KR101250463B1 - Oxygen tolerant Bifidobacterium longum from Korean neonate fecal samples and probiotic compositions produced by the same - Google Patents

Abstract

The present invention relates to a bifidus lactic acid bacteria that can be cultured without an anaerobic device, a probiotic composition using the same. Oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium) selected from neonatal feces of the present invention longum 6-T-4-55 ) can be cultured in liquid medium without anaerobic device, so it is easy to use industrially. In particular, it can grow more than 10 ⁹ CFU / mL when incubated in milk, and it can form curd. have. It is possible to produce a probiotic composition using the advantages of the strain of the present invention.

Description

Oxygen tolerant Bifidobacterium longum from Korean neonate fecal samples and probiotic compositions produced by the same}

The present invention relates to a bifidus lactic acid bacteria that can be cultured without an anaerobic device, a probiotic composition using the same.

Bifidus lactobacillus was first isolated from feces of breastfeeding infants, and it was recognized that the change in the number of bifidus lactobacillus is closely related to stress, allergy and cancer in infants and adults. It is becoming. In order to use the health contributing effect of these bifidus lactic acid bacteria, it is widely used in fermented milk, baby food, health food, formal dressings. By the way, bifidus lactic acid bacteria are a limited anaerobic bacterium that requires anaerobic conditions for cultivation, so there is a limit to industrial use.

In particular, the problem of bifidus lactic acid bacteria in fermented milk has been continuously raised by the media. This is because bifidus lactic acid bacteria do not grow well in raw material oil, and after fermented milk production, anaerobic cultured bifidus bacteria are added and distributed.In this distribution process, bifidus lactic acid bacteria are continuously killed because they cannot satisfy the anaerobic conditions, which are the growth conditions of bifidus. Therefore, the above problems arise.

Increasingly, there are increasing applications of various bioactivity of Bifidobacteria in the food industry. It has also been pointed out that it is not shown.

As a beneficial health promoting action of Bifidobacteria, various physiological activities such as resistance to infection, anticancer and antitumor effects, lowering blood cholesterol content and intestinal effects have been reported. Therefore, it is important to develop excellent strains of lactic acid bacteria such as bifidus bacteria as probiotics suitable for Koreans.

Therefore, when developing bifidus lactic acid bacteria that do not require anaerobic conditions and can grow in milk, related domestic probiotic fields such as the market of fermented milk products worth 1.3 trillion won, cheese market worth 500 billion won, and feed additive market worth 50 billion won The ripple effect is expected to be very large.

Under these technical backgrounds, the present inventors have made efforts to develop bifidus lactic acid bacteria which do not require anaerobic conditions and have excellent growth in milk.

After all, an object of the present invention is to provide a bifidus lactic acid bacteria excellent growth in more than 10 ⁹ CFU per mL when incubated in a liquid medium can be cultured in a liquid medium without an anaerobic device.

According to one aspect of the present invention, Bifidobacterium longum 6-T-4-55 ( Bifidobacterium longum 6-T-4-55) bifidus lactic acid bacteria of Accession No. KACC91563P having oxygen resistance may be provided.

According to another aspect of the present invention, a probiotic composition comprising the bifidus lactic acid bacteria or a culture solution thereof as an active ingredient may be provided.

According to another aspect of the present invention, a dairy product comprising the bifidus lactic acid bacteria or a culture solution thereof as an active ingredient may be provided.

According to one embodiment, a probiotic composition may be provided, which is used as an additive for feed or food.

According to one embodiment, a probiotic composition may be provided which is prepared in the form of a probiotic by heat drying or freeze-drying.

According to one embodiment, the probiotic composition may be a pharmaceutical composition further comprising a pharmaceutical carrier and an excipient.

According to one embodiment, the Bifidobacterium longum 6-T-4-55 Bifidobacterium longum 6-T-4-55 Bifidobacterium lactic acid bacteria of Accession No. KACC91563P, characterized in that transformed with a foreign gene having a useful function is Can be provided.

Oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium) selected from neonatal feces of the present invention longum 6-T-4-55 ) can be cultured in liquid medium without anaerobic device, so it is easy to use industrially. In particular, it can grow more than 10 ⁹ CFU / mL when incubated in milk, and it can form curd. have. It is possible to produce a probiotic composition using the advantages of the strain of the present invention.

Figure 1 shows the growth of the selected bifidus lactic acid bacteria cultured in MRS liquid medium (cysteine 0.05%) under aerobic conditions. The left side shows the addition of X-α-gal, and the right side shows the formation of blue color on the upper layer by decomposition of X-α-gal.
Figure 2 is oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium longum 6-T-4-55) shows curd formation pattern in skim milk culture.
Figure 3 is oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium longum 6-T-4-55) shows the anaerobic culture (left) and aerobic culture (right) during X-α-gal-containing MRS agar culture of curd formed by inoculating skim milk medium.
Figure 4 is oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium longum 6-T-4-55) was inoculated in 10% skim milk medium and cultured at 40 ° C.
5 is oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium longum 6-T-4-55) was inoculated in 10% skim milk medium and cultured at 37 ° C. for 24 hours and then stored at 4 ° C. for 2 weeks to show bacterial counts and pH changes.

Hereinafter, the present invention will be described in more detail.

In order to develop such a strain, the present inventors collected fecal samples of 25 newborns (male 16, female 9) within 5 days of birth recruited from all over the country (Gyeonggi, Gangwon, Chungcheong, Gyeongsang, Jeolla) by aerobic conditions The living bifidus lactic acid bacteria strains were selected by culturing. As a result, it has led to the development of a bifidus lactic acid bacteria strain which exhibits a higher number of bacteria than those contained in commercial yogurt and can form a curd alone, and shows excellent characteristics applicable to dairy products such as cheese.

According to one aspect of the present invention, Bifidobacterium longum 6-T-4-55 ( Bifidobacterium longum 6-T-4-55) bifidus lactic acid bacteria of Accession No. KACC91563P having oxygen resistance may be provided. In this case, the oxygen resistance means a characteristic that can be grown without an anaerobic device even in the presence of oxygen. The accession number was registered and issued on June 9, 2010 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and the name of the bifidus lactic acid bacteria of the present invention is named Bifidobacterium longum 6-T-4-55.

According to another aspect of the present invention, a probiotic composition comprising the bifidus lactic acid bacteria or a culture solution thereof as an active ingredient may be provided. At this time, the probiotic composition collectively refers to a microbial use product that can help improve the activity of the living body and promote health, and includes a family of products usually included as a thickener or a probiotic.

According to another aspect of the present invention, a dairy product comprising the bifidus lactic acid bacteria or a culture solution thereof as an active ingredient may be provided. The dairy product includes all dairy products that can be produced by processing milk such as milk fermented yogurt, cheese, ice cream and sauce. At this time, the addition ratio of the bifidus lactic acid bacteria is not limited, but in the case of dairy products, it is sufficient that the appropriate pH is shown within an appropriate fermentation time so as not to impair the texture. More specifically, bifidus lactic acid bacteria ( Bifidobacterium longum) according to the present invention 6-T-4-55) 10 ⁴ to 10 ⁶ When incubated in skim milk medium or milk at CFU / ml and cultured for 24 hours at 37 to 40 ° C, a curd was formed and the number of bacteria was about 2.8 × 10 ⁸ to 1.1 × 10 ⁹ CFU / mL, and the pH was about 4.74 to 4.79. Was observed. This corresponds to a very good bacterial count and proper acidity (pH) compared to commercial yogurt.

According to one embodiment of the present invention, a probiotic composition may be provided, which is used as an additive for feed or food. There is no restriction on the type of livestock when used as feed. Bifidus lactic acid bacteria according to the present invention can be grown without an anaerobic device, there is an advantage that can continue to work without losing the activity of live bacteria even when added to the feed. Taking advantage of this, it is also possible to transform the gene with a variety of genes to have additional features.

