KR20200012236A - Novel lactic acid bacteria and method of preparing fermented product having effect of anti-obesity and anti-diabets using the same - Google Patents

Abstract

According to one embodiment, the present invention relates to novel lactic acid bacteria, a fermented product of the novel lactic acid bacteria in a sweet potato medium, a composition containing the fermented product of the lactic acid bacteria as an active ingredient and having anti-diabetes and anti-obesity effects, and a method for preparing the composition. The fermented product of the lactic acid bacteria has an effect of inhibiting the activity of at least one of α-amylase, α-glucosidase and lipase in vivo, inhibits factors involved in adipocyte differentiation, such as C/εΒΡα, C/εΒΡβ, PPARγ and FABP4 in preadipocyte cells, and increases the activity of Glut4 to increase intracellular glucose inflow. Therefore, the composition containing the fermented product of the lactic acid bacteria as an active ingredient can be used as a pharmaceutical composition having an effect of preventing or treating obesity or diabetes, or as a health functional food capable of preventing or alleviating obesity or diabetes.

Description

Novel Lactic Acid Bacteria AND METHOD OF PREPARING FERMENTED PRODUCT HAVING EFFECT OF ANTI-OBESITY AND ANTI-DIABETS USING THE SAME}

The present invention relates to a composition having a anti-obesity and anti-diabetic effect obtained by fermenting novel lactic acid bacteria and sweet potatoes with the lactic acid bacteria, and a method for preparing the same.

Lactic acid bacteria are bacteria that ferment sugars to obtain energy and produce large amounts of lactic acid. It is widely distributed in the natural world such as agricultural products, foods, and human and animal bodies. It is widely used for fermentation of cheese, fermented milk, kimchi, and baking. In general, ingesting lactic acid bacteria, it is known that not only harmful bacteria among intestinal microorganisms are suppressed, but also beneficial bacteria that help digestion, absorption and decomposition of food are increased. Therefore, it is reported that ingesting lactic acid bacteria has various effects such as reducing blood cholesterol, enhancing immunity, suppressing endogenous infection, improving liver cirrhosis, and anticancer effect. In addition, research on increasing the efficacy of natural products fermented with lactic acid bacteria has recently been made.

On the other hand, sweet potatoes are a year-round root vegetable of the family buckwheat, and is a cultivated crop mainly having starch-rich and sweet hump stems. The main part of the sweet potato eats roots, and the sweet potatoes are round and large due to the accumulation of nutrients in the roots. Sweet potatoes are alkaline foods containing large amounts of fiber. That is, sweet potatoes are rich in vegetable fiber, which helps to balance constipation and blood cholesterol. In addition, it is known to be effective in blood pressure control, anti-aging, anticancer action and skin beauty. In addition, sweet potatoes are carbohydrates, most of which are energy sources except water, and are high in calorie foods and are high in vitamin C, carotenoids, anthocyanins, polyphenols, minerals, and dietary fiber.

Recently, various processed foods using sweet potatoes have been developed and marketed by paying attention to the efficacy of such sweet potatoes, but research on materials that enhance functionality by incorporating lactic acid bacteria is insignificant.

Republic of Korea Patent Publication No. 10-2001-00048371 Republic of Korea Patent Publication No. 10-2013-0021119 Republic of Korea Patent Publication No. 10-2014-0067533 Republic of Korea Patent Publication No. 10-2015-0139153

One embodiment of the present invention is invented for the prevention and treatment of obesity and diabetes, a major disease in modern society, by fermentation of sweet potato using a novel lactic acid bacteria isolated from the stool lactic acid bacteria fermentation having anti-obesity and anti-diabetic efficacy, An object of the present invention is to provide a pharmaceutical composition, a health functional food, and a method for preparing the same.

In order to achieve the above object,

One embodiment of the present invention may provide a Lactobacillus rhamnosus strain deposited with accession number KCTC 13517BP.

In addition, another embodiment of the present invention may provide a lactic acid bacteria fermented product fermented with the strain.

In addition, another embodiment of the present invention may provide a pharmaceutical composition for preventing or treating diabetes or obesity, including the lactic acid bacteria fermentation as an active ingredient.

In addition, another embodiment of the present invention can provide a dietary supplement for preventing or improving diabetes or obesity, including the lactic acid bacteria fermented product as an active ingredient.

In addition, another embodiment of the present invention may provide a method for preparing a lactic acid bacteria fermentation product comprising the step of inoculating sweet potato with the Lactobacillus rhamnosus strain deposited with accession number KCTC 13517BP.

The novel lactic acid bacteria fermentation product of sweet potatoes according to one embodiment of the present invention has the effect of inhibiting the activity of at least one of α-amylase, α-glucosidase and lipase in the body. In addition, there is an effect of inhibiting factors involved in adipocyte differentiation, such as C / EBPa, C / EBB, PPARγ, FABP4, and increasing the activity of Glut4 in adipocyte progenitor cells, thereby increasing intracellular glucose influx. Therefore, the composition containing the lactic acid bacteria fermented product according to an embodiment of the present invention as an active ingredient, a pharmaceutical composition having an effect of preventing or treating obesity or diabetes or a health functional food capable of preventing or improving obesity or diabetes It can be used as.

1 is a graph showing the survival rate of lactic acid bacteria after lyophilization of the fermented product of lactic acid bacteria fermentation product and the control YG medium, skim milk powder medium using sweet potato medium according to an embodiment of the present invention.
Figure 2 is a graph showing the total polyphenol content of lactic acid bacteria fermentation and control YG medium fermentation using sweet potato medium according to an embodiment of the present invention.
Figure 3 is a graph showing the cell survival rate when treated with a concentration and time to the lactic acid bacteria fermented product supernatant dry according to an embodiment of the present invention to the fat precursor cell 3T3-L1 cell line.
Figure 4 is a graph showing the cell survival rate when treated with a concentration and time of the lactic acid bacteria fermented product supernatant dry according to an embodiment of the present invention in a cell line differentiated into adipocytes 3T3-L1 fat cells.
Figure 5 is a graph showing the cell survival rate when treated with sterilized lactic acid bacteria fermentation in accordance with an embodiment of the present invention to the fat precursor cell 3T3-L1 cell line by concentration and time.
Figure 6 is a graph showing the cell survival rate when treated with sterilized lactic acid bacteria fermentation according to an embodiment of the present invention in a cell line differentiated into adipocytes 3T3-L1 fat cells.
Figure 7 Oil Red O staining of the distribution of fat cells in the group treated with the supernatant dry lactic acid bacteria fermented product according to an embodiment of the present invention when differentiating the progenitor cell 3T3-L1 cell line It is a photograph compared with observation under a microscope.
8 is Oil Red O staining of the distribution of fat cells of the group treated with the supernatant of lactic acid bacteria fermented product supernatant dried according to an embodiment of the present invention when differentiating progenitor cell 3T3-L1 cell line It is a graph comparing the absorbance through.
Figure 9 Western blot at the protein stage of the adipocyte differentiation factor of the group treated with the lactic acid bacteria fermented product supernatant dry according to an embodiment of the present invention when differentiating the progenitor cell 3T3-L1 cell line with adipocytes This is the result confirmed through.
10 is Oil Red O staining of the distribution of fat cells of the group treated with the sterilized lactic acid bacteria fermented product and the treated group according to an embodiment of the present invention when differentiating adipocyte 3T3-L1 cell line into adipocytes. It is a graph comparing the absorbance through.
11 is Oil Red O staining of the distribution of fat cells of the group and the group not treated with the sterilized lactic acid bacteria fermentation in accordance with an embodiment of the present invention when differentiating progenitor cell 3T3-L1 cell line It is a graph comparing the absorbance through.
Figure 12 Western blot at the protein stage of the adipocyte differentiation factor of the group treated with lactic acid bacteria fermented product supernatant dry according to an embodiment of the present invention when differentiating the progenitor cell 3T3-L1 cell line with adipocytes This is the result confirmed through.
Figure 13 is a group of cells treated with lactic acid bacteria fermented product supernatant dried according to an embodiment of the present invention in a cell line in which fat precursor cells 3T3-L1 were differentiated into adipocytes, an untreated group and a positive control group, Glucose. This is a graph comparing Glucose uptake by treating analog 2-NBDG.
Figure 14 is a group treated with lactic acid bacteria fermented product supernatant dry according to an embodiment of the present invention in a cell line differentiated into adipocytes 3T3-L1 adipocytes and a group treated with insulin as a positive control group The protein expression pattern of glucose transcription factor Glut4 was confirmed by Western blot.
Figure 15 is a group treated with sterilized lactic acid bacteria lactic acid bacteria fermentation, according to an embodiment of the present invention in a cell line differentiated into adipocytes 3T3-L1 adipocytes, in the group treated with insulin as a positive control group This is a graph comparing Glucose uptake by processing 2-NBDG which is Glucose analog.
Figure 16 is a group treated with sterilized lactic acid bacteria lactic acid bacteria fermentation, according to an embodiment of the present invention in a cell line differentiated into adipocytes 3T3-L1 adipocytes, in the group treated with insulin as a positive control group The protein expression pattern of the glucose transcription factor Glut4 was confirmed by Western blot.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention can provide a novel Lactobacillus rhamnosus strain.

