KR20170104863A

KR20170104863A - functional fermented milk with emission of cholesterol and bile acid, and method for manufacturing the same

Info

Publication number: KR20170104863A
Application number: KR1020160027833A
Authority: KR
Inventors: 손주완; 김상중; 윤석민; 양시영
Original assignee: 롯데푸드 주식회사
Priority date: 2016-03-08
Filing date: 2016-03-08
Publication date: 2017-09-18
Also published as: KR101827701B1

Abstract

The present invention relates to a functional fermented milk having a cholesterol-lowering function by containing a predetermined amount of Lactobacillus plantarum LLP5273 and a plant stanol ester which are excellent in the ability to excrete cholesterol and bile acid, and a preparation method thereof.

Description

Technical Field [0001] The present invention relates to a functional fermented milk having excellent cholesterol and bile acid excretion ability and a method for producing the fermented milk,

The present invention relates to a functional fermented milk having excellent cholesterol and bile acid excretion ability. More specifically, the present invention relates to a functional fermented milk having a cholesterol-lowering function by containing a predetermined amount of Lactobacillus plantarum LLP5273 strain and plant starol ester, And a method for producing the same.

Globally, the obesity population is rapidly increasing. Especially, the cholesterol content is increased by obesity, and the risk of heart disease, cerebrovascular disorder and hypertension is increasing. In general, drugs such as statins and niacin are widely used as therapeutic agents for lowering cholesterol.

Lovastatin, provastatin and simvastatin, statin-based compactin derivatives, are among the most commonly used drugs worldwide. These drugs inhibit the activity of HMG-CoA reductase (3-hydroxy-methyl-glutamyl coenzyme A reductase), a regulator of cholesterol biosynthesis. As a result, the amount of LDL-receptor expression is increased and the intracellular inflow of LDL is increased, thereby decreasing the cholesterol content in the blood. Despite these cholesterol-lowering functions, these drugs have side effects such as hepatotoxicity, myalgia, gastrointestinal disorders, and carcinogenicity. Therefore, rather than relying on pharmacotherapy, diuretic therapy is being performed in a direction that expects to be effective. However, dieting is not an easy method, and patients with high cholesterol levels need a different approach.

In recent years, studies on the possibility of bile acid degradation by intestinal bacteria, especially lactic acid bacteria, in the intestinal tract of mammals have been increasing in order to lower serum cholesterol levels in patients with hypercholesterolemia or to prevent hypercholesterolemia in humans with normal cholesterol levels Which increases the conversion of cholesterol to bile salts to maintain the body's homeostasis in accordance with the reduced amount of bile acid, thereby using the mechanism of lowering the blood cholesterol level in the body.

However, studies on the reduction of cholesterol in the body by using lactic acid bacteria having excellent bile acid-decomposing enzyme-releasing ability have shown that the bile acid-decomposing enzyme lactic acid bacteria are isolated from the in vitro experiment, and then fed to experimental animals, And the effect of lactic acid bacteria actually administered on biosynthetic regulators is extremely low.

Especially, it is very important to develop a lactic acid bacterium having a similar mechanism as this drug has a mechanism of inhibiting the activity of HMG-CoA reductase inhibition in order to replace statins, which is a typical cholesterol treatment drug. It is expected that verification of efficacy is very essential.

Therefore, in the previous invention, the present inventors isolated Lactobacillus plantarum LLP5273, which has high bile acid decomposing ability and excellent HMG-CoA reductase inhibiting ability, from kimchi and deposited it as a patented strain (Deposit No. KCCM11696P). In addition, we confirmed the mechanism of inhibition of HMG-CoA reductase when treated with hepatocytes, suggesting that the lactic acid bacteria may have a significant effect on the inhibition of cholesterol synthesis.

On the other hand, another method to replace drugs to lower serum cholesterol levels is to use plant stanol ester (Plant Stanol Ester). Vegetable stanol ester is produced by hydrogenation and esterification using plant sterol, which is produced in the process of producing soybean oil, and is produced by the combination of Sitostanol and Campestanol. The composition is determined according to the sum. Since vegetable stanol ester is not well absorbed in the small intestine and is characterized by similarity to cholesterol in terms of physicochemical properties, it is reported that when the vegetable stanol ester is taken together with food, the absorption rate may be lowered because the cholesterol having the same structure forms together with the crystal. . It has also been reported that it acts competingly with lipoprotein, a carrier of cholesterol in the body, thereby lowering the absorption of cholesterol through the intestines.

In fact, European Rishio Co. Ltd. (Finland) produced these vegetable stanol esters with the BeneCol brand. Since its first sale in 1995, no side effects have been reported, and many national health authorities and related organizations Are recommended to consume 2 grams of vegetable starol each day to reduce cholesterol levels.