Thus, according to another aspect of the present invention, Bifidobacterium longum 6-T-4-55 Bifidobacterium longum 6-T-4-55 Bifidobacterium lactic acid bacteria of Accession No. KACC91563P transformed with a foreign gene having a useful function This may be provided.

According to one embodiment, a probiotic composition may be provided which is prepared in the form of a probiotic by heat drying or freeze-drying.

According to one embodiment, the probiotic composition may be a pharmaceutical composition further comprising a pharmaceutical carrier and an excipient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  One. Anaerobic Device  without Cultivable Oxygen resistance  Bifidus lactic acid bacteria ( Bifidobacterium longum Identification of 6-T-4-55)

Collect fecal samples from 25 newborn babies (16 males and 9 females) within 5 days of birth, recruited from all over the country (Gyeonggi, Gangwon, Chungcheong, Gyeongsang, Jeolla) and collected them in transgalactosylated oligosaccharide (TOS) agar medium, a selection medium of bifidus lactic acid bacteria. Culture was anaerobic. 1,152 strains growing in the medium is separated by colonies. 186 strains were randomly extracted from these isolated strains and analyzed for 16S rRNA.

The extracted 186 strains were again cultured in MRS liquid medium (containing 0.05% cysteine) to select surviving strains. In the culture medium of the selected strain, X-α-gal was added to confirm that the upper layer was discolored in blue to confirm the characteristics of the bifidus lactic acid strain. This is shown in FIG. 1. The base sequence of the 16S rRNA of the selected strain is shown in SEQ ID NO: 1. This sequence was searched by GenBank database and identified as Bifidobacterium longum . The strain was deposited with the National Institute of Agricultural Science, Agricultural Genetic Resource Center (Accession Number KACC91563P, Microorganism Name: Bifidobacterium longum 6-T-4-55. In this case, the accession number is listed on the certificate as KACC91563P. (Except the last letter P of the accession number)

Example  2. Curd ( Curd A) formation confirmation

100 μl of the bifidus culture medium selected according to Example 1 was inoculated in sterile 10% skim milk broth to incubate overnight. As a result, it was confirmed that the curd (Curd) is formed (Fig. 2), curd (Curd) is a coagulant produced when the acid or renin or pepsin in milk, is used to make cheese. At this time, 1 ml of the curd formed was collected and diluted, 100 μl of the curd was smeared onto MRS agar (containing 0.05% cysteine and 50 mg / L of X-α-gal) and anaerobicly cultured, followed by exhalation of colony-formed petri dishes. It was further incubated in the state. Petri cultured in anaerobic conditions as shown in Figure 3 When the colonies of Petri dishes (which show discoloration activity against X-α-gal) were fertilized with dish colonies and aerobic conditions, it was found that the colonies of two Petri dishes matched and no growth of other strains was observed. Can be. That is, Bifidobacterium ( Bifidobacterium) isolated according to Example 1 without the influence of other various bacteria longum 6-T-4-55) shows that the curd was formed.

Test Example  One

Oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium) in 10% skim milk medium longum 6-T-4-55) was inoculated with 6.0 × 10 ⁴ CFU / mL and grown at 2.8 × 10 ⁸ CFU / mL when incubated at 40 ° C. for 24 hours, but decreased to 3.2 × 10 ⁷ CFU / mL after 48 hours of incubation. At 24 h of incubation the pH was 4.79 and curd was formed and at 48 h of incubation the pH reached 4.59.

Test Example  2

Oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium) in 10% skim milk medium longum 6-T-4-55) was inoculated with 2.1 × 10 ⁶ CFU / mL and grown at 1.1 × 0 ⁹ CFU / mL when incubated at 37 ° C. for 24 hours. Curds formed and the pH was 4.74. It was 1.3 × 10 ⁹ CFU / mL after 24 hours of cold storage at 4 ° C. for 24 hours, but it decreased to 4.3 × 10 ⁷ CFU / mL after 1 week of storage and to 1.5 × 10 ⁶ CFU / mL after 2 weeks. The pH was 4.89 even after 2 weeks storage at 4.74. Figure 5 shows the bacterial counts and pH change when stored for 2 weeks under the above conditions.

