MOON GI SEONG (KR)
| KR20030082165A | 2003-10-22 |
| Claims [Claim 1] A strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp. [Claim 2] A fermentation starter comprising, as an active ingredient, at least one selected from the group consisting of a strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth. [Claim 3] The fermentation starter of Claim 2 further comprising at least one strain selected from the group consisting of Lactococcus lactis, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei and mixtures thereof. [Claim 4] Fermented foods containing the fermentation starter of Claim 2 or 3. [Claim 5] Foods comprising, as an active ingredient, at least one selected from the group consisting of a strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth. [Claim 6] The foods of Claim 5, wherein the foods are fermented foods or fermented beverages. [Claim 7] A method for stimulating the growth of Bifidobacterium sp., the method comprising: culturing Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP) isolated from kimchi to obtain a culture broth; and using the culture broth to stimulate the growth of Bifidobacterium sp. [Claim 8] The method of Claim 7, wherein the culture of the strain is carried out in whey medium. [Claim 9] A strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp. [Claim 10] A fermentation starter comprising, as an active ingredient, at least one selected from the group consisting of a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifi- dobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth. [Claim 11] The fermentation starter of Claim 10 further comprising at least one strain selected from the group consisting of Lactococcus lactis, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus bulgaricus, Leu- conostoc mesenteroides and mixtures thereof. [Claim 12] Fermented foods containing the fermentation starter of Claim 10 or 11. [Claim 13] Foods comprising, as an active ingredient, at least one selected from the group consisting of a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth. [Claim 14] The foods of Claim 13, wherein the foods are fermented foods or fermented beverages. [Claim 15] A method for stimulating the growth of Bifidobacterium sp., the method comprising: culturing Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) isolated from cheese to obtain a culture broth; and using the culture broth to stimulate the growth of Bifidobacterium sp. [Claim 16] The method of Claim 15, wherein the culture of the strain is carried out in whey medium. |
STRAIN STIMULATING THE GROWTH OF BIFIDOBACTERIUM SP.
Technical Field
[1] The present invention relates to a novel strain of Leuconostoc mesenteroides CJNU
0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp. , a novel strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp. , and foods containing these strains. Background Art
[2] Members of Bifidobacterium sp. such as Bifidobacterium longum are beneficial bacteria that live in the human gastrointestinal tract and provide many health benefits. The Bifidobacterium sp. is known to exhibit various physiological activities, including inhibition of pathogenic microorganisms, constipation prevention, diarrhea prevention, and immune enhancement.
[3] However, it was reported that, as people become older, the ratio of Bifidobacterium sp. to the total bacteria in the human intestinal tract decreases. As a result, pathogenic bacteria prevail and cause various intestinal diseases.
[4] Methods for solving this problem are broadly classified into two categories: a method of taking exogenous Bifidobacterium sp.; and a method of stimulating the growth of Bifidobacterium sp. colonizing the intestine. The method of taking exogenous Bifidobacterium sp. is to take foods such as fermented milk products, which contain Bifidobacterium sp. However, Bifidobacterium sp. is an obligate anaerobe which is likely to be killed even in the presence of a very small amount of oxygen. For this reason, this method has a problem in that the content of living Bifidobacterium sp. in foods such as fermented milk products is significantly reduced during the distribution of the foods.
[5] Accordingly, the present inventor has conducted many studies to improve the growth of Bifidobacterium sp. As a result, the present inventor has isolated microorganisms, having an excellent ability to stimulate the growth of Bifidobacterium sp., from kimchi and cheese, and has developed foods containing the microorganisms and a method for stimulating the growth of Bifidobacterium sp. using the microorganisms. Disclosure of Invention
Technical Problem
[6] The present invention is to provide novel microorganisms, having an excellent ability to stimulate the growth of Bifidobacterium sp., and functional foods containing the microorganisms.
[7] The present invention is also to provide a strain of Leuconostoc mes enter oides CJNU
0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., and a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp.
[8] The present invention is also to provide functional foods, such as fermented foods and fermented beverages, which contain said microorganisms.
[9] The present invention is also to provide a method for stimulating the growth of Bifidobacterium sp. using said microorganisms. Solution to Problem
[10] The present invention provides a strain of Leuconostoc mesenteroides CJNU 0147
(accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., and a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp.