Korean Patent Laid-Open Publication No. 2002-379116 discloses a method for producing a complex material for inhibiting the degradation and absorption of cholesterol in blood using plant sterols and bioflavonoids, and it can be used variously in supplements, food additives, beverages, etc. There is one.

However, these vegetable starol esters are difficult to be directly ingested by themselves due to their unique flavor and odor and solid solid at room temperature. Therefore, they are being applied to various types of products and sold.

Therefore, there is a need to provide a functional food which uses vegetable stanol ester but which further improves cholesterol lowering ability without sensory difficulty.

1. Korean Patent Publication No. 2002-379116

Accordingly, the inventors of the present invention have found that, when Lactobacillus plantarum LLP5273 having excellent cholesterol lowering and excretion ability and plant stanol ester are contained in a predetermined amount, they have a remarkable cholesterol lowering ability as compared with those used alone, The present inventors have found that fermented milk can be produced by fermented milk.

Thus, the present invention provides a method of treating Lactobacillus < RTI ID = 0.0 > plantarum LLP5273 strain and vegetable stanol ester. The objective of the present invention is to provide functional fermented milk having excellent cholesterol and bile acid emission ability.

It is another object of the present invention to provide a method for producing the functional fermented milk.

It is another object of the present invention to provide a dried powder of fermented milk having excellent cholesterol and bile acid-producing ability obtained by drying the functional fermented milk.

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 provides a functional fermented milk having excellent cholesterol and bile acid excretion ability, which comprises Lactobacillus plantarum LLP5273 strain and plant stanol ester.

The term "fermented milk" as used herein refers to a fermented milk containing milk and other raw materials and added with a certain amount of the above-mentioned cells, culture liquid, concentrated liquid of the culture liquid, or dried liquid of the culture liquid.

Specifically, the above-mentioned strain used in the present invention was found to be a novel strain having a high ability to degrade bile acid and capable of inhibiting HMG-CoA reductase, and was deposited at the Korean Microorganism Conservation Center on May 7, 2015 and deposited with KCCM11696P It is a given strain.

Such a strain may be contained in the form of a cell, a culture medium, a concentrate of the culture medium or a dried product of the culture medium, more preferably a lyophilized product obtained by lyophilization using a culture medium and an excipient .

In addition, the lyophilized product preferably contains 1.0 x 10 ¹⁰ to 1.0 x 10 ¹¹ CFU / g of live cells of Lactobacillus plantarum LLP5273. Live cell number of freezing to 1.0 x 10 ¹⁰ CFU / g while the weight increase must added to ensure the effective activity or growth property and the limit occurs economically large load is less than, 1.0 x 10 ¹¹ to produce beyond the CFU / g There is a step of concentrating and culturing before drying and the time is increased and the production efficiency is lowered.

In addition, the lyophilizate preferably contains 0.001 to 0.01% by weight based on the total weight of the fermented milk. When the content is less than 0.001% by weight, there is a limit in ensuring the number of bacteria necessary for producing fermented milk. When the content is more than 0.01% by weight, there is no significant difference in fermentation ability and fermentation time.

Next, the vegetable starol ester preferably comprises 2 to 5% by weight based on the total weight of the fermented milk. In the case of less than 2% by weight, there is a limit in not sufficiently supplying the concentration known to be capable of lowering cholesterol in the existing studies, and in the case of 5% by weight, the inherent odor of vegetable stanol ester and viscosity It is preferable to use it within the above-mentioned range.

The fermented milk according to the present invention may further include skim milk powder or glucose or a mixture thereof to further improve the growth stability and proliferation of the strain, more preferably a mixture thereof.

In particular, in the case of glucose, it is preferable to use 2 to 5% by weight, more preferably 2% by weight, based on the total weight of the fermented milk. If the amount of glucose is less than 2% by weight, the effect of improving the growth stability and proliferation of the strain may not be exerted. If the amount of glucose is more than 5% by weight, the growth stability and the growth of the strain may not be improved. It is good.

In the case of skimmed milk powder, 1 to 5% by weight is preferably used. If the amount of the skimmed milk powder is less than 1% by weight, the effect of improving the growth stability and proliferation of the strain is not exerted. If the skimmed milk powder is added in an amount exceeding 5% by weight, the growth stability and symptoms of the strain may not be improved. It is good to do.

The fermented milk according to the present invention may be provided in the form of a powder obtained by drying.

