosmetics, etc. has enormous economic impact. To prevent this decay, add a biological or chemical preservative. However, in the case of chemical preservatives, safety issues such as body scaling are constantly emerging and have a negative effect on the human body depending on the type and amount of the substance. As income levels have improved, there has been a growing demand for biocompatible biological preservatives along with a health-oriented tendency and avoiding the use of chemical preservatives.
락토바실러스 루테리는 루테린으로 불리는 천연 항균성 물질 3-HPA(3-hydroxypropionaldehyde)를 생산하는 것으로 알려져 있다. 루테린은 병원성 세균뿐만 아니라 효모, 곰팡이 등에 대한 항균력을 보유하고 있어 항균 스펙트럼이 매우 넓은 특성을 가지고 있다(Doleyres et al., Appl Microbiol Biotechnol 2005, 68: 467-474). 그동안 루테린은 식품산업 및 의학치료에 적용하려는 연구들이 활발하게 진행 중이다. Lactobacillus luteri is known to produce 3-hydroxypropionaldehyde (3-HPA), a natural antimicrobial substance called ruterin. Rutherin has a broad spectrum of antimicrobial spectrum because it has antimicrobial activity against yeast, fungi as well as pathogenic bacteria (Doleyres et al., A ppl Microbiol Biotechnol 2005, 68: 467-474). Rutherin has been actively pursuing research into the food industry and medical treatment.
한편, 한국등록특허 제1483012호에서는 '재조합 대장균을 이용하여 3-히드록시프로피온산을 고수율로 생산하는 방법'이 개시되어 있고, 한국등록특허 제1505172호에서는 '3-히드록시프로피온산을 생산하는 재조합 미생물 및 이를 이용한 3-히드록시프로피온산의 생산방법'이 개시되어 있으나, 본 발명에서와 같이, '글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리 OH0335 균주 및 이의 용도'에 대해서는 밝혀진 바가 전혀 없다.Korean Patent No. 1483012 discloses a method for producing 3-hydroxypropionic acid in high yield using recombinant Escherichia coli, and Korean Patent No. 1505172 discloses a method for producing 3-hydroxypropionic acid Microbes and a method for producing 3-hydroxypropionic acid using the same, "but, as in the present invention, the lactobacillus luterian OH 0335 strain producing high levels of ruterin from glycerol and its use has never been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 천연 항균성 물질인 3-HPA(3-hydroxypropionaldehyde)로 불리는 루테린을 고생산하는 GRAS 균주인 락토바실러스 루테리 OH0335 균주(KCTC13362BP)를 새롭게 분리하였으며, 상기 균주는 540mM의 글리세롤로부터 약 300mM의 루테린을 고생산할 수 있는 점을 확인하였다. 이를 통해 글리세롤로부터 매우 높은 루테린의 생산이 가능하게 됨으로써, 본 발명을 완성하였다. SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to newly isolate Lactobacillus luterian OH 0335 strain (KCTC13362BP), a GRAS strain high in production of ruterin called 3-hydroxypropionaldehyde (3-HPA) And the strain was able to produce about 300 mM rutherin from 540 mM glycerol. This enables the production of very high levels of rutherin from glycerol, thus completing the present invention.
상기 과제를 해결하기 위해, 본 발명은 글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리(lactobacilus reuteri) OH0335 균주(KCTC13362BP)를 제공한다.In order to solve the above problems, the present invention provides a lactobacillus ( lactobacillus) reuteri ) OH0335 strain (KCTC13362BP).
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 함유하는 루테린 생산용 미생물 제제를 제공한다.The present invention also provides a microorganism preparation for producing rutherin containing the above strain or a culture solution thereof as an active ingredient.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 항균용 조성물을 제공한다.In addition, the present invention provides an antimicrobial composition comprising the strain or a culture solution thereof as an active ingredient.
또한, 본 발명은 상기 균주 또는 이의 배양액을 포함하는 항균용 사료 조성물을 제공한다.The present invention also provides an antimicrobial feed composition comprising the strain or a culture thereof.
또한, 본 발명은 상기 균주 또는 이의 배양액을 포함하는 정장용 조성물을 제공한다.The present invention also provides a dressing composition comprising the strain or a culture thereof.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 루테린을 생산하는 방법을 제공한다.The present invention also provides a method for producing rutherin comprising the step of culturing the strain.
본 발명의 락토바실러스 루테리 OH0335 균주는 글리세롤을 루테린으로 전환할 수 있는 능력이 매우 우수한 점을 확인하였다. 따라서, 본 발명에 따른 루테린 고생산 유산균 락토바실러스 루테리 OH0335 균주는 인체에 안전한 균주(GRAS, Generally Recognized As Safe)인 유산균의 일종으로, 이를 이용하여 폐글리세롤을 루테린 생산에 활용한다면 기존의 GMO 균주와 달리 안전하면서 환경적인 측면이나, 자원의 재순환 활용이라는 이점을 제공할 수 있어 관련 산업에 매우 유용하다.The Lactobacillus luteriol OH 0335 strain of the present invention was confirmed to have an excellent ability to convert glycerol to ruterin. Therefore, the lactic acid producing strain Lactobacillus luterian OH 0335 according to the present invention is a kind of lactic acid bacterium which is a generically Recognized As Safe (GRAS), and when the waste glycerol is used for the production of ruterin, Unlike strains, it is very useful in related industries because it can provide advantages such as safety, environmental aspect, and recycling of resources.
