KR102013565B1 - Lactobacillus reuteri OH0335 strain having high productivity of reuterin from glycerol and uses thereof - Google Patents

Abstract

The Lactobacillus luteri OH0335 strain of the present invention was confirmed that the ability to convert glycerol to lutein is very excellent. Therefore, the high-lactic acid bacteria Lactobacillus Lacteria OH0335 strain according to the present invention is a kind of lactic acid bacteria which are generally Recognized As Safe (GRAS), and if the waste glycerol is used for the production of lutein, the existing GMO Unlike the strain, it is very useful for related industries because it can provide the advantages of safety and environmental aspects, or recycling of resources.

Description

Lactobacillus reuteri OH0335 strain having high productivity of reuterin from glycerol and uses according to Lactobacillus reuteri OH0335 strain having high production of lutein from glycerol

The present invention relates to a Lactobacillus luteri OH0335 strain which produces lutein from glycerol and its use.

In general, microorganisms are one of the causes of corruption in food, feed, and cosmetics. In particular, toxic and carcinogenic mycotoxins produced by microorganisms are harmful to health due to various side effects when consumed by humans (Schaefer et al., Microbiology 2010, 156: 1589-1599). In addition, the corruption of food, feed, cosmetics, etc. have a huge economic impact. To prevent this rot, biological or chemical preservatives are added. However, in the case of chemical preservatives, safety-related problems such as accumulation in the body continue to emerge and adversely affect the human body depending on the type and amount of the substance used. As income levels improve, there is a growing demand for bio-friendly biological preservatives, along with health-oriented tendencies, and avoiding the use of chemical preservatives.

Lactobacillus luteri are known to produce the natural antimicrobial substance 3-HPA (3-hydroxypropionaldehyde) called luterin. Luterin possesses antibacterial activity against yeast, mold, etc. as well as pathogenic bacteria, and has a very broad antibacterial spectrum (Doleyres et al., A ppl). Microbiol Biotechnol 2005, 68: 467-474). Luterin has been actively researched for its application to the food industry and medical treatment.

Meanwhile, Korean Patent No. 1483012 discloses a method of producing 3-hydroxypropionic acid in high yield using recombinant Escherichia coli, and Korean Patent No. 1505172 discloses recombination to produce 3-hydroxypropionic acid. Microorganisms and methods for producing 3-hydroxypropionic acid using the same have been disclosed, but as described in the present invention, there is no information on 'Lactobacillus luteri OH0335 strain and its use to produce lutein from glycerol at all'.

The present invention is derived from the above requirements, and in the present invention, a new isolate of Lactobacillus luteri OH0335 strain (KCTC13362BP), which is a GRAS strain producing high lutein called 3-HPA (3-hydroxypropionaldehyde) which is a natural antimicrobial substance, is newly isolated. The strain was confirmed that the high production of about 300 mM lutein from 540 mM glycerol. This enables the production of very high lutein from glycerol, thereby completing the present invention.

In order to solve the above problems, the present invention provides a Lactobacillus reuteri OH0335 strain (KCTC13362BP) that produces high lutein from glycerol.

In addition, the present invention provides a microbial preparation for lutein production containing the strain or its culture as an active ingredient.

The present invention also provides an antimicrobial composition comprising the strain or its culture as an active ingredient.

In addition, the present invention provides an antimicrobial feed composition comprising the strain or its culture.

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

In addition, the present invention provides a method for producing lutein comprising the step of culturing the strain.

The Lactobacillus luteri OH0335 strain of the present invention was confirmed that the ability to convert glycerol to lutein is very excellent. Therefore, the high-lactic acid bacteria Lactobacillus Lacteria OH0335 strain according to the present invention is a kind of lactic acid bacteria which are generally Recognized As Safe (GRAS), and if the waste glycerol is used for the production of lutein, the existing GMO Unlike the strain, it is very useful for related industries because it can provide the advantages of safety and environmental aspects, or recycling of resources.

1 is a diagram showing the taxonomic diagram of the novel lactic acid bacteria Lactobacillus luteri OH0335 strain isolated from the present invention.

In order to achieve the object of the present invention, the present invention provides a Lactobacillus reuteri OH0335 strain (KCTC13362BP) that produces high lutein from glycerol.

It was confirmed that the Lactobacillus luteri OH0335 strain (KCTC13362BP) of the present invention can produce about 300 mM lutein from 540 mM glycerol. Therefore, the strain of the present invention can produce a lutein of at least 280mM, preferably 280-320mM, more preferably 290-310mM, even more preferably 295-305mM. The strain was deposited with the Korea Biotechnology Research Institute (KCTC) on October 13, 2017 with the accession number KCTC13362BP.

The present invention also provides a microbial agent for producing lutein containing a strain or a culture thereof as an active ingredient.