The present invention also relates to a method for preparing a starch-containing starch, comprising the steps of: 2 to 4% by weight of a vegetable starol ester is added to a main raw material containing milk, glucose or skimmed milk or a mixture thereof, followed by pressure sterilization; And pressurized milk were cooled, and then Lactobacillus plantarum The present invention also provides a method for producing fermented milk having excellent cholesterol and bile acid excretion ability by adding 0.001 to 0.01% by weight of a lyophilized product of Lactobacillus plantarum LLP5273.

, In the lyophilized product of the strain Lactobacillus plan tareom As mentioned above (Lactobacillus plantarum ) LLP5273 strain is preferably 1.0 x 10 ¹⁰ to 1.0 x 10 ¹¹ CFU / g.

The pressurized sterilization is preferably performed at a temperature of 120 to 130 ° C. for 15 to 30 minutes, and the pressurized sterilized milk is cooled to a temperature of 25 to 35 ° C. and then fermented by adding a lyophilized product of the strain. It can be changed depending on the process conditions.

The present invention shows a synergetic effect of cholesterol lowering when mixed with a Lactobacillus plantarum LLP5273 strain excellent in the ability to excrete cholesterol and bile acid and a vegetable stanol ester alone. The cholesterol lowering effect of cholesterol and cholesterol in the fermented milk Can be provided as a functional beverage which can exert an excellent bile acid excretion ability and lower cholesterol in the body.

Fig. 1 shows the mRNA expression levels of LXR-Alpha, a cholesterol regulator, and ABCG5, a cholesterol excretion factor.
FIG. 2 shows measurement of mRNA expression levels of ABCA1, which is an absorption factor of cholesterol, and ABCA1, which plays a role of transporting cholesterol out of a cell.
3 shows the growth of Lactobacillus acidophilus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacill

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention and the scope of the present invention is not limited thereto.

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 1: Using animal experiments Lactobacillus Plan Tam LLP5273 And vegetable Stanol Efficacy of Esters

(1) Preparation of experimental animals

An 8-week-old female mouse (C57BL / 6N) was purchased from Sam Taco Bio-Korea for animal experiments in the present invention, and prepared for one week based on the time of obtaining the cage for animal experiments.

(2) Preparation of samples for animal experiments

Lactobacillus plantarum LLP5273 was inoculated on MRS plate medium and activated for 24 hours at 37 ° C, and then inoculated into MRS liquid medium to prepare a sample for animal experiment. The cultured seed culture was inoculated into 800 ml of MRS liquid medium and incubated at 37 ° C for 24 hours. After completion of the cultivation, only the cells were recovered by centrifugation. The recovered cells were washed, centrifuged and recovered using 0.1 M phosphate buffer (pH 6.8 ± 0.2). This step was repeated three times to completely remove the medium components in the culture solution. The recovered cell lysate of the completed Lactobacillus plantarum LLP5273 was suspended in sterilized physiological saline and prepared to be orally challenged with a syringe at 10 ⁸ CFU per mouse per day in the experimental group.

For the feeding of vegetable stanol esters, samutako feeds which were completely removed from fats and cholesterol were purchased and were referred to as general feeds. To this feed, 21.5 wt% of fat and 1.25 wt% of cholesterol Were used as high cholesterol diet for inducing high cholesterol. The vegetable stanol ester was obtained from Benecol (Raisio co. Ltd., Finland), and 21.5 wt% fat, 1.25 wt% cholesterol and 1.6 wt% Were prepared with vegetable starol ester feed. Concentrations of Venechol were determined by reference to the previous literature that reduced blood cholesterol levels at both animal and clinical levels using Venechol.

(3) Animal experiment progress

As shown in Table 1, mice were caged for one week in the above-mentioned Examples. The mice were divided into 12 groups of 12 hours and 12 hours, And feeding of vegetable stanol ester via feed. After 6 weeks, the experimental animals were dissected with the end of the experiment to collect organs, blood and feces. The liver tissues were used for total cholesterol, triglyceride and gene analysis. Genomic analysis of the intestinal tissues and fecal analysis of cholesterol and bile acid were performed.

Experimental group Administration sample Feed feed CON 0.9% saline solution General food HCD 0.9% saline solution High cholesterol feed
(21.5 wt% fat, 1.25 wt% cholesterol) 5273 Lactobacillus plantarum LLP5273
(10 ⁸ CFU / day) High cholesterol feed
(21.5 wt% fat, 1.25 wt% cholesterol) BNC 0.9% saline solution Vegetable starol ester feed
(20.9 wt% fat, 1.25 wt% cholesterol,
1.6 wt% vegetable stanol ester) 5273 + BNC Lactobacillus plantarum LLP5273
(10 ⁸ CFU / day)

(4) Fecal cholesterol (Sterol) and bile acid analysis

After the end of the 6-week animal experiment, the fecal samples of the mice in the experimental group were collected and the total cholesterol assay kit (Cellbiolabs) was used for the analysis of cholesterol released into the feces. A certain amount of fecal samples were collected and homogenized in 1 ml of cholesterol extraction solvent according to the method of the assay kit, and centrifuged to obtain supernatant. After removal of all the solvent through nitrogen concentration, 1 ml of assay diluent was added, And the absorbance was measured at 540-570 nm using an ELISA reader (Bio-Rad 680, USA).

