KR20170043036A - Pediococcus acidilactici lrcc5307 strain having ability of choloesterol assimilation and method for manufacturing cultured butter using the same - Google Patents

Abstract

The present invention relates to a Pediococcus acidilactici LRCC5307(Accession No. KCCM11734P) strain, which is a novel lactobacillus isolated from kimchi, a production method of a cholesterol-lowering fermented butter using the same, and a cholesterol-lowering fermented butter produced by using the same.

Description

TECHNICAL FIELD The present invention relates to a method for producing cholesterol-lowering cholesterol-lowering cholesterol-lowering cholesterol-lowering cholesterol-lowering cholesterol-lowering cholesterol-lowering cholesterol lowering cholesterol lowering cholesterol lowering cholesterol lowering cholesterol lowering cholesterol lowering cholesterol lowering cholesterol lower cholesterol cholesterol lowering cholesterol

The present invention relates to Pediococcus sydyacticys LRCC5307 (Accession No. KCCM11734P) strain, a novel lactic acid bacterium isolated from kimchi, a fermentation butter using the same, and fermented butter.

Recently, as the consumption of milk, dairy products and other animal foods has increased due to the increase of western type food culture, the cholesterol content in animal foods is closely related to the disease of circulatory diseases, and thus the problem of cholesterol content is constantly emerging.

The cholesterol content of animal foods is 219mg / 100g in butter, 137mg / 100g in cream containing 36% butterfat, 105mg / 100g in cheddar cheese, and cream cheese in cheddar cheese 95mg / 100g in blue cheese, 122.9mg / 100g in fetta cheese, 112mg / 100g in lard and 1,050mg / 100g in egg yolk .

Research on the elimination of cholesterol by physical, chemical and biological methods has been actively conducted in foods containing dairy products, since it is inevitable to reduce the intake of β-cholesterol-containing foods as a means of preventing diseases caused by excessive cholesterol intake .

One of the most effective methods for removing cholesterol from dairy products is a method using an adsorbent, beta-cyclodextrin. Korean Patent Publication No. 2007-0087708 discloses a method using beta-cyclodextrin as an adsorbent. However, such an adsorbent is in contact with and binds to cholesterol to form a complex having insolubility, and cholesterol is separated and removed by precipitation in the same manner as centrifugation. However, since this beta-cyclodextrin has the advantage of removing more than 90% of cholesterol from various foods, it is only used once, which means that it is necessary to recover the waste after use together with the decrease in economic efficiency, There is a problem causing environmental pollution.

Another method of removing cholesterol in dairy products is to use cholesterol oxidase (Cholesterol Oxidase). This is an extracellular enzyme secreted by microorganisms, and can be used as a crude enzyme solution by centrifuging without destroying the cells, thereby reducing the cost in commercial use and obtaining a high yield.

However, as described above, since the cholesterol concentration in the dairy product is about 1,000 mg / kg or more and the butter is 2,000 mg / kg or more in most cases, it is very inefficient in terms of economic cost to use only cholesterol oxidizing enzyme.

Recently, interest in lactic acid bacteria has increased, and studies have been increasingly carried out to separate and apply lactic acid bacteria having the ability to lower blood cholesterol when ingested. These lactic acid bacteria are known to reduce cholesterol, which is a precursor of bile acid, by decreasing the concentration of bile acid in the body, or to act on specific genes such as liver cells to inhibit cholesterol synthesis. On the other hand, there are many studies on lactic acid bacteria that directly assimilate cholesterol differently from these mechanisms. However, there are not many studies to remove cholesterol by applying these lactic acid bacteria to dairy products.

Therefore, there is a need to study a method for lowering cholesterol content and a method for lowering cholesterol, such as butter, which has excellent ability to lower cholesterol in products having a high cholesterol concentration.

Park, Jung - Soo et al. 2008. J. Korean. Soc. Food. 37: 1507-1514

Korea Patent Publication No. 2007-0087708

Accordingly, the object of the present invention is to provide a strain Pseudococcus sydacticis LRCC5307 (Accession No. KCCM11734P) having ability to assimilate cholesterol separated from kimchi.

The present invention also relates to the use of Pediococcus acid fermented butter comprising the step of adding a seed culture to a butter by cultivating a seed culture obtained by cultivating an acidilactici LRCC5307 (KCCM11734P).

It is also an object of the present invention to provide cholesterol-reduced fermented butter produced by the above method.

The object of the present invention is not limited to the above-mentioned object. The objects of the present invention will become more apparent from the following description, which will be realized by means of the appended claims and their combinations.

In order to achieve the above object, the present invention includes the following configuration.