Specifically, the Lactobacillus rhamnosus strain was deposited on April 27, 2018 at the Korea Collection for Type Culture (KCTC), an international depository institution, and was granted accession number KCTC 13517BP.

The strain is isolated from human feces, and can be classified as a new strain belonging to Lactobacillus rhamnosus based on the phylogenetic results through sequencing.

The present inventors have isolated and identified the novel lactic acid bacteria strain. In particular, the present invention was completed by confirming that the fermented product fermented with sweet potato strain has anti-diabetic and anti-obesity effects. As used herein, the term "obesity" refers to the excessive accumulation of body fat in the body. In addition, the term "diabetes" as used herein refers to a kind of metabolic disease, such as a lack of insulin secretion or normal function that causes glucose-intolerance.

That is, another aspect of the present invention can provide a lactic acid bacteria fermented product fermented with the strain using sweet potato. The sweet potato may be used in the form of sweet potato dice, its pulverized product, sweet potato paste, sweet potato medium and the like. In addition, the sweet potato may be at least one selected from the group consisting of purple sweet potato, pumpkin sweet potato, honey sweet potato, yellow sweet potato, chestnut sweet potato, water sweet potato, sweet potato sweet potato, preferably may be chestnut sweet potato.

In addition, the lactic acid bacteria fermented product may be a fermented sweet potato medium, the sweet potato medium may include a sweet potato raw material and water in a weight ratio of 2: 1 to 1: 2. In addition, it may be fermented after further adding 1 to 3 times the water contained.

In addition, after fermenting the lactic acid bacteria YG medium, skim milk powder medium and sweet potato medium, after lyophilization of the fermented product, the survival rate of the lactic acid bacteria in the dried product was found to be 7 times to 1.4 times higher. That is, the sweet potato has an effect of improving the preservation of the lactic acid bacteria when the freeze-dried lactic acid bacteria fermented product, sweet potato may be used as a lyophilized preservative of the lactic acid bacteria.

In addition, the polyphenol content of the lactic acid bacteria fermentation may be 1.5 to 4 times the polyphenol content of the sweet potato, for example, sweet potato medium before fermentation. Polyphenols are substances that have two or more hydroxyl groups, and are known to have antioxidant and anti-inflammatory effects of removing harmful oxygen, as well as to regulate blood glucose and body metabolism. By fermenting the sweet potato medium with the novel lactic acid bacteria strain of the present invention, it is possible to effectively increase the content of polyphenols of the composition comprising sweet potatoes.

On the other hand, the lactic acid bacteria fermentation may inhibit the activity of at least one of α-amylase, α-glucosidase and lipase.

α-amylase or α-glucosidase is a carbohydrate degrading enzyme in vivo and lipase is a lipolytic enzyme. Drugs that inhibit the activity of these enzymes have been developed to improve blood sugar control and lipid metabolism in patients with diabetes or obesity, but there have been demands for natural products having similar activities due to various side effects and difficulty in synthesis. The lactic acid bacteria fermentation product according to an embodiment of the present invention has an effect of inhibiting the activity of the enzymes, and thus can be usefully used for preventing, treating and improving diabetes and obesity by directly inhibiting the decomposition of carbohydrates and fats. .

In addition, the lactic acid bacteria fermentation products are C / EBPα (CCAAT-enhancer-binding protein α), C / EBPβ (CCAAT-enhancer-binding protein β), PPARγ (peroxisome proliferator activated receptorγ), adiponectin (Adiponectin) and FABP4 (fatty acid) It inhibits the expression of at least one of binding protein 4) and increases the expression of GLUT4 (Glucose transporter type 4).

Adipocyte differentiation is a complex process that involves changes in morphology, hormone sensitivity and gene expression. Several transcription factors have been shown to play an important role in adipocyte differentiation. The major transcription factors involved in the formation of adipocytes are C / EBPa and PPARγ. In addition, PPARγ increases the expression of adifferentiation marker genes such as adiponectin or FABP4.

In addition, GULT4 is an insulin transport pathway, and when stimulation by insulin comes, intracellular GULT4 moves to the plasma membrane to promote glucose transport. Therefore, GLUT4 plays a role in lowering blood glucose by facilitating the inflow of glucose into the blood. That is, the antidiabetic effect increases as the activity and expression of GLUT4 involved in glucose transport increases.

In addition, the lactic acid bacteria fermentation may be lyophilized, it can be used as a raw material of various formulations, such as functional food by powdering the lyophilized fermentation.

Another aspect of the present invention can provide a pharmaceutical composition for preventing or treating diabetes or obesity, including the above-described lactic acid bacteria fermentation as an active ingredient.

The pharmaceutical composition may comprise the lactic acid bacteria fermentation as well as any additional or optional ingredients useful for nutritional or pharmaceutical uses not described or described herein. Many of these optional ingredients can be used in the compositions of the present invention as long as they are safe and effective for oral administration and are compatible with the essential and other optional ingredients of the compositions of the present invention.

In addition, the pharmaceutical composition may further include conventional pharmaceutically acceptable carriers and / or excipients in addition to the active ingredient, in addition to binders, disintegrating agents, coatings, lubricants, preservatives, antioxidants, buffers, other It may be formulated and formulated with a variety of pharmaceutically conventionally used optional ingredients such as pharmaceutical actives, sweeteners, colorants, flavors, flavor enhancers, thickeners and stabilizers, emulsifiers and the like.

In addition, another aspect of the present invention can provide a dietary supplement for preventing or improving diabetes or obesity, including the above-mentioned lactic acid bacteria fermented product as an active ingredient.

The health functional food may be commercialized by packaging the obtained lactic acid bacteria fermentation so as to be directly ingested, or may be provided as a functional food by adding lyophilized lactic acid bacteria fermentation as a supplementary ingredient of the food. For example, the lactic acid bacteria fermented product may be processed into a beverage form, processed into a capsule form, or processed into a tablet form to produce a product.

In addition, another aspect of the present invention, lactic acid bacteria fermentation comprising the step of inoculating a sweet potato, for example, a composition comprising sweet potato medium, Lactobacillus rhamnosus strain deposited with accession number KCTC 13517BP and fermenting the composition It is possible to provide a method for producing water. In particular, a nitrogen source and / or a carbon source may be further added for the fermentation. For example, yeast extract and / or sugar can be added.

For example, the step of inoculating may include 1) preparing a composition comprising sweet potatoes, 2) sterilizing and then adding a yeast extract to the composition, and 3) adding a composition comprising glucose to the sterilized composition. Then, it can provide a method for producing a lactic acid bacteria fermentation comprising the step of inoculating KCTC 13517BP strain.

The sweet potato composition may be a sweet potato medium prepared by pulverizing various kinds of sweet potatoes and homogenizing with water, and 10 to 40 parts by weight, 15 to 35 parts by weight, and 20 to about 30 parts by weight of sweet potato raw material. 30 parts by weight or 25 to 28 parts by weight.

In addition, the yeast extract may be added as a nitrogen source to the composition comprising sweet potatoes, wherein the yeast extract in the total composition is 2.5 g / L to 20 g / L, 5 g / L to 15 g / L or 7 g / L Can be added to a concentration of from 12 g / L. In addition, after adding the yeast extract, adjusting the pH value of the composition to about 5 to 8, 5.5 to 7.5 or 6 to 7 is most preferred in terms of organic acid production. In addition, the yeast extract may be added and then sterilized at a temperature of 100 ° C. to 130 ° C. for 10 minutes to 30 minutes.