In order to analyze the bile acid released from the feces, a certain amount of feces sample was dried at 110 ° C. for 24 hours and then weighed and added with 1 ml of potassium hydroxide (KOH, 4 g KOH in ethylene glycol) . After cooling the sample, 1 ml of 20% NaCl solution and 0.2 ml of concentrated HCl were added and mixed. Then, 6 ml of diethyl-ether was added, and the mixture was suspended for 1 minute and then centrifuged at 2,000 xg for 3 minutes The supernatant was obtained by centrifugation. Bile acid was analyzed using a mouse total bile acids assay kit (Crystal Chem, USA).

The results of analysis of cholesterol and bile acid released from feces are shown in Table 2. As shown in the results, in the group fed with high cholesterol diet, the amount of cholesterol was 116.96 ± 10.44 μmol per 1 g of feces, compared with 141.83 ± 10.91 μmol per gram of feces in the group fed LLP5273 strain and vegetable stanol ester, 138.41 ± 12.83 μmol, indicating that cholesterol emissions increased sharply. In addition, in the group fed with LLP5273 and vegetable stanol ester, 202.91 ± 14.77 μmol of cholesterol was excreted per 1 g of feces.

It is presumed that Lactobacillus plantarum LLP5273 lactic acid bacteria invented by the present inventors have an effect on the direct release of cholesterol. In addition to the bile acid degradation ability and HMG-CoA reductase inhibitory ability which have been confirmed by the efficacy of LLP5273 strain, And that there is a mechanism for discharging it to the environment. It was also confirmed that the use of the LLP5273 strain in combination with the plant stanol ester further increased the effect.

As a result of analysis of fecal bile acid emissions, 8.04 ± 1.13 μmol of LLP5273 group, 7.52 ± 1.50 μmol of vegetable starol ester group, and 7.02 ± 1.66 μmol / g fecal cholesterol group . In this case, the amount of bile acid emission increased by LLP5273 was higher than that by vegetable stanol ester. In addition, in the group fed with LLP5273 and vegetable stanol ester, it was found that 10.92 ± 1.32 μmol of bile acid was excreted per 1 g of feces, and the bile acid excretion was further increased compared with the case of feeding alone. In other words, the combination of LLP5273 and the vegetable stanol ester has excellent ability to excrete cholesterol and bile acid, and the combination of the two components has synergenic effect of cholesterol lowering.

Experimental group Liver weight
(g) Fecal cholesterol
(μmol / g feces) Fecal bile acid
(μmol / g feces) CON 0.87 ± 0.01 3.96 ± 0.23 2.93 + - 0.40 HCD 1.31 ± 0.05 116.96 ± 10.44 7.02 + 1.66 5273 1.30 ± 0.03 141.83 + - 10.91 8.04 ± 1.13 BNC 1.26 + 0.04 138.41 ± 12.83 7.52 ± 1.50 5273 + BNC 1.28 ± 0.05 202.91 + - 14.77 10.92 ± 1.32

(5) lipid analysis in liver tissue

After the end of the 6-week animal experiment, the mice were weighed and the total cholesterol assay kit (Cellbiolabs) was used for total cholesterol analysis in the liver.

After the liver tissue was weighed for measurement of triglyceride, it was immersed in 350 μl of an ethanol-potassium hydroxide solution (95% ethanol: 30% KOH = 2: 1) and treated at 55 ° C. for 24 hours. Then, 1000 μl of 50% Suspended and centrifuged (15,000 xg, 10 min). The centrifuged supernatant was transferred to a new tube, and the total volume of the supernatant was adjusted to 1,200 μl using 50% ethanol. Then, the triglyceride quantification kit (Sigma) (USA) Respectively.

