KR20190066129A - Lactobacilus reuteri OH0335 strain having high productivity of reuterin from glycerol and uses thereof - Google Patents

Abstract

The OH0335 strain of Lactobacillus reuteri of the present invention has 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, thereby being useful to the relevant industries.

Description

Lactobacillus luteriol OH 0335 strain producing high levels of rutherin from glycerol and its use {Lactobacillus reuteri OH 0335 strain having high productivity of reuterin from glycerol and uses thereof}

The present invention relates to Lactobacillus luteriol OH 0335 strain, which highly produces ruterin from glycerol, and its use.

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.

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, 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.

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.

In order to solve the above problems, 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.

In addition, 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.

1 is a diagram showing a taxonomic diagram of a novel Lactobacillus luteriol OH 0335 strain isolated in the present invention.

In order to achieve the object of the present invention, 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.

In a microbial formulation according to one embodiment of the invention, the strain is selected from the group consisting of lactobacillus reuteri strains, preferably, but not limited to, Lactobacillus luterian OH 0335 strain (KCTC13362BP).

In addition, the present invention provides an antimicrobial composition comprising the strain or a culture solution thereof as an active ingredient.

By "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.

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 .

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.

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.

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).

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, 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.

The present invention also provides a dressing composition comprising the strain or a culture thereof.

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.

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.

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).

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.

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.

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.

Example  1. New lactic acid bacteria Lactobacillus Luteri  Isolation of OH0335 strain

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 analyzed. 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.

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.

Production of ruterin from a newly isolated lactic acid bacterium Lactobacillus luterium strain Initial glycerol
(mmol / L) Cell biomass
(g DCW / L) Reaction time
(h) Reuterin
(mmol / L) 1,3-propanediol (mmol / L) 200 3 One 30.5 4.3 200 3 2 36.8 5.1

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.

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). 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.

Production of ruterin by reaction condition through fermenter culture Initial glycerol
(mmol / L) Cell biomass
(g DCW / L) Reaction time
(h) Reuterin
(mmol / L) 1,3-propanediol (mmol / L) 320 5 One 202.6 32.8 320 10 One 179.2 35.4 540 10 One 299.8 48.0

Compared to the results of other Lactobacillus lactis lactic acid bacteria reported in the literature, the isolate showed excellent lterine production.

Comparison of ruterin production of Lactobacillus luterian lactic acid bacteria Strains Initial glycerol (mmol / L) Cell biomass
(g DCW / L) Reuterin
(mmol / L) Reaction time (h) Reference L. reuteri ATCC1083 250 10 67 2 Talarico et al., (1988) L. reuteri ATCC 53608 200 30 170 2 L? Thi-Peng et al., (2002) L. reuteri ATCC 53608 400 30 235 0.75 Y.Doleyres.P
et al. (2005) L. reuteri CG001 200 25.3 195 One Chen et al. (2011) L. reuteri CGMCC 1.3264c 250 10.2 235.9 1.5 Feixia Liu et al. (2015) L. reuteri DSM 20016
(? Lr-1734) 250 12 236.4 One Marc JE et al. (2011) L. reuteri OH 0335 540 10 299.8 One Invention

Korea Biotechnology Research Institute KCTC13362BP 20171013

<110> Korea Research Institute of Bioscience and Biotechnology          ActivON Co., Ltd. <120> Lactobacillus reuteri OH0335 strain having high productivity of          reuterin from glycerol and uses thereof <130> PN17455 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1512 <212> DNA <213> Lactobacillus reuteri <400> 1 atggctcagg atgaacgccg gcggtgtgcc taatacatgc aagtcgtacg cactggccca 60 actgattgat ggtgcttgca cctgattgac gatggatcac cagtgagtgg cggacgggtg 120 agtaacacgt aggtaacctg ccccggagcg ggggataaca tttggaaaca gatgctaata 180 ccgcataaca acaaaagcca catggctttt gtttgaaaga tggctttggc tatcactctg 240 ggatggacct gcggtgcatt agctagttgg taaggtaacg gcttaccaag gcgatgatgc 300 atagccgagt tgagagactg atcggccaca atggaactga gacacggtcc atactcctac 360 gggaggcagc agtagggaat cttccacaat gggcgcaagc ctgatggagc aacaccgcgt 420 gagtgaagaa gggtttcggc tcgtaaagct ctgttgttgg agaagaacgt gcgtgagagt 480 aactgttcac gcagtgacgg tatccaacca gaaagtcacg gctaactacg tgccagcagc 540 cgcggtaata cgtaggtggc aagcgttatc cggatttatt gggcgtaaag cgagcgcagg 600 cggttgctta ggtctgatgt gaaagccttc ggcttaaccg aagaagtgca tcggaaaccg 660 ggcgacttga gtgcagaaga ggacagtgga actccatgtg tagcggtgga atgcgtagat 720 atatggaaga acaccagtgg cgaaggcggc tgtctggtct gcaactgacg ctgaggctcg 780 aaagcatggg tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgagtg 840 ctaggtgttg gagggtttcc gcccttcagt gccggagcta acgcattaag cactccgcct 900 ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg 960 gagcatgtgg tttaattcga agctacgcga agaaccttac caggtcttga catcttgcgc 1020 taaccttaga gataaggcgt tcccttcggg gacgcaatga caggtggtgc atggtcgtcg 1080 tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgttactag 1140 ttgccagcat taagttgggc actctagtga gactgccggt gacaaaccgg aggaaggtgg 1200 ggacgacgtc agatcatcat gccccttatg acctgggcta cacacgtgct acaatggacg 1260 gtacaacgag tcgcaagctc gcgagagtaa gctaatctct taaagccgtt ctcagttcgg 1320 actgtaggct gcaactcgcc tacacgaagt cggaatcgct agtaatcgcg gatcagcatg 1380 ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtttgta 1440 acgcccaaag tcggtggcct aacctttatg gagggagccg cctaagcggg acagatgact 1500 ggggtgaagt cg 1512 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacggytacc ttgttacgac tt 22

Claims (6)

Lactobacillus reuteri OH0335 strain (KCTC13362BP) which produces rutelin from glycerol. A microorganism preparation for producing ruterin comprising the strain of claim 1 or a culture thereof as an active ingredient. 11. An antimicrobial composition comprising the strain of claim 1 or a culture thereof as an active ingredient. An antimicrobial feed composition comprising the strain of claim 1 or a culture thereof. A composition for dressing comprising the strain of claim 1 or a culture thereof. A method for producing ruterin comprising culturing the strain of claim 1.

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