[11] The present invention provides a fermentation starter comprising, as an active ingredient, at least one selected from the group consisting of a strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth.
[12] In addition, the present invention provides a fermentation starter comprising, as an active ingredient, at least one selected from the group consisting of a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth.
[13] According to another aspect of the present invention, there are provided fermented foods containing said fermentation starters.
[14] According to still another aspect of the present invention, there is provided foods comprising, as an active ingredient, at least one selected from the group consisting of a strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), which is isolated from kimchi and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth.
[15] The present invention also provides foods comprising, as an active ingredient, at least one selected from the group consisting of a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), which is isolated from cheese and has the ability to stimulate the growth of Bifidobacterium sp., a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth.
[16] According to one embodiment of the present invention, the foods may be fermented foods or fermented beverages.
[17] According to yet another aspect of the present invention, there is provided a method for stimulating the growth of Bifidobacterium sp., the method comprising: culturing Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP) isolated from kimchi to obtain a culture broth; and using the culture broth to stimulate the growth of Bifidobacterium sp.
[18] In addition, the present invention provides a method for stimulating the growth of Bifidobacterium sp., the method comprising: culturing Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) isolated from cheese to obtain a culture broth; and using the culture broth to stimulate the growth of Bifidobacterium sp.
[19] According to one embodiment of the present invention, the culture of the strain can be carried out in whey medium.
Advantageous Effects of Invention
[20] The use of a strain of Leuconostoc mesenteroides CJNU 0147 (accession number:
KCTC 11475BP) according to the present invention, a culture broth of the strain, a concentrate of the culture broth and a dry matter of the culture broth can improve the growth of Bifidobacterium sp. Also, the use of a strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) according to the present invention, a culture broth of the strain, a concentrate of the culture broth, and a dry matter of the culture broth can improve the growth of Bifidobacterium sp.
[21] Furthermore, the strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), the culture broth of the strain, and the like, have the effect of selectively stimulating the growth of Bifidobacterium sp., and thus can be used to produce functional foods having a high content of Bifidobacterium sp.
[22] In addition, the strain of Leuconostoc mesenteroides CJNU 0147 (accession number:
KCTC 11475BP), the culture broth of the strain, and the like can be used to increase the content of Bifidobacterium sp. in vivo.
[23] Furthermore, the strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) according to the present invention, a culture broth of the strain, a concentrate of the culture broth and a dry matter of the culture broth have the same effects as those of the strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), the culture broth of the strain, and the like. Brief Description of Drawings
[24] FIG. 1 is a graphic diagram showing the effects of various strain samples on the growth of B. longum. Sample No. 1: a blank (RCM medium alone); sample No. 2: inoculated with only B. longum; sample No. 3: B. longum + whey; and sample Nos. 4-39: B. longum + sample (whey fermentation product).
[25] FIG. 2 is a graphic diagram showing the effect of the inventive Leuconostoc mesenteroides CJNU 0147 on the growth of B. longum, obtained by repeated experiments. Blank: RCM medium alone; B: a sample inoculated with only B. longum; B + Whey: a sample inoculated with B. longum + whey; and B + CJNU 0147: a sample inoculated with B. longum + CJNU 0147 (whey fermentation product).
[26] FIG. 3 is a graphic diagram showing the effect of the inventive Leuconostoc mesenteroides CJNU 0147 on the growth of Escherichia coli DH5a. Blank: LB medium alone; E. coli: a sample inoculated only with E. coli; E. coli + Whey: a sample inoculated with E. coli + whey; and E. coli + CJNU 0147: a sample inoculated with E. coli + CJNU 0147 (whey fermentation product).
[27] FIG. 4 is a graphic diagram showing the effect of the inventive Leuconostoc mesenteroides CJNU 0147 on the growth of Enterococcus faecalis KFRI 675. Blank: MRS medium alone; Ent: a sample inoculated only with E. faecalis; Ent + Whey: a sample inoculated with E. faecalis + whey; and Ent + CJNU 0147 is a sample inoculated with E. faecalis + CJNU 0147 (whey fermentation product).