The results of analysis of cholesterol and triglyceride in liver after 6 weeks of animal experiment are shown in Table 3. As shown in the results, the total cholesterol of the general feed group without addition of fat and cholesterol was 2.07 ± 0.38 mg / g of liver, and 5.97 ± 0.89 mg / g of liver in the high cholesterol feed group, The difference was confirmed. On the other hand, when the same high cholesterol diet was fed, the LLP5273 lactobacilli and the vegetable stanol ester were 5.60 ± 0.61 and 2.88 ± 0.47, respectively, which were found to be useful for lowering the cholesterol synthesized in the liver tissue. The effect of plant starol ester was more significant than the effect of LLP5273. In particular, considering that the total cholesterol of the general diet group without the addition of fat and cholesterol is 2.07 ± 0.38 mg per 1 g of liver, when vegetable starol ester is taken together with the high fat diet and high cholesterol diet, the amount of cholesterol Of the total population. When LLP5273 and vegetable stanol ester were fed together, 3.22 ± 0.39 mg / kg of liver was slightly higher than that of only the vegetable stanol ester.

On the other hand, in the case of triglycerides, there were significant differences among the five experimental groups and there was no significant difference in the statistical results. However, in the group fed with LLP5273 and vegetable stanol ester, a somewhat low triglyceride level was found to be 31.65 ± 8.48 g per 1 g of liver, but this was also found to be somewhat difficult to see as a significant result because there was a large difference among individuals in the experimental group .

Experimental group Liver weight
(g) Total cholesterol
(mg / g liver) Triglyceride
(mg / g liver) CON 0.87 ± 0.01 2.07 + - 0.38 48.68 + - 6.53 HCD 1.31 ± 0.05 5.97 ± 0.89 36.08 ± 19.08 5273 1.30 ± 0.03 5.60 0.61 43.56 ± 14.38 BNC 1.26 + 0.04 2.88 0.47 46.93 + - 14.68 5273 + BNC 1.28 ± 0.05 3.22 ± 0.39 31.65 + 8.48

(6) Analysis of gene expression related to cholesterol metabolism

In the cholesterol metabolism-related pathways studied, SREBP-2, HMG-CoA reductase and LDL-receptor have been known to be mainly involved in cholesterol synthesis. LCH-alpha, ABCG -5, etc. are known, and NPC1L1 is known as a cholesterol absorption factor. In addition, CYP7A1, which is the predominant factor affecting the liver, is a bile acid-producing factor. In the case of bile acids, cholesterol is synthesized as a substrate. Therefore, when the concentration of bile acid in the body is directly decreased, It is a known factor of relationship.

Therefore, we analyzed the expression patterns of genes related to cholesterol metabolism by analyzing gene expression in tissues of liver and kidney. For this purpose, the extracts of liver and intestinal tissues were extracted using an extraction kit (Easy-BLUE Total RNA Extraction Kit, iNitron, USA). In the subsequent experiments, cDNA was synthesized using cDNA synthesis kit (High Capacity cDNA Reverse Transcription Kits, AB Biosystems, USA) and the amount of gene expression was measured using RT-PCR. The confirmed mRNA concentration was calculated by relative quantification using the △ ΔCt method.

The results of analysis of the expression levels of cholesterol synthesis, excretion, and metabolism-related factors in the liver are shown in Table 4. As shown in Table 4, factors related to cholesterol synthesis, namely, HMG-CoA reductase activity and LDL-receptor and SREBP-2 in cholesterol synthesis factor were LLP5273 level and vegetable stanol ester level compared to high cholesterol level And the two groups were found to be low in the overall group, but it was difficult to see that there were significant differences. In the case of LXR-Alpha, which is a regulator of cholesterol metabolism, the amount of regulator was significantly increased in LLP5273-fed group compared with that in high-cholesterol-fed group, whereas the level of CYP7A1 as bile acid producing group was significantly higher . In the case of LXR-Alpha, which is a cholesterol regulator, LXR-Alpha increases expression and increases bile acid production factors, cholesterol is synthesized from bile acid due to the release of cholesterol in liver tissue, resulting in decreased bile acid concentration and increased bile acid production factor It can be estimated. Therefore, when LLP5273 Lactobacillus is fed, it is expected that the cholesterol regulator will increase the cholesterol level in liver tissue.

division Genetic factor mRNA expression level (Relative quantity) CON HCD 5273 BNC 5273 + BNC Cholesterol synthesis HMG-CoA
Reductase 1.00 + 0.08 0.45 ± 0.19 0.35 + 0.12 0.33 + 0.11 0.24 ± 0.63 LDL-Receptor 1.00 + - 0.11 0.42 + 0.24 0.33 ± 0.09 0.25 0.11 0.22 0.08 SREBP-2 1.02 + 0.21 0.37 + 0.17 0.37 ± 0.09 0.23 ± 0.08 0.20 ± 0.06 Cholesterol release and regulation ABGC5 1.01 ± 0.16 1.96 + - 0.23 1.71 + 0.49 0.89 ± 0.05 0.88 0.35 LXR-alpha 1.01 + - 0.12 0.59 + 0.08 0.77 + - 0.13 0.42 + 0.13 0.39 0.11 Bile acid production CYP7A1 1.13 ± 0.58 0.16 ± 0.09 0.47 + - 0.42 0.04 0.04 0.18 ± 0.11