The present invention provides a strain of sydactaci LRCC5307 (Accession No. KCCM11734P) to Pediococcus, a lactic acid bacterium having excellent cholesterol-lowering ability.

The novel Pediococcus strain Sicilactii LRCC5307 (Accession No. KCCM11734P) according to the present invention can be isolated from kimchi. From the Kimchi, the strain was identified and belonged to Pediococcus acidilactici , a kind of lactic acid bacteria.

The present invention also relates to the use of Pediococcus acid fermentation butter comprising the step of adding to the butter a culture medium of the seed culture in which an acidilactici LRCC5307 (KCCM11734P) has been cultured.

The method may further include adding 0.2 to 0.3 wt% of bile acid to the total weight of the butter. In addition, the bile acid may be at least one selected from the group consisting of bile extract, bile extract, oxgall powder, bile acid, and bile salt. The seed culture broth may include a cell lysate of the strain, a culture solution of the strain, a concentrated solution of the culture solution, and a dried product of the culture solution.

The present invention also provides cholesterol-lowering fermented butter produced by the above method.

The strain Pediococcus epidermalococci LRCC5307 (Accession No. KCCM11734P) according to the present invention is expected to be useful for cholesterol removal by applying it to various products such as cheese, cream, and butter, which are high cholesterol dairy products, because it has excellent cholesterol lowering ability.

Fig. 1 shows the result of measurement of the sugar availability of cedaractic acid LRCC5307 in Pediococcus.
Fig. 2 shows the result of 16s rDNA genetic identification of Sedilactii LRCC5307 in Pediococcus.
FIG. 3 shows the number of live bacteria, pH, and cholesterol measurement before and after cultivation of Pediococcus inoculated with Cedaractii LRCC5307 on butter.

Hereinafter, the present invention will be described in detail by way of examples. The embodiments of the present invention can be modified into various forms as long as the gist of the invention is not changed. However, the scope of the present invention is not limited to the following embodiments.

In the following description, well-known functions or constructions are not described in detail since they would obscure the invention. As used herein, " comprising "means that other elements may be included unless otherwise specified.

Example  One: Pedio Caucus Sydy Laktishi LRCC5307 Selection of

(1) Sampling and lactic acid bacteria isolation

As shown in Table 1 below, about 200 kinds of kimchi samples were collected from traditional traditional markets all over the country.

Separation area Seoul Gyeonggi-do Chungcheongnam-do Chung-cheong bukdo Jeollanam-do Sample (Kimchi) 42 56 25 26 53

Sterile distilled water was added to the sample to make a dilution, and the inside of the sample was homogenized with a homogenizer (Stomacher, Pro-Media SH-001, ELMEX).

The diluted solution was diluted stepwise with sterile saline, and 0.1 ml was taken. The cells were plated on MRS solid medium supplemented with 0.002% by weight of BCP (Bromocresol purple) and 1.5% by weight of agar, and cultured in an incubator maintained at 37 캜 for 48 hours.

After cultivation, colonies showing yellow rings were selected and cultured subculture 2-3 times in the same medium for each individual to isolate each strain pure.

The strain was inoculated into the MRS liquid medium by individual and cultivated at 37 ° C for 48 hours, and then stored at 4 ° C in a refrigerator.

(2) Cholesterol Degradability  Selective lactic acid bacteria screening

To select lactic acid bacteria having excellent cholesterol-lowering ability among the strains selected above. The strains isolated in the above examples were inoculated into MRS solid medium and activated at 30 ° C. for 24 hours. The activated colonies were collected and sterilized in 15 ml sterilized with 10 ml of MRS broth (Difco, USA) The tubes were inoculated and seeded with the same conditions. After the seed culture was completed, the culture broth was removed and centrifuged (8,000 rpm, 10 minutes) to recover only the cells. The supernatant was removed and the cells were suspended in 0.1 M phosphate buffer (pH 6.8). The same step was repeated three times. After removing the final supernatant, 0.1 M phosphate buffer corresponding to 5% by volume of the initial seed culture was added and the cells were suspended to complete the seed culture.

In addition, a culture medium capable of using only cholesterol as a carbon source was prepared, and strains having cholesterol decomposition ability were selected. For this purpose, the composition of the MRS liquid medium, which is known to be one of the best media for lactic acid bacteria growth, was partially changed and cholesterol 0.2 g / L, proteose peptone 10 g / L, Ammonium citrate 2 g / L, sodium acetate 5 g / _A medium containing 0.1 g / L of 4H ₂ O, 0.05 g / L of MnSO ₄ , ₂ g / L of K ₂ HPO ₄ and 15 g / L of agar was prepared and cultured in a sterilized 8 mm sized paper disk (Paperdisc, Advantec , Japan) were added and 10 μl of each culture broth of purely isolated strains was added and cultured at 37 ° C for 48 hours. After the completion of the culture, it was judged whether or not the cells were grown around the paper disk, and strains using cholesterol as a nutrient were firstly selected.