도 1은 본 발명에서 분리한 신규 유산균 락토바실러스 루테리 OH0335 균주의 분류학적 계통도를 나타낸 그림이다.1 is a diagram showing a taxonomic diagram of a novel Lactobacillus luteriol OH 0335 strain isolated in the present invention.
도 2는 본 발명에서 분리한 신규 유산균 락토바실러스 루테리 OH0335 균주의 1,3-프로판디올의 생산능 및 재활성화 셀을 이용한 루테린의 생산능을 나타낸 그림이다.FIG. 2 is a graph showing the production ability of 1,3-propanediol in the novel Lactobacillus luteriol OH 0335 strain isolated from the present invention and the production ability of rutherin using the reactivation cell.
본 발명의 목적을 달성하기 위하여, 본 발명은 글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리(lactobacilus reuteri) OH0335 균주(KCTC13362BP)를 제공한다.In order to achieve the object of the present invention, the present invention relates to a lactobacillus reuteri ) OH0335 strain (KCTC13362BP).
본 발명의 락토바실러스 루테리 OH0335 균주(KCTC13362BP)는 540mM의 글리세롤로부터 약 300mM의 루테린을 고생산할 수 있는 점을 확인하였다. 따라서, 본 발명의 균주는 280mM 이상, 바람직하게는 280~320mM, 더욱 바람직하게는 290~310mM, 더더욱 바람직하게는 295~305mM의 루테린을 생산할 수 있다. 상기 균주를 한국생명공학연구원(KCTC)에 기탁번호가 KCTC13362BP로 2017년 10월 13일에 기탁하였다.The Lactobacillus luterian OH0335 strain (KCTC13362BP) of the present invention was confirmed to be able to highly produce ruterin at about 300 mM from 540 mM glycerol. Therefore, the strain of the present invention can produce ruterin at 280 mM or more, preferably 280 to 320 mM, more preferably 290 to 310 mM, still more preferably 295 to 305 mM. The above strain was deposited on October 13, 2017 with the deposit number KCTC13362BP by the Korea Research Institute of Bioscience and Biotechnology (KCTC).
또한, 본 발명은 균주 또는 이의 배양액을 유효성분으로 함유하는 루테린 생산용 미생물 제제를 제공한다.The present invention also provides a microorganism preparation for producing rutherin containing a strain or a culture solution thereof as an active ingredient.
본 발명의 일 구현 예에 따른 미생물 제제에서, 상기 균주는 락토바실러스 루테리(lactobacilus reuteri) 균주일 수 있고, 바람직하게는 락토바실러스 루테리 OH0335 균주(KCTC13362BP)일 수 있으나, 이에 제한되지 않는다.In a microorganism preparation according to an embodiment of the present invention, the strain may be a lactobacillus reuteri strain, preferably, but not limited to, Lactobacillus lutea OH 0335 strain (KCTC13362BP).
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 항균용 조성물을 제공한다.In addition, the present invention provides an antimicrobial composition comprising the strain or a culture solution thereof as an active ingredient.
"항균(antimicrobial)"의 의미는 어떤 농도에서 세균의 성장 또는 생존을 감소, 방지, 억제, 또는 제거하는 능력을 의미한다.By "antimicrobial" is meant the ability to reduce, prevent, inhibit, or eliminate the growth or survival of the bacteria at any concentration.
본 발명의 락토바실러스 루테리 OH0335 균주(KCTC13362BP)는 GRAS 균주로, 루테린으로 불리는 천연 항균성 물질 3-HPA(3-hydroxypropionaldehyde)를 생산하며, 루테린은 병원성 세균뿐만 아니라 효모, 곰팡이 등에 대한 항균력을 보유하고 있어 항균 스펙트럼이 매우 넓은 특성을 가지고 있다.The Lactobacillus luterian OH0335 strain (KCTC13362BP) of the present invention produces a natural antimicrobial substance 3-hydroxypropionaldehyde called ruterin as a GRAS strain, and ruterin has antibacterial activity against yeast and mold as well as pathogenic bacteria The antimicrobial spectrum has very wide characteristics.
본 발명의 일 구현 예에 따른 항균용 조성물에서, 상기 조성물은 식품, 식품 첨가제, 사료 또는 사료첨가제 형태일 수 있고, 상기 식품은 유제품(우유, 두유, 가공우유), 발효유(액상 요구르트, 호상 요구르트), 드링크제, 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 알코올 음료 및 비타민 복합제로 구성되는 군으로부터 선택될 수 있으나, 이에 제한되지 않는다.In the antimicrobial composition according to an embodiment of the present invention, the composition may be in the form of a food, a food additive, a feed or a feed additive, and the food may be selected from the group consisting of dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, But are not limited to, be selected from the group consisting of drinks, meats, sausages, breads, chocolates, candies, snacks, confections, pizza, ramen, gums, ice cream, soups, beverages, alcoholic beverages and vitamins .
본 발명의 식품은 기능성 식품을 포함할 수 있는데, 본 발명의 기능성 식품에는 상기 유효성분 외에도 필요에 따라 다양한 보조성분을 추가로 함유할 수 있다. 본 발명의 기능성 식품의 경우, 비타민 A, 비타민 B1, 비타민 B2, 비타민 B3, 비타민 B6, 비타민 B12, 엽산 (folic acid), 비타민 C, 비타민 D3, 비타민 E 등의 비타민류와, 구리, 칼슘, 철, 마그네슘, 칼륨, 아연 등의 미네랄 또는 유산균 등을 포함할 수 있다.The food of the present invention may contain a functional food. In the functional food of the present invention, in addition to the above-mentioned active ingredients, various auxiliary ingredients may be added as necessary. In the case of the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.