The results of analysis of cholesterol metabolism related genes in the intestines are shown in FIG. 1 and FIG. 2. FIG. 1 shows the results for LXR-Alpha, a cholesterol regulator, and ABCG5, a cholesterol releasing factor. When LLP5273 was administered, the expression level of LXR-Alpha, a cholesterol regulator, was rapidly increased. ABCG5 also increased rapidly. On the other hand, in the case of the plant starch ester alone, there was no significant increase compared with the high cholesterol diet group, but the increase in the LLP5273 group was also noticeable. In contrast, NPC1L1, a factor involved in the absorption of cholesterol in the intestine, was significantly reduced in the group treated with LLP5273 and plant starol ester alone, and the effect was significantly reduced in the mixed treatment group.

In addition, ABCA1 is known to mainly synthesize high-density lipoprotein (HDL) using early absorbed cholesterol, but it was significantly increased in LLP5273-treated group. However, in the case of ABCA1, , The apparent synergistic effect is not confirmed.

In comparison with the above-mentioned gene analysis in the liver, the gene expression in the intestine was significantly higher than that in the liver when LLP5273 was treated, The results of this study are as follows.

When LLP5273 Lactobacillus acidophilus and plant starch ester were mixed, cholesterol and bile acid excretion increased significantly in the intestine rather than in the liver.

As a result of the above-mentioned examples, Lactobacillus plantarum LLP5273 Lactobacillus acidophilus and plant stanol ester have a similar mechanism or action method for lowering cholesterol in blood, but they are different from each other in mechanism, Respectively.

Therefore, LLP5273 Lactobacillus acidophilus and vegetable stanol ester can be supplemented to lower the cholesterol level, and the synergistic effect on cholesterol efflux is expected to be conducive to lowering cholesterol in the body.

Example 2: Lactobacillus Plan Tam Of LLP5273 Manufacture of freeze-dried raw materials

LLP5273 Lactobacillus fermented milk was prepared as a freeze-dried starter. Lactobacillus acidophilus L. lactobacillus LLP5273, which was cryopreserved, was activated in MRS plate culture medium. Then, a certain colony was collected and inoculated into MRS liquid culture medium and cultured at 37 ° C for 24 hours to prepare a seed culture. The colonies and seed culture that had been activated in the plate culture medium were sent to Mediogen (Chuncheon, Jecheon, Republic of Korea) and were prepared as freeze-dried materials using an edible medium and excipients. The total amount of maltodextrin and live cells in the lyophilized raw material was 1.4 × 10 ¹¹ CFU / g and the content of water was 1.6 ± 0.2%.

Example 3: Lactobacillus Plan Tam LLP5273 In milk Fermentability Confirm

(1) Confirmation of milk fermentation ability by composition

The optimum composition for fermentation of the Lactobacillus plantarum LLP5273 strain prepared in Example 1 on a milk-based medium was confirmed. Milk was purchased from Pasteur milk (Lotte Food Paste), which is commercially available on the market. In order to compare the fermentation capacity of LLP5273 strain according to presence of skimmed milk and glucose added to skim milk, 0.001 wt% of LLP5273 freeze-dried material was inoculated into each of the milk broths prepared and then incubated at 37 ° C. The results are shown in Table 1. 4% by weight of skim milk powder and 4% by weight of glucose were added. When all of them were added, 2% by weight of each was added. As shown in the results, the addition of milk alone or milk and skim milk alone did not significantly change the number of live cells until 12 hours of culture and increased about 10 ² at 24 hours of culture for LLP5273 strain. On the other hand, when glucose was added, the number of viable cells gradually increased from 6 hours after the inoculation time to 12 hours after the addition of 3.4 × 10 ⁸ glucose and skim milk powder Increased to 4.9 × 10 ⁸ and increased to 10 ⁹ at 24 hours after culture. In this case, the number of viable cells in the group supplemented with only glucose, skimmed milk and glucose was not significantly different between the two experimental groups.

These results suggest that Lactobacillus acidophilus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus isolated from Lactobacillus acidophilus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus L. Therefore, in order to prepare fermented milk using LLP5273 strain, it was considered that it would be advantageous to add glucose. In order to improve the texture and the sensory property of fermented milk when a certain amount of skim milk powder is added in the production of general fermented milk, glucose and skim milk are added together And finally decided.

