KR101907275B1 - Probiotics containing dendropanax morbifera extract and method preparing thereof - Google Patents

Abstract

The present invention relates to a lactic acid bacterium product containing a Hokkaido extract and a method for producing the same. The lactic acid bacterium product containing the extract according to the present invention exhibits an antioxidative effect, a fat accumulation inhibitory effect, an anti-inflammatory effect and an anti-hyperlipemic effect and can be easily prepared for ingestion.

Description

PROBIOTICS CONTAINING DENDROPANAX MORBIFERA EXTRACT AND METHOD PREPARING THEREOF FIELD OF THE INVENTION [0001]

The present invention relates to a probiotic agent containing an extract of Euphorbiaceae and a method for producing the same.

Lactic acid bacteria have been shown to have various physiological functions such as inhibiting intestinal decay, promoting intestinal motility, preventing constipation, increasing immunity, inhibiting carcinogenesis, and producing vitamin B group. It is also known that it produces a variety of metabolites such as acetic acid and lactic acid, organic acids such as bacteriocin, and the like to inhibit the growth of enteric bacteria and harmful pathogenic bacteria. Therefore, various studies are underway.

Probiotics (probiotics) are defined as 'live microorganisms that give health benefits to humans upon ingestion.' Lactobacillus, represented by Lactobacillus or Bifidobacterium, accounts for most of the probiotics, and in Korea It is recognized as a health functional food material that acts as a suit.

Dendropanax morbifera ) is an evergreen tree of the Araliaceae, a dicotyledonous plant native to the southern coast of Korea and Jeju Island. It is a species that does not support fallen leaves in the winter. It gives yellow resinous liquid when it bruises the bark. The physiological activities such as anticancer activity and antioxidative activity of the extract of Hwangchilchae have been reported to be limited, and they have been used in various fields such as cosmetic compositions and food additives.

Each study on probiotics products and woody trees has been continued, but it is necessary to develop a new method of manufacturing a product that is easy to ingest while exhibiting new functionality by fusing a useful woody plant extract and probiotics, and a new functional product manufactured by the method Do.

Korean Patent Publication No. 10-1544176

One aspect of the present invention is to provide a probiotic agent containing the extract of Hokkaido, and a method for producing the same.

One aspect of the present invention provides a probiotic agent containing an extract of Hokkaido tree.

One aspect of the present invention relates to a method for preparing an oil-in-water emulsion, comprising: mixing milk, Hwangryeok tree extract, glucose and skimmed milk powder; Inoculating and culturing the mixture with lactic acid bacteria; And lyophilizing the culture. The present invention also provides a method for producing a probiotic microorganism.

In one embodiment of the present invention, the concentration of the extract is 1 to 5% by weight.

In one embodiment of the present invention, the lactic acid bacteria are selected from the group consisting of Lactobacillus brevis , Lactobacillus fermentum , Lactobacillus plantarum , and combinations thereof It can be either.

One aspect of the present invention provides a probiotic agent produced by the above method.

One aspect of the present invention provides a lactic acid bacterial product comprising the above-mentioned probiotic agent.

According to the present invention, the probiotics containing Hokutogi extract exhibit an antioxidative effect, a fat accumulation inhibitory effect, an anti-inflammatory effect and an antihyperlipidemic effect, and the prophylactic agent exhibiting the above advantages according to the present invention is prepared in an easy form for ingestion can do.

Fig. 1 shows an embodiment of a method for producing a probiotic agent according to the present invention.
Fig. 2 shows the pH change of fermented broth according to the present invention with fermentation (culture) time.
Fig. 3 shows changes in acidity of the fermented broth according to the present invention with fermentation time.
FIG. 4 shows the free radical scavenging ability of the fermented broth according to the present invention according to the concentration of the extract.
FIG. 5 shows nitrite scavenging ability of the fermented product according to the present invention according to the concentration of the extract.
6 shows the cell survival rate according to the concentration of extract of Hwangchujang extract in the probiotic fermented product according to the present invention.
FIG. 7 shows the lipid content of the fermented broth according to the present invention, according to the concentration of the extract.
FIG. 8 shows changes in the amount (a) of triglyceride and the amount (b) of cholesterol according to the concentration of Hwigulak extract in the probiotic fermented product according to the present invention.
Fig. 9 shows the size of a clear zone in the fermentor product according to the present invention.

One aspect of the present invention provides a probiotic agent containing an extract of Hokkaido tree.

As used herein, the term "probiotic agent" is intended to encompass not only the lactic acid bacterium itself, but also a mixture comprising lactic acid bacteria, preferably a mixture of lactic acid bacteria and woodworm extract and a mixture comprising lactic acid bacteria and a mixture thereof, preferably a mixture of lactic acid bacteria and Water, but is not limited thereto. The probiotics may include other compounds, for example, a medium required for culturing the lactic acid bacteria, milk, milk powder, and adjuvants, but are not limited thereto.