[28] FIG. 5 is a graphic diagram showing the effect of the inventive Leuconostoc mesenteroides CJNU 0147 on the growth of Staphylococcus aureus ATCC 14458. Blank: MRS medium alone; Staphy: a sample inoculated only with S. aureus; Staphy + Whey: a sample inoculated with S. aureus + whey; and Staphy + CJNU 0147: a sample inoculated with S. aureus + CJNU 0147 (whey fermentation product).
[29] FIG. 6 is a graphic diagram showing the effect of inventive Leuconostoc mesenteroides CJNU 0147 on the growth of Listeria monocytogenes ATCC 19111. Blank: MRS medium alone; Lis: a sample inoculated only with L. monocytogenes; Lis + Whey: a sample inoculated with L. monocytogenes + whey; and Lis + CJNU 0147: a sample inoculated with L. monocytogenes + CJNU 0147 (whey fermentation product).
[30] FIG. 7 is a graphic diagram showing the effects of various strain samples on the growth of B. longum. Sample No. 1: a blank (RCM medium alone); sample No. 2: inoculated only with B. longum; sample No. 3: B. longum + whey; and sample Nos. 4-37: B. longum + sample (whey fermentation product).
[31] FIG. 8 is a graphic diagram showing the effect of the inventive Lactobacillus casei
CJNU 0588 on the growth of B. longum, obtained through repeated experiments. Blank: RCM medium alone; B. longum: a sample inoculated only with B. longum; B + Whey: a sample inoculated with B. longum + whey; and B + CJNU 0588: a sample inoculated with B. longum + CJNU 0588 (whey fermentation product).
[32] FIG. 9 is a graphic diagram showing the effect of the inventive Lactobacillus casei
CJNU 0588 on the growth of Escherichia coli DH5a. Blank: LB medium alone; E. coli: a sample inoculated only with E. coli; E. coli + Whey: a sample inoculated with E. coli + whey; and E. coli + CJNU 0588: a sample inoculated with E. coli + CJNU 0588 (whey fermentation product).
[33] FIG. 10 is a graphic diagram showing the effect of the inventive Lactobacillus casei
CJNU 0588 on the growth of Enterococcus faecalis KFRI 675. Blank: MRS medium alone; Ent: a sample inoculated only with E. faecalis; Ent + Whey: a sample inoculated with E. faecalis + whey; and Ent + CJNU 0588: a sample inoculated with E. faecalis + CJNU 0588 (whey fermentation product).
[34] FIG. 11 is a graphic diagram showing the effect of the inventive Lactobacillus casei
CJNU 0588 on the growth of Staphylococcus aureus ATCC 14458. Blank: MRS medium alone; Staphy: a sample inoculated only with S. aureus; Staphy + Whey: a sample inoculated with S. aureus + whey; and Staphy + CJNU 0588: a sample inoculated with S. aureus + CJNU 0588 (whey fermentation product).
[35] FIG. 12 is a graphic diagram showing the effect of the inventive Lactobacillus casei
CJNU 0588 on the growth of Listeria monocytogenes ATCC 19111. Blank: MRS medium alone; Lis: a sample inoculated only with L. monocytogenes; Lis + Whey: a sample inoculated with L. monocytogenes + whey; and Lis + CJNU 0588: a sample inoculated with L. monocytogenes + CJNU 0588 (whey fermentation product). Best Mode for Carrying out the Invention
[36] Leuconostoc mesenteroides CJNU 0147 of the present invention is isolated from kimchi. Specifically, freshly prepared kimchi was fermented at 10 0 C for 3 days, and then a sample was collected from the fermented kimchi. The sample was diluted, and plated on Difco™ Lactobacilli MRS solid medium. A strain was isolated from the sample, and then identified by determining its 16S rRNA gene sequence and comparing sequence homology with other published strains.
[37] As a result, the strain isolated according to the present invention was finally identified as Leuconostoc mesenteroides, a kind of lactic acid bacteria. The isolated strain was named Leuconostoc mesenteroides CJNU 0147 and deposited with the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology on March 13, 2009 under accession number KCTC 11475BP.