Test Example  3

Oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium ) with commercial yogurt starter (Christian Hansen YF-3331) 2.0 × 10 ⁷ CFU / mL in 10% skim milk medium longum 6-T-4-55) was inoculated with 1.8 × 10 ⁷ CFU / mL and incubated at 37 ° C. for 24 hours. As a result, the total number of lactic acid bacteria was 1.2 × 10 ⁹ CFU / mL, and the number of bifidus lactic acid bacteria was 2.2 × 10 ⁸ CFU / mL, and the number of bifidus bacteria was 5.4 × 10 ⁸ CFU / mL even after 2 weeks refrigerated storage at 4 ℃. It was. Table 2 below shows the pH at commercial yogurt preparation and storage.

division inoculation 1 day culture
(37 ℃) 1 week storage
(4 ℃) 2 weeks storage
(4 ℃) YF-3331 6.18 3.66 3.70 3.67 YF-3331 + B. longum 6-T-4-55 6.15 3.93 3.94 3.97

As can be seen in Table 1, the bifidus lactic acid bacteria ( Bifidobacterium) of the present invention longum When 6-T-4-55) was added in combination with a commercial yogurt starter, there was no significant difference in pH change.

Table 2 shows the total lactic acid bacteria count and the bifidus bacteria count for commercial yogurt preparation and storage.

division Number of bacteria inoculation
CFU / mL 1 day culture
(37 ℃) 1 week storage
(4 ℃) 2 weeks storage
(4 ℃) YF-3331 Total lactic acid bacteria 2.0 × 10 ⁷ 8.1 × 10 ⁸ 6.8 × 10 ⁸ 6.4 × 10 ⁸ Bifidus - - - - YF-3331 +
B. longum 6-T-4-55 Total lactic acid bacteria 2.0 × 10 ⁷ 1.2 × 10 ⁹ 1.2 × 10 ⁹ 1.2 × 10 ⁹ Bifidus 1.8 × 10 ⁷ 2.2 × 10 ⁸ 1.8 × 10 ⁸ 5.4 × 10 ⁸

Comparative example . Comparison of Bifidus Lactic Acid Bacteria in Dairy Products

In order to compare the effectiveness of the bifidus lactic acid bacteria of the present invention, 10 kinds of commercial yogurt from major dairy companies on the market were collected and the total number of lactic acid bacteria and bifidus lactic acid bacteria were measured. After diluting the yoghurt, 100 μl was anaerobicly cultured by spreading it in MRS agar (containing X-α-gal), and then described as total lactic acid bacteria, and the anaerobic removal of the colonies was indicated as bifidus lactic acid bacteria. The results are shown in Table 1.

A B C D E F G H I J Total Lactobacillus Count 1.2 × 10 ⁹ 1.4 x 10 ⁸ 1.2 × 10 ⁹ 1.1 × 10 ⁹ 6.8 × 10 ⁷ 5.0 × 10 ⁸ 1.4 × 10 ⁹ 5.3 × 10 ⁸ 8.4 × 10 ⁷ 4.5 × 10 ⁸ Bifidus lactic acid bacteria <10 ⁴ <10 ⁴ <10 ⁴ 1.0 x 10 ⁴ 6.0 × 10 ⁴ 2.5 × 10 ⁶ <10 ⁴ <10 ⁴ <10 ⁴ <10 ⁴

* Unit: CFU / mL

As shown in Table 3, the total lactic acid bacteria count of 10 commercial yogurt was 6.8 × 10 ⁷ ~ 1.4 × 10 ⁹ CFU / mL, but the number of bifidus lactic acid bacteria was 10 ⁴ CFU / mL or less and the highest product was 2.5. 10 ⁶ CFU / mL.