To the sterilized sweet potato medium composition, a composition containing sugar such as sterile glucose or sucrose may be added, wherein the composition containing sugar in the total composition is 10 g / L to 40 g / L and 15 g / L. To 30 g / L or 18 g / L to 25 g / L.

In addition, in the step of fermentation, it is possible to perform the pre-fermentation to fermentation at a temperature of 20 ℃ to 40 ℃, 35 ℃ to 39 ℃, or 36 ℃ to 38 ℃. The prefermentation may be performed for 20 to 100 hours, 40 to 90 hours, 50 hours to 80 hours, or 65 hours to 75 hours.

After the pre-fermentation process, it may further comprise a post-fermentation step of fermentation at a temperature of 1 ℃ to 10 ℃, 3 ℃ to 7 ℃ or 4 ℃ to 5 ℃. The post-fermentation may be performed for 50 to 150 hours, 70 hours to 120 hours, or 95 hours to 105 hours.

By carrying out the fermentation of the two processes of pre-fermentation and post-fermentation under different conditions as described above, it is possible to increase the content of a component that inhibits the activity of enzymes related to diabetes and obesity, thereby more effectively preventing and treating related diseases. It can provide a lactic acid bacteria fermented product.

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

Preparation Example 1 Isolation of Fermented Strains

Twenty randomly selected phosphate samples were prepared by diluting in sterile PBS in decimal. It was plated in MRS agar medium and incubated at 37 ° C. under anaerobic conditions for 24 to 48 hours. After incubation, the largest colonies were selected, inoculated in MRS broth, and cultured at 37 ° C. under anaerobic conditions for 24 hours, and strains grown at OD 1.0 or higher at 600 nm were selected. Selected strains were inoculated in skim milk medium to select strains with smooth curd. After smearing the lactic acid bacteria grown in the skim milk medium, a single colony was selected and used for the production of lactic acid bacteria sweet potato fermentation.

Production Example  2. Preparation of Sweet Potato Medium

10⁶ CFU를 10%의 부피가 되도록 접종하였다. 37℃에서 1 내지 4일간 발효 후, 4℃에서 3 내지 4일간 후발효하여 발효액을 제조하였다.Domestic sweet potatoes, such as chestnut sweet potato, pumpkin sweet potato, and flavored sweet potato, were washed and removed after peeling, and then, the sweet potato weight and the same amount of water were added to grind finely. The prepared sweet potato paste and 1 to 3 times of water were mixed and ground finely at 10,000 rpm for 10 minutes using a homogenizer. To this was mixed 2.5 to 20 g / L yeast extract. Then, the pH was adjusted to 6.5 to 8.0 using 1M HCl. Lactobacillus rhamnosus (BST.L-601) KCTC 13517BP), which was sterilized at 121 ° C. for 15 minutes and then separated into a medium containing 10 to 40 g / L of glucose solution sterilized.

10 ⁶ CFU was inoculated to a volume of 10%. After fermentation at 37 ° C. for 1 to 4 days, the fermentation broth was prepared by post-fermentation at 4 ° C. for 3 to 4 days.

Preparation Example 3 Drying of Lactic Acid Bacteria Fermentation Solution

The pH of the lactic acid bacteria fermentation broth in which the lactic acid bacteria were fermented using the sweet potato medium prepared in Preparation Example 2 was adjusted to pH 7.0 using 5N NaOH solution. Thereafter, the same amount of water was mixed and stirred for 2 hours, followed by centrifugation for 1 hour at 8,000 rpm. The supernatant was separated and freeze-dried at -70 ° C for 7 days to prepare a lyophilized powder.

Production Example  4. Lactobacillus Fermented product  Sterile

The pH of the lactic acid bacteria fermentation broth in which the lactic acid bacteria were fermented using the sweet potato medium prepared in Preparation Example 2 was adjusted to pH 7.0 using 5N NaOH solution. Thereafter, the same amount of water was mixed and stirred for 2 hours, followed by autoclaving at 121 ° C. for 15 minutes to prepare sterilized lactic acid bacteria fermented product.

Experimental Example  1. Identification of Lactic Acid Bacteria

The species of the strain was confirmed by analyzing the physiological characteristics and 16s rDNA sequence of BST.L-601 in the strain isolated in Preparation Example 1. Specifically, API kit (API CHL) was used to confirm the carbon source availability among the physiological properties, and the results were analyzed by the sugar fermentation test and the results are shown in Table 1 ((+) in Table 1 when the carbon source availability was positive). (-) Means the carbon source availability is negative).

No. Substrate BST.L-601 No. Substrate BST.L-601 0 Control - 25 Esculine + One Glycerol - 26 Salicine + 2 Ertythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose - 4 L-Arabinose - 29 Lactose + 5 Ribose + 30 Melibiose - 6 D-Xylose - 31 Saccharose - 7 L-Xylose - 32 Terhalose + 8 Adonitol - 33 Inuline - 9 β methyl-xyloside - 34 Melezitose + 10 Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycogene - 13 D-Mannose + 38 Xylitol - 14 L-Sorbose + 39 β gentiobiose - 15 Rhamnose + 40 D-Turanose + 16 Dulcitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose + 18 Mannitol + 43 D-Fucose - 19 Sorbitol + 44 L-Fucose - 20 α Methyl-D-mannoside - 45 D-Arabitol - 21 α Methyl-D-glucoside + 46 L-Arabitol - 22 N Acetyl glucosamine + 47 Gluconate - 23 Amygdaline + 48 2 ceto-gluconate - 24 Arbutine + 49 5 ceto-gluconate -

In addition, 16s rDNA analysis was commissioned by the Korea Center for Microbiological Conservation and showed 99% homology with L.rhamnosus.

Experimental Example  2. Identification of lactic acid bacteria suitable for sweet potato medium

Experimental Example  2.1. Lactobacillus Fermented product  pH measurement

The pH of the lactic acid bacteria fermentation broth fermented with lactic acid bacteria using the sweet potato medium in the same manner as in Preparation Example 2 is shown in Table 2. pH measurement was measured using a pH meter. The pH of sweet potato fermented products inoculated with BST.L-601, L.acidophilus, S.thermophilus and Kimchi lactic acid bacteria strains were compared. As a result, it was confirmed that the pH of the group inoculated with BST.L-601 and kimchi lactic acid bacteria decreased more than the group inoculated with L, acidophilus and S.tehrmophilus.

Inoculated strain pH -(Potato badge) 6.34 BST.L_601 (Manufacturing Example 2) 3.54 L. acidophilus 3.83 S. thermophilus 6.01 Kimchi lactic acid bacteria 3.51

Experimental Example 2.2. Lactobacillus viable count

Experimental Example 2.2.1. Measurement of Viable Cell Count of Lactic Acid Bacteria Fermentation

The viable cell count was confirmed by counting the number of colonies generated by culturing the fermentation broth diluted in sterile saline solution in MRS agar medium for 2 days and then culturing at 37 ° C for 2 days to confirm the number of cells present in the fermentation broth. After adjusting the entire lactic acid bacteria fermented product to pH 7.0, aseptically lyophilized, weighed about 1 g of lactic acid bacteria fermented product, diluted in saline solution, smeared on MRS agar medium, and incubated at 37 ° C for 2 days. The number of cells was confirmed.

5 g / L of the yeast extract was added in the same manner as in Preparation Example 2, 10 g / L of glucose was added to the sweet potato medium adjusted to pH 7.0, and the four lactic acid bacteria were added at 1Х10 ⁶ to 1Х10 ⁷ CFU / ml. Was inoculated at 10%. After fermentation at 37 ° C. for 3 days, the cells were aged at 4 ° C. for 3 days, and the number of viable cells was measured. The results are shown in Table 3. BST.L-601 exhibited a survival rate of about 87.23%, making it the most suitable lactic acid strain for sweet potato medium.