또한, 본 발명의 기능성 식품 중, 건강음료는 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 포함할 수 있다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 들 수 있다. 천연 탄수화물로는 포도당, 과당 등의 단당류, 말토스, 수크로오스 등의 이당류, 덱스트린, 사이클로덱스트린 등의 다당류, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올류 등을 들 수 있다.In addition, among the functional foods of the present invention, health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau martin and stevia extract, and synthetic sweetening agents such as saccharine and aspartame. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
본 발명의 균주를 배양하는 단계에서 얻어지는 상기 균주 또는 이의 배양액을 식품 첨가제로 사용할 경우, 상기 균주 또는 이의 배양액을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the strain or its culture obtained in the step of culturing the strain of the present invention is used as a food additive, the strain or the culture thereof may be added as it is, or may be used together with other food or food ingredients, . The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).
또한, 본 발명은 상기 균주 또는 이의 배양액을 포함하는 항균용 사료 조성물을 제공한다.The present invention also provides an antimicrobial feed composition comprising the strain or a culture thereof.
본 발명의 사료 첨가제는 기초사료에 일정 비율로 첨가하는 것이다. 상기 기초사료는 주성분이 옥수수, 대두박, 유청, 어분, 당밀, 소금, 비타민 프리믹스 및 미네랄 프리믹스 등으로 이루어질 수 있다. 비타민 프리믹스는 비타민 A, 비타민 D, 비타민 E, 리보프라빈 및 나이아신으로 구성될 수 있으며, 미네랄 프리믹스는 망간, 철, 아연, 칼슘, 구리, 코발트 및 셀레니늄 등으로 구성될 수 있다. 상기 사료는 가축의 사료로, 육계 사료, 양돈 사료 또는 축우사료 등을 포함할 수 있으나, 이에 제한되는 것은 아니다.The feed additive of the present invention is added to the base feed at a certain ratio. The basic diet may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, and mineral premix. The vitamin premix can be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and the mineral premix can be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium. Such feeds may include, but are not limited to, livestock feed, broiler feed, cattle feed, and the like.
또한, 본 발명은 상기 균주 또는 이의 배양액을 포함하는 정장용 조성물을 제공한다.The present invention also provides a dressing composition comprising the strain or a culture thereof.
본 발명의 조성물은 통상적인 정장용 조성물 제조방법에 따라 제조될 수 있으며, 일반적으로, 동결건조되거나 캡슐화된 형태 또는 배양현탁액이거나 건조분말 형태일 수 있다. 또한, 주성분인 상기 락토바실러스 루테리 OH0335 균주(KCTC13362BP) 또는 이의 배양액의 유효량에 1종 또는 2종 이상의 약제학적으로 허용 가능한 통상적인 담체 또는 1종 또는 2종 이상의 첨가제를 선택하여 통상적인 제형의 조성물로 제조할 수 있다.The compositions of the present invention may be prepared according to conventional methods for preparing dressing compositions and may generally be in lyophilized or encapsulated form or in a culture suspension or in the form of a dry powder. In addition, it is also possible to add one or more pharmaceutically acceptable conventional carriers or one or more additives to the effective amount of the above-mentioned Lactobacillus lutea OH0335 strain (KCTC13362BP) or a culture thereof as a main component, Can be manufactured.
담체는 희석제, 활택제, 결합제, 붕해제, 감미제, 안정제, 방부제 중에서 1종 또는 2종 이상을 선택하여 사용할 수 있으며, 첨가제로는 향료, 비타민류, 항산화제 중에서 1종 또는 2종 이상을 선택하여 사용할 수 있다.The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.
본 발명에 있어서, 담체 및 첨가제는 약제학적으로 허용 가능한 것은 모두 사용이 가능하며, 구체적으로는 희석제로는 유당(lactose monohydrate), 트레할로스(Trehalose), 옥수수 전분(corn starch), 콩기름(soybean oil), 미세결정 셀룰로오스(microcrystalline cellulose) 또는 만니톨(D-mannitorl)이 좋고, 활택제로는 스테아린산 마그네슘(magnesium stearate) 또는 탈크(talc)가 바람직하며, 결합제로는 폴리비닐피롤리돈(PVP: polyvinyipyrolidone) 또는 하이드록시프로필셀룰로오스(HPC: hydroxypropylcellulose) 중에서 선택함이 바람직하다. 또한, 붕해제로는 카르복시메칠셀룰로오스칼슘(Ca-CMC: carboxymethylcellulose calcium), 전분글리콜산나트륨(sodium starch glycolate), 폴라크릴린칼륨(polacrylin potassium) 또는 크로스포비돈(cross-linked polyvinylpyrrolidone) 중에서 선택함이 바람직하고, 감미제로는 백당, 과당, 소르비톨(sorbitol) 또는 아스파탐(aspartame) 중에서 선택되고, 안정제로는 카르복시메칠셀룰로오스나트륨(Na-CMC: carboxymethylcellulose sodium), 베타-시클로덱스트린(β-cyclodextrin), 백납(white bee's wax) 또는 잔탄검(xanthan gum) 중에서 선택되며, 방부제로는 파라옥시안식향산메칠(methyl p-hydroxy benzoate, methlparaben), 파라옥시안식향산프로필(propyl p-hydroxybenzoate, propylparaben), 또는 소르빈산칼륨(potassium sorbate) 중에서 선택하는 것이 바람직하다.In the present invention, the carrier and the additive may be any pharmaceutically acceptable ones. Specific examples of the diluent include lactose monohydrate, trehalose, corn starch, soybean oil, Microcrystalline cellulose or mannitol is preferred and the lubricant is preferably magnesium stearate or talc and the binder may be polyvinylpyrrolidone (PVP) or polyvinylpyrrolidone And hydroxypropylcellulose (HPC). The disintegrant may be selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone And the sweetener is selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta-cyclodextrin) white bee's wax or xanthan gum and the preservative is selected from the group consisting of methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate).