As can be seen in the above test examples and comparative examples, according to the present invention, oxygen-resistant bifidus lactic acid bacteria ( Bifidobacterium) selected from the feces of newborns longum 6-T-4-55) can grow more than 10 ⁹ CFU / mL when cultivated in milk, showing the growth rate exceeding the number of lactic acid bacteria of commercial yogurt on the market and forming curd alone without affecting other bacteria. In addition, the pH formed during manufacture and storage also appears to be a suitable level for drinking, it can be seen that the production of dairy products such as fermented milk using this can be any amount. In addition, since it can be cultured in a liquid medium without an anaerobic device, industrial utilization utilizing this advantage can be made very broadly.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

In Figure 5 ◆ indicates CFU, ■ indicates pH.

National Institute of Agricultural Sciences KACC91563P 20100609

                         SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION   <120> Oxygen tolerant Bifidobacterium longum 6-T-4-55 from Korean        neonate fecal samples and probiotic compositions produced by the        same <130> NPF18242 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 1394 <212> DNA <213> Bifidobacterium longum <400> 1 ttagacggct catcccacaa ggggttaggc caccggcttc gggtgctgcc cactttcatg 60 acttgacggg cggtgtgtac aaggcccggg aacgcattca ccgcgacgtt gctgattcgc 120 gattactagc gactccgcct tcacgcagtc gagttgcaga ctgcgatccg aactgagacc 180 ggttttcagg gatccgctcc gcgtcgccgc gtcgcatccc gttgtaccgg ccattgtagc 240 atgcgtgaag ccctggacgt aaggggcatg atgatctgac gtcatccccc accttcctcc 300 gagttaaccc cggcggtccc ccgtgagttc ccggcataat ccgctggcaa cacggggcga 360 gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga cgacgaccat 420 gcaccacctg tgaacccgcc ccgaagggaa gccgtatctc tacgaccgtc gggaacatgt 480 caagcccagg taaggttctt cgcgttgcat cgaattaatc cgcatgctcc gccgcttgtg 540 cgggcccccg tcaatttctt tgagttttag ccttgcggcc gtactcccca ggcgggatgc 600 ttaacgcgtt agctccgaca cggaacccgt ggaacgggcc ccacatccag catccaccgt 660 ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720 cgtcagtaac ggcccagaga cctgccttcg ccattggtgt tcttcccgat atctacacat 780 tccaccgtta caccgggaat tccagtctcc cctaccgcac tcaagcccgc ccgtacccgg 840 cacggatcca ccgttaagcg atggactttc acaccggacg cgacgaaccg cctacgagcc 900 ctttacgccc aataattccg gataacgctt gcaccctacg tattaccgcg gctgctggca 960 cgtagttagc cggtgcttat tcaacgggta aactcactct cgcttgctcc ccgataaaag 1020 aggtttacaa cccgaaggcc tccatccctc acgcggcgtc gctgcatcag gcttgcgccc 1080 attgtgcaat attccccact gctgcctccc gtaggagtct gggccgtatc tcagtcccaa 1140 tgtggccggt cgccctctca ggccggctac ccgtcgaagc cacggtgggc cgttaccccg 1200 ccgtcaagct gataggacgc gaccccatcc cataccgcga aagctttccc agaagaccat 1260 gcgatcaact ggagcatccg gcattaccac ccgtttccag gagctattcc ggtgtatggg 1320 gcaggtcggt cacgcattac tcacccgttc gccactctca ccaccaagca aagcctgatg 1380 gatcccgttc gact 1394

Claims (7)

Bifidobacterium longum 6-T-4-55 ( Bifidobacterium longum 6-T-4-55) bifidus lactic acid bacterium having oxygen resistance and capable of forming a curd alone.
Probiotic composition comprising the bifidus lactic acid bacteria of claim 1 or a culture thereof as an active ingredient.
Dairy products comprising the bifidus lactic acid bacteria of claim 1 or a culture thereof as an active ingredient.
The probiotic composition according to claim 2, which is used as an additive for feed or food.
The probiotic composition according to claim 2, wherein the probiotic composition is prepared in the form of a probiotic by heat drying or freeze-drying.
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