Inoculated strain name Fermentation
Viable cell count (CFU / g) Post fermentation
Viable cell count (CFU / g) Survival rate (%) BST.L_601 (Manufacturing Example 2) 4.7 x ^{10 11} 4.1 x ^{10 11} 87.23 L. acidophilus 1.1 x ^{10 8} 6.3 x 10 ⁵ 0.57 S. thermophilus 5.5 x 10 ⁷ 1.5 x 10 ⁷ 27.27 Kimchi lactic acid bacteria 2.9 x ^{10 9} 1.1 x ^{10 9} 37.93

Experimental Example 2.2.2. Measurement of viable cell count after lyophilization of lactic acid bacteria

In the same manner as Preparation Example 2 YG (5% yeast extract and 10% glucose) medium, skim milk powder (10% skim milk powder and 10% glucose) and sweet potato medium (5 g / L of yeast extract, pH 7.0 After the addition of 10 g / L of glucose to the sweet potato medium adjusted to, the new lactic acid bacteria (BST.L-601) was inoculated 10% in each medium at 1Х10 ⁶ to 1Х10 ⁷ CFU / ml. After fermentation at 37 ° C. for 3 days, the cells were aged at 4 ° C. for 3 days, and the number of viable cells was measured after lyophilization. The results are shown in FIG. After lyophilization, the survival rate of lactic acid bacteria was about 79% in sweet potato medium, 58% in skim milk powder medium and 11% in YG medium. Therefore, it was confirmed that the sweet potato medium may play a role of a lyophilized preservative during lyophilization after fermentation using sweet potato medium.

Experimental Example  2.3. Lactobacillus Fermented product  characteristic

Experimental Example 2.3.1. Measurement of Organic Acid of Lactic Acid Bacteria Fermentation

Four kinds of sweet potato medium were prepared in the same manner as in Experiment 2.2, except that 2.5 g / L of the yeast extract was added during the preparation of the sweet potato medium.

The amount of organic acid present in the fermentation broth was measured by gas chromatography (GC). CP-Sil 5 CB (25x0.53x0.7) column was used for the analysis, and the split ratio was 10: 1. Flow rate was adjusted to carrier gas (N ₂ ) 3ml / min, H2 65ml / min, Air 450ml / min, Makeup Flow 30ml / min. In addition, the content of lactic acid was analyzed under the conditions of Injector 225 ℃, column 40 ℃ (rate 1 min), 10 ℃ / min, 180 ℃, FID Detector 250 ℃.

As shown in Table 4, the concentration of lactic acid was produced significantly higher in the novel strain BST.L_601 of the present invention.

Inoculated strain Lactic acid (mg / ml) L. acidophilus 13.361 BST.L-601 38.417 S. thermophilus 10.74 Kimch 11.675

Experimental Example  2.3.2. Lactobacillus Fermented product  Measurement of α-amylase Inhibitory Activity

Α-amylase inhibitory activity of the fermentation was measured by 250 μl of standard α-amylase enzyme solution diluted to 13 Unit / ml in 0.02M phosphate buffer solution (pH 6.9) and 250 μl of sample or control (1mM Acarbose) and blank test (distilled water). Put in. This was reacted for 10 minutes at 37 ° C. Then, 250 μl of 1% starch solution was mixed and reacted at 37 ° C. for 10 minutes. 500 μl of DNS reagent was added to the reaction solution and the mixture was boiled at 100 ° C. for 5 minutes. After cooling, the absorbance was measured at 520 nm to calculate the inhibitory activity as follows. The results are shown in Table 5.

Ctrl: Absorbance (520 nm) of control (1 mM Acarbose) reaction solution

SPL: absorbance of sample reaction solution (520 nm)

As a result, as shown in Table 5, the α-amylase inhibitory activity of BST.L_601 was the highest.

sample α-amylase inhibitory activity (%) Control 1mM acarbose 71.774 Lactobacillus species L. acidophilus 11.507 BST.L-601 24.940 S. thermophilus 20.534 Kimch 6.739

Experimental Example  2.3.3. Lactobacillus Fermented product  Measurement of α-glucosidase inhibitory activity

To measure the α-glucosidase inhibitory activity of the lactic acid bacteria fermentation, 10 μl of standard α-glucosidase enzyme solution diluted to 1 Unit / ml in 0.05 M phosphate buffer solution (pH 6.8) and a sample or control (1 mM Acarbose), 20 μl of blank test (distilled water) and 50 μl of buffer were added to a 96well microplate. This was reacted for 5 minutes at 37 ° C. Then, 20 μl of 1 mM PNPG solution was mixed and enzymatically reacted at 37 ° C. for 30 minutes. Further, 50 µl of 1 M sodium carbonate solution was added to terminate the reaction. The absorbance was measured at 405 nm, and the inhibitory activity was calculated as shown in Table 6 below.

Blank test: Absorbance of blank test solution (405 nm)

Sample: Absorbance of Sample Reaction Liquid (405 nm)

sample α-glucosidase inhibitory activity (%) Control 1mM acarbose 57.417 Lactobacillus species L. acidophilus 8.572 BST.L-601 26.447 S. thermophilus 16.931 Kimch 6.849

As a result, as shown in Table 6, the α-glucosidase inhibitory activity of BST.L_601 was the highest.

Based on the results of Experimental Example 2, it was confirmed that BST.L_601 lactic acid bacteria is the strain producing the best fermented product.

Experimental Example 3. Exploration of Fermentation Conditions for Increasing Efficacy of Lactic Acid Bacteria Fermentation

Experimental Example 3.1. Confirmation of Lactic Acid Bacteria Fermentation According to the Concentration of Sweet Potato Medium

In order to confirm the change in activity of lactic acid bacteria fermentation according to the concentration of sweet potato medium, the stock solution of sweet potato medium was diluted 1.5 to 3 times. Then, 2.5 g / L yeast extract was added and adjusted to pH 7.0. After sterilizing this composition, sterilized glucose was added to 10 g / L. Herein, the selected lactic acid bacteria BST.L-601 was inoculated and fermented at 37 ° C. for 3 days, and then aged at 4 ° C. for 3 days to prepare a lactic acid bacteria fermented product. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 7 to 9, respectively. 1 mM acarbose was used as a control.

Sweet Potato Exclusion
(Dilution drainage) Lactic acid (mg / ml) One 20.861 1.5 28.319 2 18.104 2.5 18.684 3 17.296

Sweet potato badge
(Dilution drainage) α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 One 9.790 1.5 24.221 2 25.429 2.5 14.113 3 17.143

Sweet potato badge
(Dilution drainage) α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 One 16.784 1.5 29.373 2 24.694 2.5 13.458 3 9.857

As shown in Table 7, it can be seen that the most lactic acid was produced at 28 mg / ml when using a 1.5-fold diluted sweet potato medium. In addition, in Table 8 and Table 9, the highest inhibitory activity of α-amylase and α-glucosidase was confirmed using the 1.5-fold diluted sweet potato medium.

Experimental Example 3.2. Confirmation of Lactic Acid Bacteria Fermentation by Fermentation

The stock solution of sweet potato medium was diluted 1.5-fold. Then, 2.5 g / L yeast extract was added and adjusted to pH 7.0. After sterilizing the composition, sterilized glucose was added to 10 g / L. Here, the selected lactic acid bacteria BST.L-601 was inoculated. Four experimental groups were designed by fermentation at 37 ° C. for 1 day, 2 days, 3 days and 4 days, respectively. Then, aged for 3 days at 4 ℃ to prepare a lactic acid bacteria fermented product. Its lactic acid content and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 10 to 12, respectively. At this time, 1mM acarbose was used as a control.

Effective date (day) Lactic acid (mg / ml) One 23.081 2 21.662 3 22.185 4 22.101

Effective date (day) α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 One 17.484 2 18.297 3 23.774 4 24.006

Effective date (day) α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 One 22.096 2 22.488 3 33.544 4 25.867

As shown in Table 10, lactic acid production showed no significant difference. However, referring to Table 11, it can be seen that the fermentation for 3 days or more significantly increases the inhibitory activity of α-amylase. In addition, looking at Table 12, it was confirmed that the fermentation for three days showed a higher α-glucosidase inhibitory activity than when fermented for four days (33.54% (25.84%).

Experimental Example 3.3. Confirmation of Lactic Acid Bacteria Fermentation by Aging

The stock solution of sweet potato medium was diluted 1.5-fold. Then, 2.5 g / L yeast extract was added and adjusted to pH 7.0. After sterilizing this composition, sterilized glucose was added to 10 g / L. Here, the selected lactic acid bacteria BST.L-601 was inoculated. And it fermented at 37 degreeC for 3 days. Thereafter, three lactic acid bacteria fermented products were prepared by aging at 4 ° C. for 3 days, 4 days, and 5 days, respectively. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 13 to 15, respectively. At this time, 1mM acarbose was used as a control.