또한, 상기 락토바실러스 루테리 OH0335 균주(KCTC13362BP)를 접종하여 배양시켜 얻어지는 균주 배양물에 프로바이오틱 활성을 갖는 미생물을 추가로 접종하여 배양시킨 균주 배양물은 프로바이오틱 활성을 갖는 생균제로 동물에 투여되거나 또는 식품, 의약품, 동물약품 또는 유산균제와 함께 동물에 투여될 수 있으며 바람직하게는 가축의 사료 첨가제 또는 보조사료로 사용되며, 상기 생균제를 양돈에 급여시 초유에 함유된 풍부한 영양소와 유용 생리활성물질로 양돈의 증체율 증가 및 이유 자돈의 설사발생을 없게 할 수 있다.Further, the strain culture obtained by inoculating and culturing the strain culture obtained by inoculating and culturing the Lactobacillus luteriol OH 0335 strain (KCTC13362BP) with a microorganism having a probiotic activity is administered to an animal as a probiotic activity- Or may be administered to an animal together with food, medicines, animal drugs or lactic acid bacteria, and is preferably used as a feed additive or supplementary feed for livestock, and the nutrient rich in colostrum and useful physiological activity It is possible to increase the rate of growth of swine and eliminate the occurrence of diarrhea in pigs.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 루테린을 생산하는 방법을 제공한다.The present invention also provides a method for producing rutherin comprising the step of culturing the strain.
본 발명의 상기 루테린을 생산하는 방법은 균주의 배양액으로부터의 루테린을 회수하는 단계를 추가로 포함할 수 있다. 상기 균주의 배양액으로부터의 루테린을 회수하는 단계는 통상적인 분리 기술, 예를 들어 증류, 전기투석, 투과증발, 크로마토그라피, 용매추출, 반응추출 등을 이용할 수 있으며, 통상적으로 순도가 높은 물질을 분리하기 위하여 이들을 조합하여 이용할 수 있다.The method of producing the rutherin of the present invention may further comprise the step of recovering rutherin from the culture broth of the strain. The step of recovering the ruthenin from the culture broth of the strain can be carried out by conventional separation techniques such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction and the like, They can be used in combination to separate them.
본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법에 따라 배양할 수 있으며, 특별한 방법에 한정되는 것은 아니다.The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and is not limited to a specific method.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예Example 1. 신규 유산균  1. New lactic acid bacteria 락토바실러스Lactobacillus 루테리Luteri OH0335 균주의 분리 Isolation of OH0335 strain
전라북도 익산시의 돼지 도축장에서 돼지 소장 및 십이지장 시료를 채취하고, 잘게 파쇄한 1g의 시료에 생리식염수 10mL을 가하여 현탁한 후 1×10-4배까지 희석하여 브로모크레졸 퍼플이 함유된 MRS 고체배지(10g/L 프로테오스 펩톤 NO.3, 10g/L 쇠고기 추출물, 5g/L 효모 추출물, 20g/L 덱스트로스, 1g/L 폴리솔베이트 80, 2g/L 암모늄 시트레이트, 5g/L 소듐 아세테이트, 0.1g/L 마그네슘 설페이트, 0.05g/L 망가니즈 설페이트, 2g/L 디포타슘 포스페이트, 0.04g/L 브로모크레졸 퍼플, 15g/L 아가)에 각각 100㎕씩 도말 후, 37℃에서 Bactron Anaerobic Chamber(SHEL LAB, Cornelius, OR·USA.)를 이용하여 24시간 동안 혐기 상태로 배양하여 얻어진 콜로니들을 브로모크레졸 퍼플이 함유된 MRS 배지에 재접종하여 순수분리한 후 MALDI Biotyper(Bruker Daltonik, Billerica, MA USA)로 81종의 락토바실러스 루테리 균주를 분리하였다. 3mL MRS 배지(15mL 팔콘 튜브)에 접종하여 37℃에서 Bactron Anaerobic Chamber를 이용하여 24시간 동안 혐기 상태로 전배양 후, 50mL 유산균 배양 배지 (10g/L 프로테오스 펩톤 NO.3, 10g/L 쇠고기 추출물, 5g/L 효모 추출물, 20g/L 덱스트로스, 1g/L 폴리솔베이트 80, 2g/L 암모늄 시트레이트, 5g/L 소듐 아세테이트, 0.1g/L 마그네슘 설페이트, 0.05g/L 망가니즈 설페이트, 2g/L 디포타슘 설페이트)(250mL 둥근 플라스크)에 초기 접종 OD 0.5로 37℃에서 Bactron Anaerobic Chamber를 이용하여 24시간 동안 혐기상태로 배양하였다. 