Incubation time
(time) Number of viable cells (CFU / ml) Milk alone Milk + skim milk powder Milk + glucose milk
Skim milk powder + glucose 0 2.7 x 10 ⁶ 2.2 x 10 ⁶ 1.9 x 10 ⁶ 2.3 x 10 ⁶ 2 2.2 x 10 ⁶ 1.8 x 10 ⁶ 1.8 x 10 ⁶ 2.1 x 10 ⁶ 4 2.2 x 10 ⁶ 1.4 x 10 ⁶ 2.4 x 10 ⁶ 2.3 x 10 ⁶ 6 1.9 x 10 ⁶ 2.5 x 10 ⁶ 5.1 x 10 ⁷ 4.4 x 10 ⁷ 8 2.5 x 10 ⁶ 1.9 x 10 ⁶ 1.4 x 10 ⁸ 1.9 x 10 ⁸ 10 2.6 x 10 ⁶ 2.3 x 10 ⁶ 2.9 x 10 ⁸ 2.2 x 10 ⁸ 12 3.1 x 10 ⁶ 2.2 x 10 ⁶ 3.4 x 10 ⁸ 4.9 x 10 ⁸ 24 5.5 x 10 ⁸ 6.3 x 10 ⁸ 1.6 x 10 ⁹ 1.3 x 10 ⁹

(2) Confirmation of milk fermentation ability by glucose concentration

The effect of glucose addition on the fermentation ability of the milk medium was investigated. 2, 3, 4, and 5% by weight of glucose were added to a milk medium supplemented with 4% by weight of skimmed milk powder by the same method as in the above examples, and the number of live cells was measured according to the time of culture, . As shown in the results, when 1 wt% of glucose was added, it was confirmed that the level of 10 ¹ was increased in the number of viable cells at 10 hours after the inoculation, and it was 8.3 x 10 ⁸ CFU / g at 24 hours. This means that the fermentation time and the number of viable cells were somewhat improved as compared with the experimental group in which glucose was not added at all in the above example, but it is difficult to see the result as a significant increase. On the other hand, when 2 to 5 wt% of glucose was added, the number of viable cells was increased at a similar level. This is presumably because there is no significant difference in availability when a carbon source of a certain concentration or more is added and it is a similar result considering that a carbon source is added at a level of 2% by weight (20 g / L) to the medium for the growth of lactic acid bacteria .

Therefore, the concentration of glucose for growth of LLP5273 lactic acid bacteria is considered to be advantageous from 2 to 5% by weight, and it is considered that 2% by weight is most suitable in view of economical efficiency.

Incubation time
(time) Number of viable cells (CFU / ml) Glucose 1 wt% Glucose 2 wt% Glucose 3 wt% Glucose 4 wt% Glucose 5 wt% 0 1.4 x 10 ⁶ 1.6 x 10 ⁶ 1.8 x 10 ⁶ 1.8 x 10 ⁶ 1.8 x 10 ⁶ 2 1.7 x 10 ⁶ 1.9 x 10 ⁶ 2.3 x 10 ⁶ 2.2 x 10 ⁶ 1.7 x 10 ⁶ 4 1.7 x 10 ⁶ 2.9 x 10 ⁶ 3.1 x 10 ⁶ 2.6 x 10 ⁶ 2.2 x 10 ⁶ 6 1.8 x 10 ⁶ 3.6 x 10 ⁷ 5.8 x 10 ⁷ 6.2 x 10 ⁷ 2.8 x 10 ⁷ 8 3.2 x 10 ⁶ 4.1 x 10 ⁸ 1.9 x 10 ⁸ 2.3 x 10 ⁸ 5.3 x 10 ⁸ 10 1.1 x 10 ⁷ 7.4 x 10 ⁸ 5.3 x 10 ⁸ 4.5 x 10 ⁸ 6.7 x 10 ⁸ 12 5.4 x 10 ⁷ 7.6 x 10 ⁸ 7.2 x 10 ⁸ 5.7 x 10 ⁸ 6.2 x 10 ⁸ 24 8.3 x 10 ⁸ 1.5 x 10 ⁹ 1.5 x 10 ⁹ 1.6 x 10 ⁹ 1.8 x 10 ⁹

Example 4: Vegetable Stanol Optimization of ester concentration

In the case of vegetable stanol esters, which are well known for lowering blood cholesterol levels, many national health authorities and researchers recommend that you take 2 to 3 g / day or more. If you are overseas, you may be consumed in fermented milk, soy milk or butter many. The concentration of vegetable stanol ester suitable for fermented milk production was determined using Lactobacillus plantarum LLP5273, and the effect of each concentration on the growth of lactic acid bacteria was examined.