[38] The strain of Leuconostoc mesenteroides CJNU 0147 has an excellent effect of selectively stimulating the growth of particularly Bifidobacterium sp. Thus, the strain can be used to produce functional foods having a high content of Bifidobacterium sp. Also, the strain can be used to increase the content of Bifidobacterium sp. in vivo.
[39] In addition, Lactobacillus casei CJNU 0588 of the present invention is isolated from cheese. Specifically, a sample was collected from natural cheese provided by Namyang Dairy Products Co., Ltd. (Korea). The sample was diluted and plated on Difco™ Lactobacilli MRS solid medium, and then a strain was isolated from the sample and identified by determining its 16S rRNA gene sequence and comparing sequence homology with other published strains.
[40] In the present invention, processed cheese and natural cheese can both be used, but natural cheese is preferably used. As used herein, the term natural cheese refers to cheese made by fermenting milk without any additive. The term processed cheese refers to cheese containing additives such as a preservative.
[41] As a result, the strain isolated according to the present invention was finally identified as Lactobacillus casei, a kind of lactic acid bacteria. The strain was named Lactobacillus casei CJNU 0588 and deposited with the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology on March 13 under accession number KCTC 11474BP.
[42] The Lactobacillus casei CJNU 0588 has an excellent effect of selectively stimulating the growth of particularly Bifidobacterium sp. Thus, the strain can be used to produce functional foods having a high content of Bifidobacterium sp. Also, the strain can be used to increase the content of Bifidobacterium sp. in vivo.
[43] The above strains can be used as fermentation starters. Specifically, in the present invention, a substance comprising, as an active ingredient, at least one selected from the group consisting of the Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), a culture broth of the strain, a concentrate of the strain, and a dry matter of the culture broth, can be used as a fermentation starter. Moreover, the case of the Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) can also be used as a fermentation starter, like the case of the Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP).
[44] The fermentation starters may be used in a mixture and may contain at least one strain selected from the group consisting of Lactococcus lactis, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus bulgaricus, and mixtures thereof, but the scope of the present invention is not limited thereto.
[45] The composition ratio of the strains in the strain mixture may be the same and is not particularly limited. [46] The present invention can provide fermented foods containing the fermentation starters.
[47] Furthermore, according to the present invention, foods comprising, as an active ingredient, the strain of Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), a culture broth of the strain, a concentrate of the strain and a dry matter of the culture broth can be prepared.
[48] In addition, according to the present invention, foods comprising, as an active ingredient, the strain of Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP), a culture broth of the strain, a concentrate of the strain and a dry matter of the culture broth can be prepared. The foods may be fermented foods or fermented beverages.
[49] As used herein, the term culture medium means a solid or liquid material containing nutrients necessary for the growth of animal or plant cells or microorganisms. The term culture broth refers to one obtained by inoculating and culturing the strain in the liquid medium and is meant to include a culture filtrate which is a supernatant obtained by removing the strain from the liquid culture broth. As used herein, the term concentrate of the culture broth refers to one obtained by concentrating the culture broth, and the term dry matter of the culture broth refers to one obtained by removing water from the culture broth.
[50] The present invention provides fermented foods containing the fermentation starters.
As used herein, the term fermented foods refers to foods made by adding one or two or more microorganisms such as lactic acid bacteria or yeasts and performing fermentation by the microorganisms. Specifically, the term refers to foods made by adding the fermentation starters to food bases which are then incubated.
[51] The fermented foods include all unpasteurized open-fermented foods, such as alcoholic beverages, breads, kimchi, salted-fermented sea foods, soybean paste, cheese, yogurt and the like. The amount of fermentation starter used is suitably determined depending on the kind of fermented food and the kind of fermentation starter. Specifically, the fermentation starter is inoculated into a food base in an amount of 0.0001-0.5 parts by weight based on 100 parts by weight of the food base, but the scope of the present invention is not limited thereto.
[52] As used herein, the term functional foods refers to: a group of foods obtained by providing value-added features to foods by physical, biochemical and biotechnological techniques so as to exhibit the functions of the foods depending on the intended use thereof; or processed foods designed such that food ingredients may fully perform their functions for the regulation of biorhythm, and regulation of physical condition relating to the prevention of and recovery from diseases.