배양 후, 배양액을 13,000 rpm으로 5분간 원심분리한 후 셀을 회수하여 50mM 소듐 포스페이트 버퍼(pH 7.5)에 2회 세척하였다. 세척된 셀은 200mM 글리세롤 용액에 재현탁하여 30℃ 배양기에서 정치반응하며 1시간 간격으로 시료를 채취하였다. 반응 시료는 13,000 rpm으로 5분간 원심분리한 후에 분석에 사용하였다. 루테린 정량 분석은 아크롤레인(acrolein)을 표준물질로 사용하였다. 루테린 시료 1mL, 37% HCl 3mL, 10mM 트립토판-HCl 용매를 0.75mL를 혼합하여 37℃에서 20분간 반응하였다. 반응 후, ELISA 리더를 이용하여 560nm에서 반응액의 흡광도를 측정하여 루테린의 생산량을 정량하였다.Pig small intestine and duodenum samples were collected from a pig slaughterhouse in Iksan, Jeollabuk-do, and 1 g of finely crushed samples were suspended in 10 mL of physiological saline, diluted to 1 × 10 -4 times, and cultured in MRS solid medium containing bromocresol purple 5 g / L of yeast extract, 20 g / L dextrose, 1 g / L of polysorbate 80, 2 g / L of ammonium citrate, 5 g / L of sodium acetate, 10 g / L of protease peptone NO.3, 10 g / L of beef extract, 100 μl each was plated on each of the wells at a temperature of 37 ° C, and the plate was immersed in a Bactron Anaerobic Chamber (Bruker Daltonik, Billerica, CA, USA) for 24 hrs. The colonies were re-inoculated on the MRS medium containing bromocresol purple and purified by MALDI Biotyper (Bruker Daltonik, Billerica, MA USA), 81 strains of Lactobacillus luterium It was isolated. 3 mL of MRS medium (15 mL falcon tube) was pre-cultured for 24 hours in an anaerobic condition using a Bactron Anaerobic Chamber at 37 ° C, and then cultured in 50 mL of lactic acid culture medium (10 g / L Protease Peptone NO.3, 10 g / L beef L of yeast extract, 20 g / L dextrose, 1 g / L polysorbate 80, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 g / L dipotassium sulfate) (250 mL round-bottomed flask) was incubated in an anaerobic condition for 24 hours at 37 ° C in an initial inoculation OD of 0.5 using a Bactron Anaerobic Chamber. After the culture, the culture was centrifuged at 13,000 rpm for 5 minutes, and the cells were recovered and washed twice with 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in a 200 mM glycerol solution, allowed to stand in a 30 ° C incubator, and sampled at intervals of 1 hour. The reaction samples were centrifuged at 13,000 rpm for 5 minutes and then used for analysis. Quantitative analysis of ruterin used acrolein as a standard. 1 mL of a rutin sample, 3 mL of 37% HCl and 0.75 mL of a 10 mM tryptophan-HCl solvent were mixed and reacted at 37 DEG C for 20 minutes. After the reaction, the absorbance of the reaction solution was measured at 560 nm using an ELISA reader to quantitate the amount of ruthenin produced.
그 결과, 분리된 140 균주 중 루테린 생산이 우수한 락토바실러스 루테리 균주 1종을 최종 선정하였다. 반응 1시간째에 글리세롤 200mM로부터 30mM의 루테린을 생산하였다.As a result, one strain of Lactobacillus luterius having excellent ruterin production among the 140 strains isolated was finally selected. One hour after the reaction, ruterin was produced from 200 mM glycerol to 30 mM rutherin.