However, it is known that many plant-derived lipid components have a possibility of inhibiting the growth of microorganisms. Therefore, the inventors do not inhibit the growth of LLP5273 by the plant stanol ester, It is essential to establish an optimal dosage level for which it is possible.

Therefore, when Lactobacillus plantarum LLP5273 and 0, 1, 2, 3, 4, 5 and 10% by weight of vegetable stanol ester were added, the growth performance in milk medium was examined. The final weight of the milk medium was 100 g, and 4 wt% of skimmed milk powder and 2 wt% of glucose were added thereto, and the vegetable stanol ester was added by weight. The growth of LLP5273 lactic acid bacteria according to the concentration of the added vegetable stanol ester is shown in FIG.

As shown in FIG. 3, even when the concentration of the plant starch ester was increased, the growth of LLP5273 lactic acid bacteria was not significantly changed, and it was not found that the plant stanol ester had a growth inhibitory effect. However, when the concentration of the vegetable stanol ester was increased, especially when 5 wt% or more was added, the fragrance specific to the raw material was increased, resulting in a sensual problem, and it was confirmed that the viscosity increased and the smoothing improved. Previous studies related to vegetable stanol ester have shown that a daily intake of about 2 to 3 g may have a significant effect on cholesterol. Thus, the optimal concentration of vegetable stanol ester is 2 to 4 wt% .

Manufacturing example One: LLP5273 And vegetable Stanol Production of fermented milk using ester

The fermented milk was to be prepared based on the results confirmed in the above Examples. 25 g of glucose and 40 g of skim milk powder were added to 1 kg of commercially available milk (Pasteur, Korea), 30 g of vegetable stanol ester was added, autoclaved at 121 ° C. for 20 minutes, and then cooled to 30 ° C. . 0.02 g of LLP5273 Lactobacillus freeze-dried product (1.4 x 10 ¹¹ CFU / g based on viable cell count) was added to the prepared milk medium and then fermented in an incubator maintained at 37 ° C for 24 hours and then recovered and left in a refrigerator maintained at 4 ° C for 8 hours The The final recovered fermented milk was 985 g, and the number of viable cells in the fermented milk was found to be 1.3 x 10 ⁹ CFU / g and the final pH was 4.1.

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.

Claims

Lactobacillus plantarum) LLP5273 strain and plant stanol ester functional fermented milk superior ability to cholesterol and bile acids, characterized in comprising a discharge.

The method according to claim 1,
The strain may be selected from the group consisting of Lactobacillus plantarum LLP5273 culture medium, wherein the fermented milk is 0.001 to 0.01% by weight based on the total weight of the fermented milk,
Wherein the vegetable starol ester comprises 2 to 4% by weight based on the total weight of the fermented milk, wherein the fermented milk has excellent cholesterol and bile acid excretion ability.

3. The fermented milk according to claim 2, wherein the lyophilized product of Lactobacillus plantarum LLP5273 has a viable cell count of 1.0 x 10 ¹⁰ to 1.0 x 10 ¹¹ CFU / g.

The method according to claim 1, wherein the strain has excellent cholesterol and bile acid releasing ability in the intestines,
LXR-Alpha, a cholesterol regulator, and ABCG5, a cholesterol-releasing factor,
Wherein the amount of mRNA expression of NPC1L1, which is a cholesterol absorption factor, is decreased, and a functional fermented milk excellent in the ability to excrete cholesterol and bile acid.

The functional fermented milk according to any one of claims 1 to 4, wherein the fermented milk further comprises glucose or skim milk or a mixture thereof.

The functional fermented milk according to claim 5, wherein the glucose is present in an amount of 2 to 5% by weight based on the total weight of the fermented milk.

[Claim 6] The functional fermented milk according to claim 5, wherein the skim milk powder is 1 to 5 parts by weight based on the total weight of the fermented milk.

Adding 2 to 4% by weight of a vegetable stanol ester to a raw material containing milk, glucose or skimmed milk or a mixture thereof, followed by pressure sterilization; And
Adding 0.001 to 0.01% by weight of a lyophilized product of Lactobacillus plantarum LLP5273 and fermenting it after cooling the pressure sterilized milk;
Wherein the fermented milk has excellent cholesterol and bile acid emission ability.

[Claim 9] The lyophilized product of claim 8, wherein the number of viable cells of Lactobacillus plantarum LLP5273 is 1.0 x 10 ¹⁰ to 1.0 x 10 ¹¹ CFU / g. Gt;

A dry powder of fermented milk having excellent cholesterol-producing ability and bile acid excretion obtained by drying the fermented milk of any one of claims 1 to 4.