[53] As used herein, the term beverages collectively refers to beverages which are used to quench thirst or enjoy the taste. Examples of the beverages include refreshing beverages, water, syrup, tea, coffee, fruit beverages. The term beverages is meant to include fermented beverages. The term fermented beverages refers to beverages obtained by culturing lactic acid bacteria, performing lactic acid fermentation using the cultured lactic acid bacteria and adding sugars and spices thereto.
[54] As used herein, the term foods refers to natural materials or processed products, which contain at least one nutrient. There is no limitation on the kind of food of the present invention, and there is no particular limitation on the addition of components other than the food comprising, as an active ingredient, at least one selected from the group consisting of the Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP), a culture broth of the strain, a concentrate of the strain and a dry matter of the culture broth (this similarly applies to the case of the Lactobacillus casei CJNU 0588).
[55] The fermented foods and/or foods of the present invention may contain, in addition to the active ingredient, various flavoring agents or carbohydrates.
[56] The Leuconostoc mesenteroides CJNU 0147 (accession number: KCTC 11475BP) and Lactobacillus casei CJNU 0588 (accession number: KCTC 11474BP) of the present invention have an excellent effect of stimulating the growth of Bifidobacterium sp.
[57] Specifically, Bifidobacterium sp. can be grown using a culture broth obtained by culturing the Leuconostoc mesenteroides CJNU 0147. The definition of the culture broth includes all a culture broth (including a culture filtrate) of the above-mentioned Leuconostoc mesenteroides CJNU 0147, a concentrate of the culture broth, and a dry matter of the culture broth.
[58] Also, specifically, Bifidobacterium sp. can be grown using a culture broth obtained by culturing the Lactobacillus casei CJNU 0588. The definition of the culture broth includes all a culture broth (including a culture filtrate) of the Lactobacillus casei CJNU 0588, a concentrate of the culture broth, and a dry matter of the culture broth.
[59] The culture of the Leuconostoc mesenteroides CJNU 0147 or the culture of the Lactobacillus casei CJNU 0588 can be carried out in whey medium. The whey is a byproduct generated in a process of producing cheese from milk and contains various nutrients (lactose, proteins, minerals, vitamins, etc.).
[60] In the present invention, when whey whose price is low is used as medium, the production cost can be reduced, thus ensuring the price competitiveness of the product. Also, various functional proteins (β-lactoglobulin, α-lactalbumin, immunoglobulin, lactoferrin, lactoperoxidase, etc.) which are contained in whey can contribute to the added value of the culture broth.
[61] The whey fermentation product of the Leuconostoc mesenteroides CJNU 0147 or the Lactobacillus casei CJNU 0588 may directly be used without further processing, because it has an excellent effect of stimulating the growth of Bifidobacterium sp. and is beneficial to the human body.
[62] Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
[63]
[64] Example 1
[65] 1-1: Isolation and identification of Leuconostoc mesenteroides CJNU 0147
[66] (1) Isolation of strain having excellent effect of stimulating the growth of BiR- dobacteήum sp.
[67] Lactic acid bacteria were isolated from kimchi in order to examine their ability to stimulate the growth of Bifidobacterium sp. First, freshly prepared kimchi was fermented at 10 0 C for 3 days, and then a sample (10 g) was collected from the fermented kimchi, aseptically placed in a filter bag, mixed with 90 mL of peptone water (0.1% w/v), and then homogenized. Next, the sample was diluted using the 10-fold dilution method and plated on Difco™ Lactobacilli MRS solid medium (10.0 g/L proteose peptone No. 3, 10.0 g/L beef extract, 5.0 g/L yeast extract, 20.0 g/L dextrose, 1.0 g/L polysorbate 80, 2.0 g/L ammonium citrate, 5.0 g/L sodium acetate, 0.1 g/L magnesium sulfate, 0.05 g/L manganese sulfate, 2.0 g/L dipotassium phosphate, and 15 g/L agar). The medium was incubated at 37 0 C for 24 hours, and then the formed colonies were picked randomly and cultured in 5 mL of Difco™ Lactobacilli MRS liquid medium (having the same composition as that of the above- mentioned solid medium except that it contained no agar). The cultured strains were isolated, purified twice by three-phase partitioning, and stored as glycerol stocks in an ultra-low-temperature freezer at -76 0 C.