신규 분리 유산균 락토바실러스 루테리 균주의 루테린 생산Production of ruterin from a newly isolated lactic acid bacterium Lactobacillus luterium strain
Initial glycerol(mmol/L)Initial glycerol (mmol / L) Cell biomass(g DCW/L)Cell biomass (g DCW / L) Reaction time (h)Reaction time (h) Reuterin (mmol/L)Reuterin (mmol / L) 1,3-propanediol (mmol/L)1,3-propanediol (mmol / L)
200200 33 1One 30.530.5 4.34.3
200200 33 22 36.836.8 5.15.1
최종적으로 루테린 생산이 가장 우수한 유산균 락토바실러스 루테리 OH0335 균주의 분자생물학적 동정을 위하여 16S rRNA 유전자 서열을 분석하였다. 하나의 콜로니로부터 게놈 DNA 추출 키트(Invitrogen, Germany)를 이용하여 염색체 DNA를 분리한 후, 이로부터 락토바실러스 16S rRNA 유전자 증폭용 프라이머 5'-AGAGTTTGATCMTGGCTCAG-3'(27f, 서열번호 2)와 5'-TACGGYTACCTTGTTACGACTT-3'(1492r, 서열번호 3)을 이용하여 PCR법으로 16S rRNA 유전자 DNA를 증폭하였다. PCR 증폭은 Ex Taq 폴리머라제(Takara)(2.5U), 폴리머라제 버퍼, dNTP 혼합물(각 1mM), 각 프라이머(100pmol) 1㎖, 주형 DNA 500ng을 함유하는 PCR 반응용액(50㎕)을 준비한 후, 유전자 증폭기(Takara, Japan)로 96℃ 30초, 50℃ 1분, 72℃ 2분 조건으로 30회간 수행하였다. PCR 반응액을 1% 아가로즈겔에서 전기영동하여 예상되는 크기의 DNA 단편이 증폭된 것을 확인하고 pGEM-TEasy 벡터(Promega, USA)를 이용하여 대장균 EPI300으로 형질전환 하였다. 형질전환된 재조합 대장균들로부터 플라스미드 DNA를 추출(Qiagen, USA)하고, 제한효소 EcoRI을 처리하여 원하는 크기의 DNA 단편이 클로닝된 것을 확인하였으며, 염기서열을 결정하였다(서열번호 1). 염기서열의 상동성 분석을 실시한 결과를 토대로 분리한 유산균을 락토바실러스 루테리(Lactobacillus reuteri) OH0335로 명명하였다.Finally, 16S rRNA gene sequence was analyzed for molecular biology identification of Lactobacillus luterian OH0335 strain, which has the highest production of ruterin. The chromosomal DNA was isolated from a single colony using a genomic DNA extraction kit (Invitrogen, Germany), and the primer 5'-AGAGTTTGATCMTGGCTCAG-3 '(27f, SEQ ID NO: 2) for amplifying the Lactobacillus 16S rRNA gene and 5' 16S rRNA gene DNA was amplified by PCR using -TACGGYTACCTTGTTACGACTT-3 '(1492 r, SEQ ID NO: 3). PCR amplification was carried out by preparing a PCR reaction solution (50 μl) containing Ex Taq polymerase (Takara) (2.5 U), polymerase buffer, dNTP mixture (1 mM each), 1 ml of each primer (100 pmol) and 500 ng of template DNA , And gene amplification (Takara, Japan) for 30 times at 96 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 2 minutes. The PCR reaction solution was electrophoresed on 1% agarose gel, and E. coli EPI300 was transformed with pGEM-TEasy vector (Promega, USA) after confirming amplification of the DNA fragment of the expected size. The plasmid DNA was extracted from the transformed recombinant E. coli (Qiagen, USA) and the restriction enzyme Eco RI was treated to confirm that the DNA fragment of the desired size was cloned and the nucleotide sequence was determined (SEQ ID NO: 1). Based on the result of homology analysis of the nucleotide sequence, the isolated lactic acid bacterium was named Lactobacillus reuteri OH 0335.
실시예Example 2. 발효조 배양을 통한 신규 분리 유산균  2. New isolated lactic acid bacteria by fermentation 락토바실러스Lactobacillus 루테리Luteri OH0335 균주의  Of the strain OH0335 루테린Ruterin 생산 production
5L 배양기에서 바실러스 루테리 OH0335 균주를 이용하여 MRS 배지(10g/L 프로테오스 펩톤 NO.3, 10g/L 쇠고기 추출물, 5g/L 효모 추출물, 20g/L 덱스트로스, 1g/L 폴리솔베이트 80, 2g/L 암모늄 시트레이트, 5g/L 소듐 아세테이트, 0.1g/L 마그네슘 설페이트, 0.05g/L 망가니즈 설페이트, 2g/L 디포타슘 설페이트)에서 배양 후 배양액을 13,000 rpm으로 5분간 원심분리한 후 셀을 회수하여 50mM 소듐 포스페이트 버퍼(pH 7.5)에 2회 세척하였다. 세척된 셀은 200mM 글리세롤 용액에 재현탁하여 30℃ 배양기에서 정치반응하며 1시간 간격으로 시료를 채취하였다. 그 결과, 아래 표 2에 나타낸 바와 같이 셀 농도 10g DCW/L, 글리세롤 540mM에서 반응 1시간째 299.8mM의 루테린을 생산하였다. 5 g / L yeast extract, 20 g / L dextrose, 1 g / L Polysorbate 80, and 10 g / L protein extract were added to the MRS medium (10 g / L Proteose Peptone NO.3, 10 g / L beef extract, L of ammonium citrate, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 2 g / L of dipotassium sulfate) and centrifuged at 13,000 rpm for 5 minutes. Was collected and washed twice with 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in a 200 mM glycerol solution, allowed to stand in a 30 ° C incubator, and sampled at intervals of 1 hour. As a result, ruterin was produced at a cell concentration of 10 g DCW / L and 540 mM glycerol at 299.8 mM for 1 hour as shown in Table 2 below.
발효조 배양을 통한 반응 조건별 루테린 생산Production of ruterin by reaction condition through fermenter culture
Initial glycerol(mmol/L)Initial glycerol (mmol / L) Cell biomass(g DCW/L)Cell biomass (g DCW / L) Reaction time (h)Reaction time (h) Reuterin (mmol/L)Reuterin (mmol / L) 1,3-propanediol (mmol/L)1,3-propanediol (mmol / L)
320320 55 1One 202.6202.6 32.832.8
320320 1010 1One 179.2179.2 35.435.4
540540 1010 1One 299.8299.8 48.048.0
문헌상으로 보고된 다른 락토바실러스 루테리 유산균의 결과들과 비교해 볼 때, 분리 균주는 우수한 루테린 생산량을 보이는 것으로 나타났다.Compared to the results of other Lactobacillus lactis lactic acid bacteria reported in the literature, the isolate showed excellent lterine production.