Experimental group No. Ripening period (day) Lactic acid (mg / ml) Ripening period (day) One 3 23.386 2 4 28.461 3 5 28.461

Ripening period (day) α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 3 25.429 4 37.042 5 36.173

Ripening period (day) α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 3 29.373 4 34.712 5 34.770

As shown in Table 13, lactic acid production showed no significant difference. However, referring to Table 14 and Table 15, it can be seen that the high inhibitory activity of α-amylase and α-glucosidase when aged for 4 days or more.

Experimental Example  3.4. Lactobacillus by Sweet Potato Species Fermented product  Check active

Four kinds of sweet potato medium were prepared using chestnut sweet potato, pumpkin sweet potato, taste gel sweet potato, and mixtures thereof. It was diluted 1.5 fold. Then, 2.5 g / L yeast extract was added and adjusted to pH 7.0. After sterilizing this composition, sterilized glucose was added to 10 g / L. Here, the selected lactic acid bacteria BST.L-601 was inoculated. After fermentation for 3 days at 37 ℃, aged for 4 days at 4 ℃ to prepare a lactic acid bacteria fermented product. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 16 to 18, respectively.

Sweet potato varieties Lactic acid (mg / ml) Sweet potato 21.189 Pumpkin sweet potato 20.399 Flavored Sweet Potato 18.394 mix 21.711

sample α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 Sweet potato 19.986 Pumpkin sweet potato 14.858 Flavored Sweet Potato 9.140 mix 14.018

sample α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 Sweet potato 33.312 Pumpkin sweet potato 32.529 Flavored Sweet Potato 23.294 mix 26.586

Looking at Table 16, the most delicious amount of lactic acid was produced when using only sweet potato (18.39 mg / ml), and the highest amount of about 21 mg / ml when sweet potato was used and chestnut sweet potato was used. Produced lactic acid.

In addition, as shown in Table 17, α-amylase inhibitory activity was the highest when the night sweet potato was used (19.98%). In addition, as shown in Table 18, the inhibition activity of α-glucosidase showed the highest inhibitory activity of 33.31% and 32.53%, respectively, when chestnut and pumpkin sweet potatoes were used.

Experimental Example  3.5. Lactobacillus by Addition of Nitrogen Source Fermented product  Check active

The stock solution of sweet potato medium was diluted 1.5-fold. To this, four experimental groups were designed by adding 2.5 g / L, 5 g / L, 10 g / L, and 20 g / L yeast extracts, respectively. Then, its pH was adjusted to 7.0. After sterilizing this composition, sterilized glucose was added to 10 g / L. Here, the selected lactic acid bacteria BST.L-601 was inoculated. Here, the selected lactic acid bacteria BST.L-601 was inoculated and fermented at 37 ° C. for 3 days and then fermented at 4 ° C. for 4 days to prepare a lactic acid bacteria fermented product. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 19 to 21, respectively.

Nitrogen source Lactic acid (mg / ml) Non nitrogen source 20.813 2.5 g / L Yeast 19.375 5 g / L yeast 21.233 10 g / L yeast 23.754 20 g / L yeast 22.407

Nitrogen source α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 Non nitrogen source 23.037 2.5 g / L Yeast 23.938 5 g / L yeast 23.466 10 g / L yeast 19.648 20 g / L yeast 20.077

Nitrogen source α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 Non nitrogen source 34.489 2.5 g / L Yeast 34.489 5 g / L yeast 35.152 10 g / L yeast 39.907 20 g / L yeast 38.981

Looking at Table 19, it can be seen that the highest amount of organic acid was produced at 23.75 mg / ml when 10 g / L yeast extract was added. In addition, as shown in Table 20, a slight difference, but the highest α-amylase inhibitory activity of about 23.5% when 5 g / L Yeast was added. In addition, as shown in Table 21, when the α-glucosidase inhibitory activity was measured, the highest inhibitory activity was found to be about 39% when fermented with 10 g / L yeast.

Experimental Example  3.6. Carbon source  Lactobacillus by Type and Amount Fermented product  Check active

The stock solution of sweet potato medium was diluted 1.5-fold. 10 g / L yeast extract was added thereto and the pH was adjusted to 7.0. After sterilizing the composition, six experimental groups were designed with 5 g / L, 10 g / L and 20 g / L of sterilized glucose or sucrose, respectively. Here, the selected lactic acid bacteria BST.L-601 was inoculated and fermented at 37 ° C. for 3 days and then fermented at 4 ° C. for 4 days to prepare a lactic acid bacteria fermented product. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 22 to 24, respectively.

Carbon source Lactic acid (mg / ml) Non carbon source 17.901 5 g / L glucose 20.398 10 g / L glucose 22.42 20 g / L glucose 27.222 5 g / L Sucrose 19.126 10 g / L Sucrose 19.898 20 g / L Sucrose 20.771

Carbon source α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 Non carbon source 20.979 5 g / L glucose 21.266 10 g / L glucose 20.318 20 g / L glucose 16.435 5 g / L Sucrose 23.141 10 g / L Sucrose 23.186 20 g / L Sucrose 23.715

Carbon source α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 Non carbon source 22.573 5 g / L glucose 33.576 10 g / L glucose 36.590 20 g / L glucose 41.635 5 g / L Sucrose 29.775 10 g / L Sucrose 23.679 20 g / L Sucrose 32.319

Looking at Table 22, the addition of 20 g / L glucose produced the most organic acid of 27 mg / ml. In addition, as shown in Table 23, in the case of α-amylase inhibitory activity, the difference between the fermented product and the fermented product without a carbon source was not large. However, as shown in Table 24, it was confirmed that the highest inhibitory activity as 41.6% in lactic acid bacteria fermentation of sweet potato medium containing 20 g / L glucose.

Experimental Example  3.7. Lactobacillus by Initial pH Fermented product  Check active

The stock solution of sweet potato medium was diluted 1.5-fold. To this 10 g / L yeast extract was added. Thereafter, sweet potato medium having no pH adjusted and sweet potato medium having pH adjusted to 6.5, 7.0, 7.5, and 8.0 were prepared using 5N NaOH. After sterilizing each medium, 20g / L sterile glucose was added. Here, the selected lactic acid bacteria BST.L-601 was inoculated and fermented at 37 ° C. for 3 days and then fermented at 4 ° C. for 4 days to prepare a lactic acid bacteria fermented product. Its content of lactic acid and the inhibitory activity of α-amylase and α-glucosidase were measured by the method described in Experimental Example 2. The results are shown in Tables 25 to 27, respectively.

PH before sterilization Lactic acid (mg / ml) Not control to pH 30.134 pH 6.5 30.952 pH 7.0 27.56 pH 7.5 26.461 pH 8.0 25.363

PH before sterilization α-amylase inhibitory activity (%) Control: 1 mM acarbose 71.774 Not control to pH 21.96 pH 6.5 25.33 pH 7.0 26.46 pH 7.5 19.14 pH 8.0 8.87

PH before sterilization α-glucosidase inhibitory activity (%) Control: 1 mM acarbose 57.417 Not control to pH 55.17 pH 6.5 53.62 pH 7.0 50.87 pH 7.5 50.67 pH 8.0 49.38

As a result, as shown in Table 25, the content of the organic acid was the highest as 30 mg / ml or more at pH 6.5 or less. In addition, in Table 26 and Table 27 it was confirmed that α-amylase and α-glucosidase inhibitory activity was the highest at pH 6.5, about 25% and 53%, respectively.

Experimental Example 4. Confirmation of the lactic acid bacteria fermentation product after drying

Experimental Example  4.1. Lactobacillus Fermented product  Lactobacillus by Lyophilization and Culture Stage Fermented product  Efficacy Check

Experimental Example  4.1.1. α-amylase inhibitory activity

YG medium was prepared by adding only 10g / L yeast extract and 20g / L glucose without adding sweet potato medium. In addition, three experimental groups in which YG medium, sweet potato medium was added thereto, and lyophilized were set. In addition, all of these were pre-fermented at 37 ° C. before BST.L_601 inoculation for 3 days, and then after 4 days at 4 ° C. for post-fermentation, α-amylase inhibitory activity was measured. In order to confirm the effect of the organic acid was also measured α-amylase inhibitory activity of lactic acid of 40mg / ml. The results are shown in Table 28.

sample α-amylase inhibitory activity (%) Control: 1 mM Acarbose 67.69 YG-control 0.94 YG-Full Fermentation 4.88 YG-post fermentation 1.78 Lactic Acid Bacteria Fermentation-control 6.52 Lactic Acid Bacteria Fermentation-Pre Fermentation 11.59 Lactic Acid Bacteria Fermentation-Post Fermentation 16.52 Lyophilized products of lactic acid bacteria fermentation-control 2.21 Lyophilized Products of Lactic Acid Bacteria Fermentation-Prefermentation 5.19 Lyophilized Lactic Acid Bacteria Fermentation-Post Fermentation 23.82 40mg / ml Lactic acid pH 7.0 0.65

As shown in Table 28, the α-amylase inhibitory activity was not observed even in YG even after the post-fermentation. Lactic acid also had no α-amylase inhibitory activity. However, in the case of lactic acid bacteria fermented products, α-amylase inhibitory activity was about 16.52% when inoculated before BST.L_601 inoculation and after fermentation, and there was almost no difference in α-amylase inhibitory activity even after lyophilization.