(3) Cholesterol Degradability  Final Selection of Excellent Lactic Acid Bacteria

And to select the lactic acid bacteria having the best cholesterol-lowering ability among the strains selected above. For this, a liquid medium except for agar was prepared in the cholesterol lowering ability selection medium described above, and the primary selected lactic acid bacterium was cultured in the same manner as described above. Then, 1.0% by volume of the culture medium was added, Time culture.

After the incubation, add 10 ml of isopropyl alcohol for 5 minutes, centrifuge (4,000 rpm, 5 minutes), collect 4 ml of supernatant, add 100 μl of 4 M potassium hydroxide (KOH) solution and calcium chloride NaCl) and 4 ml of distilled water. After centrifugation (4,000 rpm, 5 minutes), the supernatant was collected and quantitatively analyzed using a GC-MS / MS system (Gas Chromatography-Tandem Mass Spectrometer System). After the quantitative analysis, the strain with the highest amount of cholesterol removed was selected in consideration of the concentration of cholesterol before culturing, and the cholesterol lowering ability of the present invention was finally selected.

Example  2: Characterization and Identification of Strain

(1) Characteristic analysis

The morphological and biochemical characteristics of the selected strains were analyzed. Referring to Table 2 below, the strain was identified as Gram-positive, spore-forming ability and catalase-negative as a bacillus.

Characteristic Results analysis Colony shape Small round shape Colony color Light yellow Cell shape Coccus Gram staining Gram positive Expiratory condition Flowable anaerobic Endospore voice Spore shape voice Catalase test voice

(2) Identification of strain

The sugar availability of the strain was analyzed using API 50 CHL kit (Biomerieux France). Referring to Table 3 and FIG. 1, 99.9% of Pediococcus acidilactici was found to be identical to Pediococcus acidilactici , but the availability for Rhamnose and Salicin was 75% Respectively.

Strip 0-19
Tube / substrate +/- Strips 20-39
Tube / substrate +/- Strip 40-49
Tube / substrate +/- 0 control group - 20 [alpha] -methyl-D-mannoside
(Mannoside) - 40 D-Turanos
(TURANose) - 1 glycerol - 21 [alpha] -methyl-D-glucoside - 41 D-lyros
(LYXose) - 2 Erythritol
(ERYthritol) - 22 N-acetyl-glucosamine + 42 D-tagatose
(TAGatose) + 3 D-arabinose
(Arabinose) - 23 Amigalline
(AMYgdalin) - 43 D-Fucos
(FUCose) - 4 L-arabinose + 24 Arbutin (ARButin) - 44 L-Fuchos - 5 Ribose + 25 Esculin + 45 D-arabitol
(ArabitoL) - 6 D-xylose
(XYLose) + 26 Salicin (Salicin) - 46 L-Alvitol - 7 L-xylose - 27 Cellobios
(CELobiose) + 47 Gulou Konate
(GlucoNaTe) - 8 Adonitor
(ADOnitol) - 28 MALtose - 48 2-geton-gluconate - 9? -Methyl-D-xylose - 29 Lactose (LACtose) + 49 5-Geto-gluconate - 10 galactose
(GALactose) + 30 Melibiose
(MELibiose) - 11 glucose
(GLUcose) + 31 Sus cross
(Sucrose) - 12 fructose
(FRUctose) + 32 Trehalose
(TREhalose) + 13 MANOS + 33 INULINE - 14 Srobos
(SroBose) - 34 Meligitos
(MeLeZitose) - 15 Lambos
(RHAmnose) - 35 Raffinos
(RAFfinose) - 16 Dulcitol - 36 starches - 17 Inositol
(INOsitol) - 37 glycogen
(GLYcoGen) - 18 MANITOL - 38 xylitol
(XyLiTol) - 19 Sorbitol - 39 Zentio Bios
(GENtiobiose) +

The 16s rRNA gene sequence of the strain is shown in SEQ ID NO: 1. As a result of the nucleotide sequence analysis, the strain has a partial sequence of up to 99% with pseudokocus with the sydactactyss genus. This can be seen in FIG.

Therefore, the present inventors identified the strain as a new Pediococcus Sedilactii strain and named it Pediococcus as Sicilactii LRCC5307 and deposited it on July 22, 2015 (Deposit No. KCCM11734P) at the Korean Microorganism Conservation Center. .