락토바실러스 루테리 유산균 균주의 루테린 생산 비교Comparison of ruterin production of Lactobacillus luterian lactic acid bacteria
StrainsStrains Initial glycerol(mmol/L)Initial glycerol (mmol / L) Cell biomass(g DCW/L)Cell biomass (g DCW / L) Reuterin (mmol/L)Reuterin (mmol / L) Reaction time(h)Reaction time (h) ReferenceReference
L. reuteri ATCC1083L. reuteri ATCC1083 250250 1010 6767 22 Talarico et al., (1988)Talarico et al., (1988)
L. reuteri ATCC53608L. reuteri ATCC 53608 200200 3030 170170 22 Luthi-Peng et al., (2002)Luthi-Peng et al., (2002)
L. reuteri ATCC53608L. reuteri ATCC 53608 400400 3030 235235 0.750.75 Y.Doleyres.P et al. (2005)Y.Doleyres.P et al. (2005)
L. reuteri CG001L. reuteri CG001 200200 25.325.3 195195 1One Chen et al. (2011)Chen et al. (2011)
L. reuteri CGMCC 1.3264cL. reuteri CGMCC 1.3264c 250250 10.210.2 235.9235.9 1.51.5 Feixia Liu et al. (2015)Feixia Liu et al. (2015)
L. reuteri DSM 20016(Δlr-1734)L. reuteri DSM 20016 (? Lr-1734) 250250 1212 236.4236.4 1One Marc J A et al.(2011)Marc JE et al. (2011)
L. reuteri OH0335L. reuteri OH 0335 540540 1010 299.8299.8 1One 본 발명Invention
실시예Example 3. 발효조를 이용한  3. Using a fermentor 유가식Oil price formula 배양을 통한 신규 분리 유산균  New isolated lactobacilli through culture 락토바실러스Lactobacillus 루테리Luteri OH0335OH0335 균주의 1,3- The 1,3- 프로판디올Propanediol 및  And 루테린Ruterin 생산 production
5L 배양기에서 락토바실러스 루테리 OH0335 균주를 이용하여 글리세롤이 포함된 MRS 배지(20g/L 글리세롤, 10g/L 프로테오스 펩톤 NO.3, 10g/L 쇠고기 추출물, 5g/L 효모 추출물, 20g/L 덱스트로스, 1g/L 폴리솔베이트 80, 2g/L 암모늄 시트레이트, 5g/L 소듐 아세테이트, 0.1g/L 마그네슘 설페이트, 0.05g/L 망가니즈 설페이트, 2g/L 디포타슘 설페이트)에서 유가식 배양 중 1,3-프로판디올 및 부산물의 생산량을 조사하였다. 그 결과, 도 2에 개시한 바와 같이 최종적으로 배양 72시간째에 45.3g/L의 1,3-프로판디올을 생산하였으며, 글리세롤로부터 1,3-프로판디올의 생산수율과 시간당 생산성은 0.760g/g과 629.2mg/L/h를 나타내었다. 이때 부산물의 생산량은 34.2g/L의 젖산, 21.7g/L의 아세트산(데이터 미제시) 및 4.1g/L의 에탄올(데이터 미제시)로 나타났다.In a 5 L culture, Lactobacillus luteriol OH 0335 strain was used to culture the MRS medium (20 g / L glycerol, 10 g / L protease peptone NO.3, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L deck L diatomite sulfate, 1 g / L polysorbate 80, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1,3-propanediol and by-products were investigated. As a result, as shown in Fig. 2, at the 72 hours after cultivation, 45.3 g / L of 1,3-propanediol was produced. From the glycerol, 1,3-propanediol production yield and hourly productivity were 0.760 g / g and 629.2 mg / L / h, respectively. The yield of byproducts was 34.2 g / L of lactic acid, 21.7 g / L of acetic acid (data not shown) and 4.1 g / L of ethanol (data not shown).
또한, 상기 72시간 동안 배양한 셀을 13,000rpm으로 5분간 원심분리한 후 회수하여 5L 배양기에서 3L MRS 배지에 재현탁하여 4~6시간 동안 셀을 재활성화하였다. 재활성화 배양액을 13,000rpm으로 5분간 원심분리한 후 셀을 회수하여 50mM 소듐 포스페이트 버퍼(pH 7.5)에 2회 세척하였다. 세척된 셀은 540mM 글리세롤 용액에 셀 농도 10g DCW/L로 재현탁하여 30℃ 배양기에서 정치반응하며 1시간 간격으로 시료를 채취하였다. 그 결과, 하기 표 4에 개시한 바와 같이 6시간 동안 재활성화하였을 때, 반응 2시간째에 244.5mM의 루테린을 생산하는 것을 확인하였다.The cells cultured for 72 hours were centrifuged at 13,000 rpm for 5 minutes, collected, re-suspended in a 3 L MRS medium in a 5 L incubator, and reactivated for 4 to 6 hours. The reactivation culture was centrifuged at 13,000 rpm for 5 minutes, and the cells were recovered and washed twice with 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in 540 mM glycerol solution at a cell concentration of 10 g DCW / L, subjected to a stationary reaction at 30 ° C in an incubator, and samples were collected at intervals of 1 hour. As a result, when reactivated for 6 hours as shown in Table 4 below, it was confirmed that ruterin was produced at 244.5 mM 2 hours after the reaction.