Experimental Example  4.1.2. α- glucosidase  Confirmation of inhibitory activity

YG medium was prepared by adding only 10 g / L yeast extract and 20 g / L glucose without adding sweet potato medium. In addition, three experimental groups in which YG medium, sweet potato medium was added thereto, and lyophilized were set. In addition, all of the α-glucosidase inhibitory activity was measured before the BST.L_601 inoculation, 3 days after inoculation at 37 ° C. and then 4 days at 4 ° C. after fermentation. In order to confirm the influence of organic acid, the α-glucosidase inhibitory activity of lactic acid of 40 mg / ml was also measured. The results are shown in Table 29.

sample α-Glucosidase Inhibitory Activity (%) Control: 1 mM Acarbose 69.44 YG-control 2.75 YG-Full Fermentation 0.35 YG-post fermentation 8.15 Lactobacillus fermentation control 26.17 Lactic Acid Bacteria Fermentation 30.74 After fermentation of lactic acid bacteria fermentation product 51.18 Lyophilized products of lactic acid bacteria fermentation-control 42.98 Lyophilized Products of Lactic Acid Bacteria Fermentation-Prefermentation 41.11 Lyophilized Lactic Acid Bacteria Fermentation-Post Fermentation 54.81 40mg / ml Lactic acid pH 7.0 5.54

As shown in Table 29, when inoculated with BST.L_601 in YG medium, α-Glucosidase inhibitory activity was increased to about 8% during post-fermentation, but the effect was minimal. In addition, about 25% of α-Glucosidase inhibitory activity in lactic acid bacteria fermented sweet potato medium without BST.L-601 inoculation, however, α-Glucosidase inhibitory activity is about 51% and 20% when it proceeds to post-fermentation. It was confirmed that the degree increased.

Experimental Example  5. Lactobacillus Fermented product  Determination of Total Polyphenol Content

The determination of the total polyphenol content was carried out in the following manner. 0.1 ml of the sample was prepared in a 15 ml conical tube, and 2 ml of 7% Na ₂ CO ₃ solution was added to each tube and reacted for 3 minutes at room temperature. Thereafter, 0.1 ml of 1N Folin-Ciocalteu's phenol reagent was added and reacted at room temperature for 30 minutes. The absorbance was measured at 750 nm and the total polyphenol content was measured by substituting the calibration curve prepared as a standard. At this time, gallic acid was used as a standard of total polyphenol content. BST.L-601 was inoculated into YG medium and the same composition as Preparation Example 2, followed by pre-fermentation (fermentation at 37 ° C. for 3 days) and post-fermentation (fermentation at 4 ° C. for 4 days), followed by total polyphenol content. Was measured. The results are shown in FIG.

Referring to FIG. 2, the content of YG medium was generally low, but the total polyphenol content was increased by about 2 times compared to the control and pre-fermentation in the post-fermentation. In addition, as a result of comparing the total polyphenol content of the fermented product after control and pre-fermentation and post-fermentation of BST.L-601 in the sweet potato medium, the sweet potato medium itself contained about 1,000 μg / ml of total polyphenols. And it was confirmed that the total polyphenol content was similar until the pre-fermentation. On the other hand, when proceeding to the post-fermentation it was confirmed that the total polyphenol content is increased by about 2.5 times to about 2,500 ㎍ / ml.

Experimental Example 6. Measurement of lipase inhibitory activity of lactic acid bacteria fermentation

Lipase inhibitory activity of the lactic acid bacteria fermentation can be measured by the following method. First, 10 μl of the enzyme solution prepared by diluting the lipase stock solution 1000 times in 0.1 mM potassium buffer solution (pH 6.0), 100 μg / ml in distilled water, and 50 μl of the control group (100 μg / ml orlistat) and blank test (distilled water) 30 μl of 0.1 mM potassium buffer solution (pH7.2, containing 0.1% tween 80) was added to a 96well microplate and reacted at 30 ° C. for 1 hour. Next, 10 μl of a 10 mM p-NPC (para-nitrophenyl caprylate) solution is mixed and enzymatically reacted at 30 ° C. for 5 minutes. Thereafter, the absorbance was measured at 405 nm and the inhibitory activity was calculated as follows in comparison with the control group. The results are shown in Table 30.

Blank test: Absorbance of blank test solution (405 nm)

Sample: Absorbance of Sample Reaction Liquid (405 nm)

sample Lipase Inhibitory Activity (%) Orlistat (100µg / ml) 36.657 YG (stock) 0 Lactic Acid Bacteria Fermentation (100mg / ml) 34.778

As a result of measuring the lipase inhibitory activity of lactic acid bacteria sweet potato fermentation, 100 ㎍ / ml Orlistat showed a lipase inhibitory activity of about 36.65%. There was no. However, it was confirmed that the lactic acid bacteria fermentation product of 100mg / ml showed lipase inhibitory activity of about 34.77%. In other words, it has a lipase inhibitory activity similar to that of Orlistat of 100 µg / ml as a control group.

Experimental Example  7. Lactobacillus Fermented products  Supernatant dry matter and Fermented product On fat precursor cells  For cytotoxicity check

Experimental Example  7.1. Lactobacillus Fermented products  Before eruption of supernatant dry matter On fat precursor cells  For cytotoxicity check

10³ cell/well로 분주하였다. 제조예 3의 유산균 발효물 상등액 건조물을 상기의 동일한 배지에 100, 50, 25, 12.5, 6.25, 3.13, 1.56 mg/ml로 용해하여 0.22㎛ filter로 여과하였다. 24, 48, 72시간 동안 처리한 뒤, MTT assay를 통해 세포 독성을 확인하였다. 그 결과를 도 3에 나타내었다.3T3-L1 adipocytes were treated with DMEM (Dulbecco's modified Eagle's medium) medium containing 10% Fetal bovine serum (FBS) and 1% Penicillin-streptomycin (P / S) at 37 ° C and 5% CO ₂ . The incubator was incubated. Then, 3T3-L1 adipocytes in a 96 well plate 1

Aliquots were performed at 10 ³ cell / well. Lactic acid bacteria fermented product supernatant of Preparation Example 3 was dissolved in 100, 50, 25, 12.5, 6.25, 3.13, 1.56 mg / ml in the same medium and filtered with a 0.22㎛ filter. After 24, 48, and 72 hours of treatment, cytotoxicity was confirmed by MTT assay. The results are shown in FIG.

As can be seen in Figure 3, in the non-differentiated 3T3-L1 adipocytes treated with lactic acid bacteria fermented product supernatant dry 24 hours after 25 mg / ml began to appear, after 48 hours even at 1.56 mg / ml Cell death of 20% was observed, showing cytotoxicity in a concentration- and time-dependent manner.

Experimental Example  7.2. Lactobacillus Fermented products  After eruption of supernatant dry matter On fat precursor cells  For cytotoxicity check

10³ cell/well로 분주하였다. 2일 후 배지를 교환하여 1-2일간 배양하여 세포가 완전히 융합되어 자랐을 때, 10% FBS와 MDI solution (0.5mM 3-iso butyl-1-methylxanthine, 1μM dexamethasone, 5 ㎍/㎖ insulin)을 포함하는 DMEM 배지로 배지를 교환하여 분화 유도를 시작하였다. 분화 유도 시작 2일 후 10% FBS와 insulin이 포함된 DMEM 배지를 2일 간격으로 배지를 교환하였다. 이후, 6일간 더 배양하여 지방세포로 분화를 유도하였다. 지방세포로 분화한 3T3-L1 세포에 유산균 발효물 상등액 건조물을 100, 50, 25, 12.5, 6.25, 3.13, 1.56 mg/ml로 24, 48, 72시간동안 처리하였다. MTT assay를 통해 세포 독성을 확인하고 그 결과를 도 4에 나타내었다.To determine the cytotoxicity of 3T3-L1 differentiated into adipocytes, 3T3-L1 cells were collected in 96 well plates.