[68] 400 strains isolated from kimchi as described above were activated in Difco™ Lactobacilli MRS liquid medium, and then inoculated into whey medium (10% w/v whey powder) at a ratio of 1: 100. After culture for 24 hours, each of the strains was cen- trifuged to precipitate bacteria and insoluble solids, and each of the supernatants (culture filtrates) was collected and filtered through a 0.2-/M filter to eliminate bacteria. The filtrates (including the fermentation products) passed through the filter were used as samples.
[69] 50 L of each of the samples was added to 5 mL of Difco™ reinforced clostridial medium (5.0 g/L pancreatic digest of casein, 5.0 g/L proteose peptone No. 3, 10.0 g/L beef extract, 3.0 g/L yeast extract, 5.0 g/L dextrose, 5.0 g/L sodium chloride, 1.0 g/L soluble starch, 0.5 g/L cysteine hydrochloride, 3.0 g/L sodium acetate, and 0.5 g/L agar) inoculated with a Bifidobacterium longum strain at a ratio of 1:100. As a control, a non-fermented whey filtrate was used.
[70] The culture of Bifidobacterium sp. was carried out in an anaerobic incubator at 37 0 C for 10 hours. After completion of the culture, 1 mL of each sample was pipetted into a disposable cuvette and measured for absorbance (OD 600 ) using a spectrophotometer (BioPhotometer, Eppendorf). On the basis of the measured absorbance, the growth rates of the samples were compared with each other, and the measurement results are shown in FIG. 1.
[71] From the results in FIG. 1, it could be seen that the sample represented by bar graph
No. 16 showed a very high growth rate of Bifidobacterium sp. The sample No. 16 was first named CJNU 0147. FIG. 2 shows the results obtained by three repeated experiments performed to reconfirm the effect of CJNU 0147 on the growth of Bifidobacterium sp. From the results in FIG. 2, the effect of CJNU 0147 on the growth of Bifidobacterium sp. was significantly demonstrated.
[72]
[73] (2) Identification of strain
[74] The isolated CJNU 0147 strain was identified by analyzing the nucleotide sequence of the 16S rRNA gene. Specifically, genomic DNA was extracted from the isolated strain using a genomic DNA preparation kit (Promega), and its 16S rRNA gene was amplified by PCR using universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The amplified product was purified using acetate and 70% ethanol, and then PCR amplification was performed using the purified product as a template with sequencing primers 518F (5'-CCA GCA GCC GCG GTA ATA CG-3') and 800R (5'-TAC CAG GGT ATC TAA TCC-3') and a sequencing kit (ABI PRISM BigDye Terminator Cycle Sequencing Kits). Then, the amplified product was purified with ethanol, and the nucleotide sequence thereof was analyzed using an ABI PRISM 3730XL Analyzer (96 capillary type).
[75] The 16S rDNA nucleotide sequence of CJNU 0147 is shown in SEQ ID NO: 1. The
16S rRNA gene sequence of the isolated strain was aligned and compared with the sequences of reference strains from the EMBL/DDBJ/PDB/GenBank sequence databases to determine sequence homology.
[76] As a result, the sequence of the isolated CJNU 0147 strain showed a homology of
99% with Leuconostoc mesenteroides (GenBank accession no. AB362705.1). The CJNU 0147 strain was named Leuconostoc mesenteroides CJNU 0147 and deposited with the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology on March 13, 2009 under accession number KCTC 11475BP.
[77] [78] 1-2: Examination of effect of stimulating the growth of Bifidobacterium sp.
[79] In order to confirm if the culture broth (whey fermentation product) of CJNU 0147 selectively stimulates the growth of Bifidobacterium sp., the effects of the culture broth on the growth of typical intestinal bacterial strains (Escherichia coli DH5, and Ente- rococcus faecalis KFRI 675) and pathogenic microbial strains (Staphylococcus aureus ATCC 14458, and Listeria monocytogenes ATCC 19111) were examined. Specifically, CJNU 0147 was cultured in whey medium in the same manner as in Example l-l-(l), and the culture broth was added to a medium inoculated with each of the above strains. The growth rate of each of the strains was compared with a control. Herein, Escherichia coli DH5 was shaking-cultured in 5 mL of LB medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract, pH 7.0) at 37 0 C, and the remaining strains were stationary-cultured in 5 mL of Difco™ Lactobacilli MRS liquid medium at 37 0 C.