락토바실러스 루테리 유산균 균주의 루테린 생산 비교Comparison of ruterin production of Lactobacillus luterian lactic acid bacteria
Reactivation time(h)Reactivation time (h) Reaction time(h)Reaction time (h) Reuterin(mmol/L)Reuterin (mmol / L) 1,3-propanediol(mmol/L)1,3-propanediol (mmol / L)
44 1One 108.7108.7 18.418.4
22 150.5150.5 25.225.2
66 1One 187.3187.3 30.330.3
22 244.5244.5 41.241.2
[수탁번호][Access number]
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC13362BPAccession number: KCTC13362BP
수탁일자 : 20171013Checked on: 20171013
Figure PCTKR2018014545-appb-I000001
Figure PCTKR2018014545-appb-I000001

Claims (6)

  1. 글리세롤로부터 루테린을 고생산하는 락토바실러스 루테리(lactobacilus reuteri) OH0335 균주(KCTC13362BP). Lactobacillus reuteri OH0335 strain (KCTC13362BP) which produces rutelin from glycerol.
  2. 제1항의 균주 또는 이의 배양액을 유효성분으로 함유하는 루테린 생산용 미생물 제제.A microorganism preparation for producing ruterin comprising the strain of claim 1 or a culture thereof as an active ingredient.
  3. 제1항의 균주 또는 이의 배양액을 유효성분으로 포함하는 항균용 조성물.11. An antimicrobial composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
  4. 제1항의 균주 또는 이의 배양액을 포함하는 항균용 사료 조성물.An antimicrobial feed composition comprising the strain of claim 1 or a culture thereof.
  5. 제1항의 균주 또는 이의 배양액을 포함하는 정장용 조성물.A composition for dressing comprising the strain of claim 1 or a culture thereof.
  6. 제1항의 균주를 배양하는 단계를 포함하는 루테린을 생산하는 방법.A method for producing ruterin comprising culturing the strain of claim 1.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112493377A (en) * 2020-11-27 2021-03-16 广东省微生物研究所（广东省微生物分析检测中心） Application of lactobacillus reuteri microbial inoculum containing reuterin in preparation of preparation for promoting metabolism of glycolipid of pelteobagrus fulvidraco
CN114806929A (en) * 2022-03-29 2022-07-29 山东凤凰生物科技股份有限公司 Lactobacillus reuteri LR4009 capable of highly producing reuterin and application thereof
WO2024077181A1 (en) * 2022-10-06 2024-04-11 Microbyre, Inc. Methods of producing reuterin and propenoic acid using an isolated strain of lactobacillus reuteri

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102588346B1 (en) * 2020-11-27 2023-10-11 코오롱인더스트리(주) Novel strain of Lactobacillus reuteri and use thereof
KR102394120B1 (en) 2021-10-26 2022-05-04 코오롱인더스트리(주) Antimicrobial composition comprising alkanediol and reuterin and use thereof
KR20230137014A (en) 2022-03-21 2023-10-04 코오롱인더스트리 주식회사 Antimicrobial composition comprising preservative compound and reuterin and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR19990022307A (en) * 1995-06-07 1999-03-25 보 몰스탬 How to improve animal health
KR20150025897A (en) * 2013-08-30 2015-03-11 삼성종합화학주식회사 Microbial conversion of glycerol

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR19990022307A (en) * 1995-06-07 1999-03-25 보 몰스탬 How to improve animal health
KR20150025897A (en) * 2013-08-30 2015-03-11 삼성종합화학주식회사 Microbial conversion of glycerol

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DOLEYRES, Y: "Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 68, 2005, pages 467 - 474, XP019331934 *
KRAUTER, H: "Production of high amounts of 3-hydroxypropionaldehyde from glycerol by Lactobacillus reuteri with strongly increased biocatalyst lifetime and productivity", NEW BIOTECHNOLOGY, vol. 29 2, 15 January 2012 (2012-01-15), pages 211 - 217, XP002658362 *
LACTOBACILLUS REUTERI, vol. 37, no. 2, 2005, pages 318 - 320 *
LIU, F: "Efficient production of reuterin from glycerol by magnetically immobilized Lactobacillus reuteri", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 99, 2015, pages 4659 - 4666, XP035502956 *
LUTHI-PENG, Q: "Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 60, 2002, pages 73 - 80, XP55615194 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112493377A (en) * 2020-11-27 2021-03-16 广东省微生物研究所（广东省微生物分析检测中心） Application of lactobacillus reuteri microbial inoculum containing reuterin in preparation of preparation for promoting metabolism of glycolipid of pelteobagrus fulvidraco
CN112493377B (en) * 2020-11-27 2023-09-29 广东省科学院微生物研究所(广东省微生物分析检测中心) Application of lactobacillus reuteri agent containing reuterin in preparation of preparation for regulating glycolipid metabolism of pelteobagrus fulvidraco
CN114806929A (en) * 2022-03-29 2022-07-29 山东凤凰生物科技股份有限公司 Lactobacillus reuteri LR4009 capable of highly producing reuterin and application thereof
CN114806929B (en) * 2022-03-29 2023-10-31 山东凤凰生物科技股份有限公司 Lactobacillus reuteri LR4009 with high yield of reuterin and application thereof
WO2024077181A1 (en) * 2022-10-06 2024-04-11 Microbyre, Inc. Methods of producing reuterin and propenoic acid using an isolated strain of lactobacillus reuteri

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