In the microbial preparation according to an embodiment of the present invention, the strain may be a Lactobacillus reuteri strain, preferably, may be a Lactobacillus luster OH0335 strain (KCTC13362BP), but is not limited thereto.

The present invention also provides an antimicrobial composition comprising the strain or its culture as an active ingredient.

By “antimicrobial” is meant the ability to reduce, prevent, inhibit or eliminate the growth or survival of bacteria at any concentration.

The Lactobacillus luteri OH0335 strain (KCTC13362BP) of the present invention is a GRAS strain, which produces a natural antimicrobial substance 3-HPA (3-hydroxypropionaldehyde) called lutein, and lutein possesses antibacterial activity against yeast, mold, etc. as well as pathogenic bacteria. The antibacterial spectrum has a very wide characteristic.

In the antimicrobial composition according to an embodiment of the present invention, the composition may be in the form of food, food additives, feed or feed additives, the food may be dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, staple yogurt) ), Drink, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soups, beverages, alcoholic beverages and vitamin complexes, but is not limited thereto. .

The food of the present invention may include a functional food. The functional food of the present invention may further contain various auxiliary ingredients as necessary in addition to the active ingredient. In 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, vitamin E, copper, calcium, Minerals such as iron, magnesium, potassium, zinc, or lactic acid bacteria, and the like.

In addition, among the functional food of the present invention, the health beverage may include various flavors or natural carbohydrates, etc. as additional components, as in the usual beverage. Flavoring agents include natural sweeteners such as taumartin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. Examples of the natural carbohydrate 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 using the strain or its culture obtained in the step of culturing the strain of the present invention as a food additive, the strain or its culture may be added as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method. Can be. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment).

In addition, the present invention provides an antimicrobial feed composition comprising the strain or its culture.

The feed additive of the present invention is to be added to the basic feed at a predetermined ratio. The basic feed may be made of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix and mineral premix. Vitamin premixes may consist of vitamin A, vitamin D, vitamin E, riboprabin and niacin, and mineral premixes may consist of manganese, iron, zinc, calcium, copper, cobalt and selenium. The feed is a livestock feed, but may include broiler feed, pig feed or cattle feed, but is not limited thereto.

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

The composition of the present invention may be prepared according to a conventional method for preparing a formal composition, and generally, may be in lyophilized or encapsulated form, culture suspension or dry powder form. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives may be selected as an effective amount of the Lactobacillus luterii OH0335 strain (KCTC13362BP) or a culture medium thereof, which is a main component, to form a composition of a conventional formulation. It can manufacture.

The carrier may be used by selecting one or two or more from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of the fragrances, vitamins, and antioxidants. Can be used.

In the present invention, the carrier and the additive may be used in all pharmaceutically acceptable, and specifically, as a diluent, lactose monohydrate, trehalose, corn starch, soybean oil. , Microcrystalline cellulose or mannitol (D-mannitorl) is preferable, and magnesium stearate or talc is preferable as a lubricant, and polyvinylpyrrolidone (PVP: polyvinyipyrolidone) or It is preferable to select from hydroxypropyl cellulose (HPC: hydroxypropyl cellulose). In addition, the disintegrant may be selected from among carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polyacrylin potassium or cross-linked polyvinylpyrrolidone. Preferably, the sweetening agent is selected from sucrose, fructose, sorbitol or aspartame, and the stabilizer is carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (β-cyclodextrin) or white lead. (white bee's wax) or xanthan gum, and preservatives include methyl p-hydroxy benzoate (methhlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate).

In addition, the strain culture obtained by inoculating the strain culture obtained by inoculating the Lactobacillus ruteri OH0335 strain (KCTC13362BP) further inoculated with microorganisms having probiotic activity was administered to the animal as a probiotic having probiotic activity. Or can be administered to animals together with foods, medicines, veterinary drugs or lactic acid bacteria, and preferably used as feed additives or supplementary feed for livestock, and the rich nutrients and useful physiological activity contained in colostrum when feeding the probiotics to pigs. Substances can increase pig growth rates and prevent diarrhea in weaned piglets.

In addition, the present invention provides a method for producing lutein comprising the step of culturing the strain.

The method of producing lutein of the present invention may further comprise the step of recovering lutein from the culture of the strain. Recovering lutein from the culture medium of the strain may use a conventional separation technique, for example, distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, etc. These may be used in combination to separate them.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1. New Lactic Acid Bacteria Lactobacillus Lutheri  Isolation of OH0335 Strain