[80] The results of the cultures are shown in FIGS. 3 to 6. From the results in FIGS. 3 to
6, it can be seen that the whey fermentation product of CJNU 0147 has no significant effect on the growth of E. coli DH5, Enterococcus faecalis KFRI 675, Staphylococcus aureus ATCC 14458 and Listeria monocytogenes ATCC 19111. Namely, it can be seen that the whey fermentation product of CJNU 0147 selectively stimulates the growth of Bifidobacterium sp.
[81]
[82] 1-3: Preparation of yogurt using Leuconostoc mesenteroides CJNU 0147
[83] Raw milk having fat content of 3.2% (a skimmed solid content of 8.5%) was adjusted to a skimmed solid content of 12% by adding skimmed milk powder thereto, and then it was heated, homogenized and sterilized, thus preparing a medium. Then, the medium was inoculated with a culture broth of Leuconostoc mesenteroides CJNU 0147 cultured in whey medium (10% w/v whey powder), and it cultured at 37 0 C. The culture process was carried out until a pH of 4.5 was reached. Then, fruit jam as a flavoring agent was added thereto, thus preparing yogurt.
[84]
[85] Example 2
[86] 2-1: Isolation and identification of Lactobacillus casei CJNU 0588 strain
[87] (1) Isolation of strain having excellent effect of stimulating the growth of Bifidobacterium sp.
[88] Lactic acid bacteria were isolated from natural cheese in order to examine their ability to stimulate the growth of Bifidobacterium sp. First, a natural cheese sample (10 g) was collected from natural cheese provided by Namyang Dairy Products Co., Ltd. (Korea), and it was aseptically placed in a filter bag, mixed with 90 mL of peptone water (0.1% w/v), homogenized, and then diluted using the 10-fold dilution method. The dilution was plated on Difco™ Lactobacilli MRS solid medium (10.0 g/L proteose peptone No. 3, 10.0 g/L beef extract, 5.0 g/L yeast extract, 20.0 g/L dextrose, 1.0 g/L polysorbate 80, 2.0 g/L ammonium citrate, 5.0 g/L sodium acetate, 0.1 g/L magnesium sulfate, 0.05 g/L manganese sulfate, 2.0 g/L dipotassium phosphate, and 15 g/L agar). After culture at 37 0 C for 24 hours, the formed colonies were picked randomly and cultured in 5 mL of Difco™ Lactobacilli MRS liquid medium (having the same composition as that of the above-mentioned solid medium except that it contained no agar). The cultured strains were isolated, purified twice by three-phase partitioning, and stored as glycerol stocks in an ultra-low-temperature freezer at -76 0 C.
[89] Three hundred strains isolated from cheese as described above were activated in
Difco™ Lactobacilli MRS liquid medium, and then inoculated into whey medium (10% (w/v) whey powder) at a ratio of 1: 100. After culture for 24 hours, each of the cultures was centrifuged to precipitate bacteria and insoluble solids, and each of the su- pernatants (culture filtrates) was collected and filtered through a 0.2-βia filter to eliminate bacteria. The filtrates (including the fermentation products) passed through the filter were used as samples.
[90] 50 L of each of the samples was added to 5 mL of Difco™ reinforced clostridial medium (5.0 g/L pancreatic digest of casein, 5.0 g/L proteose peptone No. 3, 10.0 g/L beef extract, 3.0 g/L yeast extract, 5.0 g/L dextrose, 5.0 g/L sodium chloride, 1.0 g/L soluble starch, 0.5 g/L cysteine hydrochloride, 3.0 g/L sodium acetate, and 0.5 g/L agar) inoculated with a Bifidobacterium longum strain at a ratio of 1:100. As a control, a non-fermented whey filtrate was used.
[91] The culture of Bifidobacterium sp. was carried out in an anaerobic incubator at 37 0 C for 10 hours. After completion of the culture, 1 mL of each sample was pipetted into a disposable cuvette and measured for absorbance (OD 600 ) using a spectrophotometer (BioPhotometer, Eppendorf). On the basis of the measured absorbance, the growth rates of the samples were compared with each other, and the measurement results are shown in FIG. 7.