Aliquots were performed at 10 ³ cell / well. After 2 days, medium was exchanged and cultured for 1-2 days. When cells were fully fused and grown, they contained 10% FBS and MDI solution (0.5mM 3-iso butyl-1-methylxanthine, 1μM dexamethasone, 5 ㎍ / ml insulin). Induction of differentiation was initiated by changing the medium with DMEM medium. Two days after the differentiation induction, DMEM medium containing 10% FBS and insulin was exchanged every two days. After that, the cells were further cultured for 6 days to induce differentiation into adipocytes. 3T3-L1 cells differentiated into adipocytes were treated with lactic acid bacteria fermented product supernatant dried at 100, 50, 25, 12.5, 6.25, 3.13, 1.56 mg / ml for 24, 48 and 72 hours. The cytotoxicity was confirmed by MTT assay and the results are shown in FIG. 4.

Similar to undifferentiated 3T3-L1 cells as in FIG. 4, toxicity began to appear from 25 mg / ml after 24 hours. Although cytotoxicity was shown to be concentration-dependent, the cytotoxicity was no longer increased after 48 hours, and cell viability of 51.15% was shown at a concentration of 25 mg / ml even after 72 hours of treatment.

Experimental Example  7.3. Sterilized Lactobacillus Fermented product  Before eruption On fat precursor cells  For cytotoxicity check

The sterilized lactic acid bacteria fermentation product prepared in Preparation Example 4 was dissolved in DMEM medium at 50, 10, 5, 1, 0.5, 0.25 mg / ml. The 3T3-L1 adipocytes were treated for 24 hours to confirm the toxicity of each cell in the same manner as in Experimental Example 7.1. The results are shown in FIG.

In the case of non-fermented sweet potato medium, the treatment of 5 mg / ml or less did not show cytotoxicity, and the lactic acid bacteria fermentation did not show cytotoxicity when treated under 1 mg / ml in pre- and post-fermented products. .

Experimental Example  7.4. Sterilized Lactobacillus Fermented product  After eruption On fat precursor cells  For cytotoxicity check

The sterilized lactic acid bacteria fermented product prepared in Preparation Example 4 was dissolved in DMEM medium at 50, 10, 5, 1, 0.5, 0.25 mg / ml. This was treated with differentiated 3T3-L1 adipocytes for 24 hours to confirm the toxicity to each cell in the same manner as in Experimental Example 7-2. The result is shown in FIG.

As shown in Figure 6, when treated with the non-fermented sweet potato medium and pre-fermentation and post-fermentation it was confirmed that the treatment does not exhibit cytotoxicity to 10mg / ml or less.

Experimental Example  8. Lactobacillus Fermented products  Supernatant dry matter ( Production Example  3) and sterile lactic acid bacteria Of fermented product (manufacturing example 4)  Anti-obesity effect confirmed

Based on the results of Experimental Example 7, a test for confirming the effect of fat formation of lactic acid bacteria fermentation was performed.

Experimental Example  8.1. Lactobacillus through Oil Red O staining Fermented products  Confirmation of Adipocyte Differentiation Inhibitory Activity of Dried Supernatant (Microscope)

The supernatant powder of the lyophilized lactic acid bacteria fermentation in Preparation Example 3 was dissolved in DMEM medium at 12.5 mg / ml and treated together when the cells were differentiated to differentiate. Cultures were removed from 3T3-L1 that induced differentiation and washed twice with PBS. Next, 4% paraformaldehyde was treated for 60 minutes at room temperature to fix cells. After the fixation of the cells, paraformaldehyde was removed and washed again with PBS three times. Oil red O solution dissolved in 0.5% in 60% isopropanol was treated. After staining the cells by treatment for 1 hour at 37 ℃, washed again three times with PBS and observed under a microscope.

As a result of observing the cells not treated with the lactic acid bacteria fermented product and the treated cells under a microscope 100 times, as shown in FIG. 7, the cells not treated with the lactic acid bacteria fermented product supernatant were generally differentiated, but the lactic acid bacteria fermentation was large. In the case of cells treated with water supernatant dry matter, the number of differentiated cells was relatively low.

Experimental Example  8.2. Lactobacillus through Oil Red O staining Fermented products  Adipocyte Differentiation of Supernatant Dried Inhibitory ability  Confirmation (absorbance)

In addition, after the microscopic observation in Experimental Example 8.1, 100% isopropanol was treated to lyse the cells. In comparison with the absorbance at 500 nm, the ability to inhibit adipocyte differentiation is shown in FIG.

As a result, it can be seen that the fat cells treated with the lactic acid bacteria fermented product supernatant dried by about 30% less, the lactic acid bacteria fermented product supernatant dried inhibits the formation of fat cells, which has an anti-obesity effect.

Experimental Example  8.3. Lactobacillus Fermented products  Determination of the Lipid Formation Factor Inhibitory Effect of the Supernatant Dried

, PPARγ, FABP4의 발현을 비교하였다.Adipocytes cultured by inducing differentiation in Experimental Example 8.1 were washed once with PBS. Then, all cells were recovered and suspended in RIPA buffer. After eluting for 30 minutes, the mixture was centrifuged at 15,000 rpm for 30 minutes. The supernatant was quantified by Bradford assay and the same amount was loaded by 30 ㎍. C / EBPa, C / EBP via electrophoretic western blots on SDS PAGE

, PPARγ, FABP4 expression was compared.

, PPAR

, FABP4 등의 발현 억제를 확인할 수 있었다. 이는 유산균 발효물 상등액이 지방세포의 분화를 억제함으로써 궁극적으로 항비만 효과를 나타낼 수 있는 것을 확인한 것이다.As shown in Figure 9, when treated with the lactic acid bacteria fermented supernatant, fat cell differentiation factors C / EBPa, C / EBP

, PPAR

, FABP4 and other expression inhibition was confirmed. This confirms that the lactic acid bacteria fermented supernatant can ultimately have an anti-obesity effect by inhibiting the differentiation of fat cells.

Experimental Example  8.4. Lactobacillus sterilized by Oil Red O staining Fermented product  Adipocyte differentiation Inhibitory ability  OK (microscope)

The sterilized lactic acid bacteria fermented product prepared in Preparation Example 4 was performed in the same manner as in Experiment 8.1 except that 0.5 mg / ml and 0.25 mg / ml were dissolved in DMEM medium. Cells treated with lactic acid bacteria fermentation at 0.5 mg / ml were observed under a microscope.

As shown in FIG. 10, cells that did not process the sterilized lactic acid bacteria fermentation were generally differentiated, but in the case of cells treated with the sterilized lactic acid bacteria fermentation, the number of differentiated cells was relatively small. there was.

Experimental Example  8.5. Lactobacillus sterilized by Oil Red O staining Fermented product  Adipocyte differentiation Inhibitory ability  Confirmation (absorbance)

After observing the microscope in Experimental Example 8.4, cells were lysed by treatment with 100% isopropanol. In comparison with the absorbance at 500 nm, the ability to inhibit adipocyte differentiation is shown in FIG.

As a result, it was confirmed that the cells differentiated by treating the sterilized lactic acid bacteria fermentation at 0.5 mg / ml inhibited the differentiation of about 29% adipocytes compared to the cells which were not treated. In addition, it was confirmed that the cells differentiated by treating the sterilized lactic acid bacteria fermented product at 0.25 mg / ml exhibited about 15% adipocyte differentiation inhibitory ability.

Experimental Example  8.6. Lactobacillus Fermented product  Confirmation of inhibitory effect on the expression of fat forming factor

Western blot was performed in the same manner as Experimental Example 8.3 for adipocytes cultured by inducing differentiation in Experimental Example 8.4.

As shown in Figure 12, when the lactic acid bacteria fermented product supernatant of Preparation Example 3, it was confirmed that the suppression of the expression of fat cell differentiation factors C / EBPa, C / EBB, PPARγ, FABP4. This is similar to the lactic acid bacteria fermentation supernatant, it can be seen that even when the sterilized lactic acid bacteria fermentation product of Production Example 4, that is, the whole of the lactic acid bacteria fermentation, the differentiation of adipocytes is similarly suppressed.