Example  3: Pedio Caucus Sydy Laktishi LRCC5307 Of cholesterol Degradability

To investigate the cholesterol lowering ability of ciprofloxacin LRCC5307 in a novel lactic acid bacterium Pediococcus. The Lactobacillus acidophilus ATCC43121 strain, which has been most studied for the cholesterol-lowering effect, was used as a control group for comparing the cholesterol-lowering ability of the LRCC5307 strain with the American National Biological Resources Center (ATCC, USA) The experiment was carried out.

(1) Cholesterol in the medium Degradability

In order to confirm the cholesterol lowering ability of the LRCC5307 strain and the ATCC43121 strain of the present invention, they were inoculated into MRS solid medium and incubated at 37 ° C for 24 hours for activation, and then inoculated into the MRS liquid medium, To prepare a culture solution. The seed culture was inoculated into a liquid medium containing cholesterol and cultured at 37 ° C for 48 hours. After completion of the culture, the culture solution was recovered, and cholesterol was quantitatively analyzed in the same manner as in Example 1, and the results are shown in Table 4. As shown in the results, in the ATCC 43121 strain, about 21% by weight of the cholesterol before cultivation was reduced and decreased, while LRCC5307 showed a decrease of about 27% by weight of cholesterol before the incubation.

division Cholesterol (mg / L) LRCC5307 ATCC43121 Before culture 198.3 196.6 After incubation 143.9 154.9 Decrease (%) -27.4 -21.2

(2) Bile acid  Cholesterol by type of addition Degradability

Recently, studies on lactic acid bacteria for lowering cholesterol have been carried out to increase cholesterol lowering ability by adding various kinds of bile (bile). Therefore, when the LRCC5307 strain of the present invention is cultured, a certain amount of bile salt is added to test the ability to lower cholesterol.

The cholesterol-added medium was prepared in the same manner as in Example 1, and then added with various kinds of bile acids. Sigma, USA), bile acid (bile salt, Sigma, USA), bile acid (Sigma, USA) and bile acid (Sigma, USA) 0.1% by weight, respectively, and the pH was adjusted to 6.8 ± 0.2 using 1N sodium hydroxide (NaOH) solution.

LRCC5307 strain and ATCC43121 strain were cultured in the same manner as in the above example. 1% by volume of seed culture was added to the prepared bile acid-supplemented medium, followed by culturing at 37 ° C for 24 hours. Cholesterol was quantitatively analyzed according to the method described in Example 1, and the results are shown in Table 5. As shown in the results, the addition of various kinds of bile acids significantly increased cholesterol lowering ability compared to the non-added cholesterol group. Especially, the highest cholesterol lowering ability was confirmed by addition of bile salt.

Type of bile acid Cholesterol (mg / L) LRCC5307 ATCC43121 Control group (no inoculation) 201.4 199.5 Blank 146.2 155.2 Even Extract 94.6 110.0 Oxgall 105.3 113.6 Even Acid 108.1 114.3 Even Salt 64.2 83.5

(3) Bile salt  Cholesterol by concentration addition Degradability

In order to establish the most optimal concentration of bile acid when the bile salt (bile salt) identified in the above examples was added by concentration. The cholesterol-supplemented medium was prepared in the same manner as in the above examples, and bile salts were added at 0, 0.1, 0.2, and 0.3 wt%, followed by inoculation with 1.0 vol.% Of LRCC5307 and ATCC43121. The results of measurement of cholesterol concentration are shown in [Table 6]. As shown in the results, the LRCC5307 strain showed the highest cholesterol lowering ability when 0.2 wt% of bile salt was added, but it was confirmed that there was no significant difference with the addition of 0.3 wt%. On the other hand, in case of ATCC43121 strain, the addition of 0.3% by weight showed a higher ability to degrade compared to other concentrations, but it was not found to increase with the increase of bile salt. Therefore, the optimum concentration of the bile salt added to the LRCC5307 strain of the present invention was determined to be 0.2% by weight.

Concentration of bile salt (%) Cholesterol (mg / L) LRCC5307 ATCC43121 Control group (no inoculation) 199.2 202.7 Blank 141.3 149.2 0.1 68.5 85.8 0.2 51.7 62.1 0.3 54.4 58.5

Example  4: Pedio Caucus Sydy Laktishi LRCC5307 Fermented butter production

In order to remove cholesterol by applying ciprofloxacin LRCC5307 to butter to a novel lactic acid bacterium Pediococcus of the present invention. In the case of the LRCC5307 strain, the seed culture broth was carried out in the same manner as in the above example, and washed in phosphate buffer to complete the broth culture.