[92] From the results in FIG. 7, it could be seen that the sample represented by bar graph
No. 33 showed a very high growth rate of Bifidobacterium sp. The sample No. 33 was first named CJNU 0588.
[93] FIG. 8 shows the results obtained by three repeated experiments performed to reconfirm the effect of CJNU 0588 on the growth of Bifidobacterium sp. From the results in FIG. 8, the effect of CJNU 0588 on the growth of Bifidobacterium sp. was significantly demonstrated.
[94]
[95] (2) Identification of strain
[96] The isolated CJNU 0588 strain was identified by analyzing the nucleotide sequence of the 16S rRNA gene. Specifically, genomic DNA was extracted from the isolated strain using a genomic DNA preparation kit (Promega), and its 16S rRNA gene was amplified by PCR using universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The amplified product was purified using acetate and 70% ethanol, and then PCR amplification was performed using the purified product as a template with sequencing primers 518F (5'-CCA GCA GCC GCG GTA ATA CG-3') and 800R (5'-TAC CAG GGT ATC TAA TCC-3') and a sequencing kit (ABI PRISM BigDye Terminator Cycle Sequencing Kits). Then, the amplified product was purified with ethanol, and the nucleotide sequence thereof was analyzed using an ABI PRISM 3730XL Analyzer (96 capillary type).
[97] The 16S rDNA nucleotide sequence of CJNU 0588 is shown in SEQ ID NO: 2. The
16S rRNA gene sequence of the isolated strain was aligned and compared with the sequences of reference strains from the EMBL/DDBJ/PDB/GenBank sequence databases to determine sequence homology.
[98] As a result, the sequence of the isolated CJNU 0588 strain showed a homology of
99% with & Lactobacillus casei strain (GenBank accession no. CP000423.1). The CJNU 0588 strain was named Lactobacillus casei CJNU 0588 and deposited with the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology on March 13, 2009 under accession number KCTC 11474BP.
[99]
[100] 2-2: Examination of effect of stimulating the growth of Bifidobacterium sp.
[101] In order to confirm if the culture broth (whey fermentation product) of CJNU 0588 selectively stimulates the growth of Bifidobacterium sp., the effects of the culture broth on the growth of typical intestinal bacterial strains (Escherichia coli DH5, and Ente- rococcus faecalis KFRI 675) and pathogenic microbial strains (Staphylococcus aureus ATCC 14458, and Listeria monocytogenes ATCC 19111) were examined. Specifically, CJNU 0588 was cultured in whey medium in the same manner as in Example 2-l-(l), and the culture broth was added to a medium inoculated with each of the above strains. The growth rate of each of the strains was compared with a control. Herein, Escherichia coli DH5 was shaking-cultured in 5 mL of LB medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract, pH 7.0) at 37 0 C, and the remaining strains were stationary-cultured in 5 mL of Difco™ Lactobacilli MRS liquid medium at 37 0 C.
[102] The results of the culture are shown in FIGS. 9 to 12. From the results in FIGS. 9 to 12, it can be seen that the whey fermentation product of CJNU 0588 has no significant effect on the growth of E. coli DH5, Enterococcus faecalis KFRI 675, Staphylococcus aureus ATCC 14458 and Listeria monocytogenes ATCC 19111. Namely, it can be seen that the whey fermentation product of CJNU 0588 selectively stimulates the growth of Bifidobacterium sp. [103] 2-3: Preparation of yogurt using Lactobacillus casei CJNU 0588
[104] Raw milk having fat content of 3.2% (a skimmed solid content of 8.5%) was adjusted to a skimmed solid content of 12% by adding skimmed milk powder thereto, and then it was heated, homogenized and sterilized, thus preparing a medium. Then, the medium was inoculated with a culture broth of Lactobacillus casei CJNU 0588 cultured in whey medium (10% (w/v) whey powder), and it cultured at 37 0 C. The culture process was carried out until a pH of 4.5 was reached. Then, fruit jam as a flavoring agent was added thereto, thus preparing yogurt.
[105] Although the preferred embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
[106]
[107]