Experimental Example  9. Lactobacillus Fermented products  Supernatant dry matter ( Production Example  3) and sterile lactic acid bacteria Of fermented product (manufacturing example 4) Antidiabetic  Check the effect

On the basis of the results of Experimental Example 7 was confirmed the antidiabetic effect of the lactic acid bacteria fermentation.

Experimental Example  9.1. Lactobacillus through Glucose uptake Fermented products  Supernatant Antidiabetic  Check the effect

10³ cell/well로 분주하였다. 2일 후 배지를 교환하여 1-2일간 배양하여 세포가 완전히 융합되어 자랐을 때, 10% FBS와 MDI solution (0.5mM 3-iso butyl-1-methylxanthine, 1μM dexamethasone, 5 ㎍/㎖ insulin)을 포함하는 DMEM 배지로 배지를 교환하여 분화 유도를 시작하였다. 분화 유도 시작 2일 후 10% FBS와 insulin이 포함된 DMEM 배지를 2일간격으로 배지를 교환하여 6일간 더 배양하여 지방세포로 분화를 유도하였다. Incubator of 3T3-L1 adipocytes with 37 ° C and 5% CO2 in DMEM (Dulbecco's modified Eagle's medium) medium with 10% Fetal bovine serum (FBS) and 1% Penicillin-streptomycin (P / S) Incubated at. To induce differentiation of 3T3-L1 adipocytes, the cells were placed on 96 well plates.

Aliquots were performed at 10 ³ cell / well. After 2 days, medium was exchanged and cultured for 1-2 days. When cells were fully fused and grown, they contained 10% FBS and MDI solution (0.5mM 3-iso butyl-1-methylxanthine, 1μM dexamethasone, 5 ㎍ / ml insulin). Induction of differentiation was initiated by changing the medium with DMEM medium. Two days after the induction of differentiation, DMEM medium containing 10% FBS and insulin was exchanged for 2 days and cultured for another 6 days to induce differentiation into adipocytes.

_ex=460 nm,

_em=530 nm에서 형광도를 측정(2-NBDG)하여 glucose uptake를 도 12에 나타내었다.Differentiated adipocytes were cultured for 4 hours by replacing the medium with DMEM medium that did not contain glucose. Here, the lactic acid bacteria fermented product supernatant was mixed at 50 mg / ml, 25 mg / ml, and 12.5 mg / ml, respectively. Glucose-containing DMEM medium was treated for 14 hours. As a control, 100 uM of insulin was treated for 30 minutes. Each cell was washed twice with PBS followed by 100 μl / well of 50 uM 2-NBDG. Then, after incubation for 30 minutes in a CO ₂ incubator at 37 ℃, washed three times with cold PBS. 70 μl of 0.1 M potassium phosphate buffer (pH 10) containing 1% triton X100 was added to each well for 10 minutes. Next, 30 μl of DMSO was added to digest the cells by pipetting.

_ex = 460 nm,

_The glucose uptake is shown in FIG. 12 by measuring the fluorescence at _em = 530 nm (2-NBDG).

As can be seen in Figure 13,  25 mg / ml lactic acid bacteria fermented product supernatant It can be seen that the average appeared about 210% at 14 hours after treatment. This is similar to the glucose uptake of about 198.6% in the control group (insulin). After 24 hours, about 270% glucose uptake was confirmed to have a relatively high antidiabetic effect.

Experimental Example  9.2. Glut4  Lactobacillus through the difference in expression Fermented products  Supernatant Antidiabetic  Check the effect

Adipocytes cultured by differentiation were treated with a supernatant of 25 mg / ml lactic acid bacteria fermented product for 24 hours. After washing once with PBS, all the cells were recovered and suspended in RIPA buffer. After eluting for 30 minutes, it was centrifuged for 30 minutes at 15,000 rpm. Supernatants were quantified via a Bradford assay. Equivalent amounts of 30 μg were compared by expression of GLUT4 via electrophoretic western blot on SDS PAGE.

As shown in FIG. 14, Glut4 was expressed higher than general differentiated cells. This resulted in the expression of a large amount of Glut4 compared to the insulin treated as a control, and reconfirmed the antidiabetic effect of the dried supernatant of lactic acid bacteria.

Experimental Example  9.3. Lactobacillus sterilized by Glucose uptake Fermented product Antidiabetic  Check the effect

Experiments were carried out in the same manner as in Experiment 9.1 except that the sterilized lactic acid bacteria fermented product prepared in Preparation Example 4 were 3T3-L1 adipocytes induced by differentiation by mixing 10, 5, and 1 mg / ml in DMEM medium, respectively. Was performed.

As a result, as shown in Figure 15, when treated with 10 mg / ml sterilized lactic acid bacteria fermentation was an average of about 230% glucose uptake after 14 hours, it was confirmed that up to 250% glucose after 24 hours. When treated with 5 mg / ml and 1 mg / ml, glucose uptake was not significantly increased until 14 hours, but glucose uptake was observed up to about 170-200% after 24 hours. This is a similar effect to about 250% uptake when treated with 100 nM insulin as a control for 30 minutes, and it was confirmed that it has a relatively high antidiabetic effect.

Experimental Example  9.4. Glut4  Sterilized Lactobacillus with Different Expressions Fermented product Antidiabetic  Check the effect

The experiment was conducted in the same manner as Experimental Example 9.4, except that the sterilized lactic acid bacteria fermented product of 10mg / ml was treated for 24 hours.

As shown in FIG. 16, as a result of comparing the expression of Glut4, a glucose transporter, Glut4 was expressed higher than that of normal differentiated cells. That is, the antidiabetic effect of the sterilized lactic acid bacteria fermentation was reconfirmed by confirming that Glut4 was expressed in an amount similar to that of the control treated with insulin.

Korea Research Institute of Bioscience and Biotechnology KCTC13517BP 20180427

Claims (17)

Lactobacillus rhamnosus strain deposited with accession number KCTC 13517BP.
Lactic acid bacteria fermented product fermented with the strain of claim 1.
The method of claim 2,
The lactic acid bacteria fermented product is a fermented sweet potato, lactic acid bacteria fermented product.
The method of claim 3,
The sweet potato is to improve the preservation of lactic acid bacteria when lyophilized, lactic acid bacteria fermented product.
The method of claim 3,
The polyphenol content of the lactic acid bacteria fermentation is lactic acid bacteria fermentation 1.5 times to 4 times the polyphenol content of sweet potato before fermentation.
The method of claim 2,
The lactic acid bacteria fermented product will inhibit the activity of at least one of α-amylase, α-glucosidase and lipase, lactic acid bacteria fermented product.
The method of claim 2,
The lactic acid bacteria fermentation product is C / EBPα (CCAAT-enhancer-binding protein α), C / EBPβ (CCAAT-enhancer-binding protein β), PPARγ (peroxisome proliferator activated receptorγ), adiponectin (FAdi4) and FABP4 (fatty acid binding protein) To inhibit the expression of at least one of 4), lactic acid bacteria fermentation.
The method of claim 2,
The lactic acid bacteria fermentation is to increase the expression of GLUT4 (Glucose transporter type 4), lactic acid bacteria fermentation.
The method of claim 2,
The lactic acid bacteria fermented product is lyophilized, lactic acid bacteria fermented product.
The method of claim 2,
The lactic acid bacteria fermentation is sterile, lactic acid bacteria fermentation.
A pharmaceutical composition for preventing or treating diabetes or obesity comprising the lactic acid bacteria fermentation product according to any one of claims 2 to 10 as an active ingredient.
A health functional food for preventing or improving diabetes or obesity, comprising the fermented lactic acid bacterium according to claim 2 as an active ingredient.
Inoculating a composition comprising sweet potatoes with the Lactobacillus rhamnosus strain deposited with accession number KCTC 13517BP; And
Comprising the step of fermenting the composition, method for producing lactic acid bacteria fermentation.
The method according to claim 13,
Further comprising the step of sterilization before inoculation, the production method of the lactic acid bacteria fermentation.
The method of claim 14,
PH value before sterilization of the composition is 5 to 8, the production method of the lactic acid bacteria fermentation.
The method of claim 13,
The fermentation step, the fermentation step of the fermentation at a temperature of 20 ℃ to 40 ℃, the production method of the lactic acid bacteria fermentation.
The method of claim 13,
The fermentation step further comprises a post-fermentation step of fermentation at a temperature of 1 ℃ to 10 ℃, method of producing a lactic acid bacteria fermentation.

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