In the case of butter, 500 g of sam- ple-free unsalted butter was used. The butter was dissolved in a constant-temperature water bath maintained at 90 ° C and stored in an incubator maintained at 40 ° C. Cooled to the possible temperature. The butter was cooled to 40 캜 or lower, and 0.2% by weight of bile acid was added thereto. The mixture was stirred and 1.0% by volume of the culture medium of the LRCC5307 strain was inoculated and cultured at 37 캜 for 48 hours.

The number of live bacteria, pH and cholesterol before and after fermentation after 48 hours of culture were measured and shown in FIG. The pH was decreased from about 6.61 before culture to 5.43 after culturing and the number of viable cells increased from 7.53 log CFU / g to 8.74 log CFU / g. The pH and the number of viable cells were found to be lower than 1 log CFU / g in the case of the viable cell count and pH compared with the culture using the general MRS liquid culture medium, and this was compared with the culture medium optimized for the growth of lactic acid bacteria It is estimated to be due to insufficient nutrients and environment in the butter (such as excessively high fat content). In the case of cholesterol, it decreased to about 1,870 mg / L after culturing at about 2,100 mg / L before culturing, and it was found to be decreased by about 230 mg / L. In general butter, it is known that cholesterol is present from 1,800 mg / L to 2,300 mg / L.

Therefore, the LRCC5307 lactic acid bacteria of the present invention can assimilate the cholesterol in the butter to remove a certain portion thereof, thereby providing a fermented butter having reduced cholesterol.

The present invention has been described in detail. However, the scope of rights of the present invention is not limited thereto, but is defined by the following claims.

Korea Microorganism Conservation Center (overseas) KCCM11734P 20150722

<110> LOTTE FOODS CO., LTD. <120> Pediococcus acidilactici LRCC5307 strain having ability of          cholesterol assimilation and method for manufacturing cultured          butter using the same <130> DP-2015-0389 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1440 <212> DNA <213> Pediococcus acidilactici LRCC5307 <400> 1 ggctagctcc taaaaggtta ccccaccggc tttgggtatt acaaactctc atggtgtgac 60 gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 120 gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggttttaa 180 gagattagct aaacctcgcg gtttcgcaac tcgttgtacc atccattgta gcacgtgtgt 240 agcccaggtc ataaggggca tgatgatttg acgtcgtccc caccttcctc cggtttgtca 300 ccggcagtct cactagagtg cccaactgaa tgctggcaac tagtaataag ggttgcgctc 360 gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420 cattctgtcc ccgaagggaa cgcctaatct cttaggttgg cagaagatgt caagacctgg 480 taaggttctt cgcgtagctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540 tcaattcttt tgagtttcaa ccttgcggtc gtactcccca ggcggattac ttaatgcgtt 600 agctgcagca ctgaagggcg gaaaccctcc aacacttagt aatcatcgtt tacggcatgg 660 actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720 gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780 acatggagtt ccactgtcct cttctgcact caagtctccc agtttccaat gcacttcttc 840 ggttgagccg aaggctttca cattagactt aaaagaccgc ctgcgctcgc tttacgccca 900 ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960 gtggctttct ggttaaatac cgtcactggg tgaacagtta ctctcaccca cgttcttctt 1020 taacaacaga gctttacgag ccgaaaccct tcttcactca cgcggcgttg ctccatcaga 1080 cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtctg ggccgtgtct 1140 cagtcccaat gtggccgatt accctctcag gtcggctacg catcatcgcc ttggtgagcc 1200 gttacctcac caactagcta atgcgccgcg ggtccatcca gaagtgatag cagagccatc 1260 ttttaaaaga aaaccaggcg gttttctctg ttatacggta ttagcatctg tttccaggtg 1320 ttatcccctg cttctgggca ggttacccac gtgttactca cccgtccgcc actcacttcg 1380 tgttaaaatc tcattcagtg caagcacttc ctgatcaatt aacggaagtt cgttcgactg 1440                                                                         1440

Claims (5)

Pediococcus derived from kimchi with excellent cholesterol-lowering ability ( Pediococcus acidilactici ) LRCC5307 (KCCM11734P).
Pediococcus acidilactici ) LRCC5307 (KCCM11734P).
Pediococcus acidic lactic acid LRCC5307 (KCCM11734P) cells to a butter.
4. The method of claim 3, further comprising adding 0.2 to 0.3% by weight of bile acid to the total weight of the butter.
A cholesterol reduced fermented butter produced by the method of claim 3 or 4.

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