Pig small and duodenal samples were taken from a pig slaughterhouse in Iksan-si, Jeollabuk-do, and suspended in 10 g of physiological saline to finely crushed 1 g of the sample, diluted to 1 × 10 ^-4 times, and then containing MRS solid medium containing bromocresol purple ( 10 g / L proteose peptone NO.3, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L dextrose, 1 g / L polysorbate 80, 2 g / L ammonium citrate, 5 g / L sodium acetate, 100 μl of 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 g / L dipotassium phosphate, 0.04 g / L bromocresol purple, 15 g / L agar) and smear Bactron Anaerobic Chamber at 37 ° C. Colonies obtained by culturing in anaerobic condition for 24 hours using (SHEL LAB, Cornelius, OR.USA.) Were re-inoculated in MRS medium containing bromocresol purple, and then purely separated, followed by MALDI Biotyper (Bruker Daltonik, Billerica, 81 Lactobacillus ruteri strains It was isolated. Inoculated in 3mL MRS medium (15mL Falcon tube) and pre-incubated in anaerobic condition for 24 hours using Bactron Anaerobic Chamber at 37 ° C, and then 50mL lactic acid bacteria culture medium (10g / L proteose peptone NO.3, 10g / L beef Extract, 5 g / L 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 flask) was incubated anaerobicly for 24 hours using an Bactron Anaerobic Chamber at 37 ° C. with an initial inoculation of OD 0.5. After incubation, the culture was centrifuged at 13,000 rpm for 5 minutes, and then the cells were recovered and washed twice in 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in 200mM glycerol solution and allowed to stand in a 30 ° C. incubator and samples were taken at 1 hour intervals. The reaction sample was centrifuged at 13,000 rpm for 5 minutes and then used for analysis. Luterin quantitative analysis used acrolein as a standard. 0.75 mL of 1 mL of lutein sample, 3 mL of 37% HCl, and 10 mM tryptophan-HCl were mixed and reacted at 37 ° C. for 20 minutes. After the reaction, the absorbance of the reaction solution was measured at 560 nm using an ELISA reader to quantify the yield of lutein.

As a result, one of Lactobacillus luteri strains excellent in lutein production among 140 isolates was finally selected. One hour after the reaction, 30 mM of lutein was produced from 200 mM of glycerol.

Luterin Production of Newly Isolated Lactobacillus Lacteri Strains 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 of the Lactobacillus Lactobacillus luteri OH0335 strain with the best lutein production. Chromosomal DNA was isolated from a colony using a genomic DNA extraction kit (Invitrogen, Germany), and then the primers 5'-AGAGTTTGATCMTGGCTCAG-3 '(27f, SEQ ID NO: 2) and 5' were used for amplifying the Lactobacillus 16S rRNA gene. 16S rRNA gene DNA was amplified by PCR using -TACGGYTACCTTGTTACGACTT-3 '(1492r, 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. , 30 times with a genetic amplifier (Takara, Japan) at 96 ℃ 30 seconds, 50 ℃ 1 minutes, 72 ℃ 2 minutes conditions. The PCR reaction solution was electrophoresed on 1% agarose gel to confirm that the DNA fragment of the expected size was amplified and transformed into E. coli EPI300 using a pGEM-TEasy vector (Promega, USA). 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 results of homology analysis of the nucleotide sequence, the isolated lactic acid bacteria was named Lactobacillus reuteri OH0335.

Example  2. Newly Isolated Lactic Acid Bacteria through Fermenter Culture Lactobacillus Lutheri  OH0335 strain Lutherin  production

MRS medium (10 g / L proteose peptone NO.3, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L dextrose, 1 g / L polysorbate 80, using Bacillus luteri OH0335 strain in a 5 L incubator) 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), followed by centrifugation of the culture medium at 13,000 rpm for 5 minutes and then It was recovered and washed twice in 50 mM sodium phosphate buffer (pH 7.5). The washed cells were resuspended in 200mM glycerol solution and allowed to stand in a 30 ° C. incubator and samples were taken at 1 hour intervals. As a result, as shown in Table 2 below, a cell concentration of 10 g DCW / L and glycerol at 540 mM produced 299.8 mM lutein at 1 hour of reaction.

Lutein Production 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 luteric lactic acid bacteria reported in the literature, the isolated strains were shown to show good lutein production.

Comparison of Luterin Production by Lactobacillus Luterry Lactobacillus Strains 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 ATCC53608 200 30 170 2 L-thi-Peng et al., (2002) L. reuteri ATCC53608 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
(Δlr-1734) 250 12 236.4 One Marc J A et al. (2011) L. reuteri OH0335 540 10 299.8 One The present invention

Korea Research Institute of Bioscience and Biotechnology KCTC13362BP 20171013

<110> Korea Research Institute of Bioscience and Biotechnology          ActivON Co., Ltd. <120> Lactobacilus reuteri OH0335 strain having high productivity of          reuterin from glycerol and uses <130> PN17455 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1512 <212> DNA <213> Lactobacilus 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) to produce lutein from glycerol. A microbial agent for producing lutein, comprising the strain of claim 1 or a culture thereof as an active ingredient. Antimicrobial composition comprising the strain of claim 1 or a culture thereof as an active ingredient. Feed composition for antimicrobial comprising the strain of claim 1 or a culture thereof. delete delete

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