One aspect of the present invention relates to a method for preparing an oil-in-water emulsion, comprising: mixing milk, Hwangryeok tree extract, glucose and skimmed milk powder; Inoculating and culturing the mixture with lactic acid bacteria; And lyophilizing the culture. The present invention also provides a method for producing a probiotic microorganism.

More particularly, one aspect of the present invention relates to a method of making a milk powder, comprising: mixing milk, woodchuck extract, glucose and skimmed milk powder; Sterilizing the mixture; Cooling the sterilized mixture with ice, and then inoculating the lactic acid bacteria; And culturing the mixture inoculated with the lactic acid bacterium.

As used herein, the term " extract of Hwangchu-myeon tree "may be any one selected from the group consisting of roots, stems, flowers, leaves, fruits, and combinations thereof, or a stem, flower, , Fruit, and a combination thereof. The extract may be one selected from the leaves of Hwangchujang. In addition, the extract may be an extract or a fraction prepared to contain the active ingredient of Huaculi according to an extraction method or a fractionation method known in the art, and may be extracted with water, a lower alcohol or a mixed solvent thereof. And preferably water, more preferably purified water, but is not limited thereto. In the present specification, the Hokkaido extract can be extracted by adding 5 to 10 times of purified water to a dried weedy tree of a predetermined weight. In one embodiment of the present invention, the concentration of the extract is 1 to 10% by weight, preferably 1 to 5% by weight.

As used herein, the term "extract" means a preparation obtained by extracting a herbal medicine with an appropriate extraction solvent and concentrating the product by evaporating the extraction solvent. The extract is obtained by extracting, diluting or concentrating the extract, These can be controlled products or refined products. The extract according to the present invention can be prepared by using common extraction, isolation and purification methods known in the art. Examples of the extraction method include, but are not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

The lactic acid bacteria described herein may be of the genus Lactobacillus or Bifidobacterium and preferably include Lactobacillus brevis , Lactobacillus fermentum and Lactobacillus plantarum . , But is not limited thereto.

One aspect of the present invention provides a probiotic agent produced by the above method.

One aspect of the present invention provides a lactic acid bacterial product comprising the above-mentioned probiotic agent. The lactic acid bacteria product includes all forms such as food composition, functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

The "functional food" described in the present specification can be prepared by preparing the probiotics of the present invention in the form of tea, juice and drink, drinking them, granulating, encapsulating and pulverizing them, desirable. In addition, the probiotics of the present invention may be mixed with other active ingredients to form a composition.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.) by adding the probiotics of the present invention.

In order to use the probiotics of the present invention in the form of food additives, they may be used in the form of powders or concentrates.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these embodiments are illustrative of one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example 1. Preparation of extracts of Woodenwood

The wood is mixed with water and 1: 1 to 1:10 parts by weight. The mixture is placed in a high pressure autoclave and heated at a temperature of 80 to 120 ° C for 5 to 30 minutes, Extracted for 13 hours, filtered and concentrated.

Example 2. Establishment of optimal production conditions for probiotics

2.1. Cultivation of lactic acid bacteria

Lactobacillus brevis , Lactobacillus fermentum and Lactobacillus plantarum were inoculated on the MRS agar and MRS broth medium of the composition shown in Table 1 to obtain 37 And cultured in an incubator for 24 hours.

* Final pH (25): 6.5 ± 0.2

2.2. Production of fermented milk and freeze-drying of fermented product

1% of glucose and 1%, 3%, and 5% of sterilized Hwangchujang extract were added to 500 ml of milk, and three kinds of lactic acid bacteria were inoculated and cultured for 48 hours in an incubator of 37 to prepare an active fermented product.

Freeze-drying of the fermented product was carried out under the conditions shown in Table 2 below.

2.3. Observation of pH change

Table 3 shows the results of measurement of the pH change of the fermented broth fermented product after the addition of the extract. The fermentation time of the probiotics decreased with increasing incubation time, and the pH of the fermented broth was lower than that of the control. The pH changes of the strains used showed that the L. brevis and L. fermentum strains had shorter fermentation times than those of the L. plantatum strains (see FIG. 2).

2.4. Observation of acidity change

Table 4 shows the results of measurement of the change in titratable acidity with time of the fermented product of the probiotics prepared by adding the extract of Tochigi keitii. As the pH was changed, the titratable acidity increased with the incubation time, and the higher the acidity, the higher the acidity was. L. fermentum showed the highest acidity, while L. brevis and L. plastarium showed no significant difference. Therefore, it was confirmed that the addition of the extract of Hwangchujang extract in the production of the probiotic fermented product improves the growth of lactic acid bacteria and the production of lactic acid, thereby shortening the culturing time and advantageously producing useful metabolites (see FIG. 3).

2.5. Homogenate Fermentation product  Yield measurement

Table 5 shows the results of the lyophilization after the lyophilized fermented product with the extract of Hwangchujang extract. That is, the yield of the fermented product obtained by culturing L. plutarium was highest at 13.96% by adding 5% extract, but the fermented product was not different from the fermented product of L. brevis and L. fermentum. Therefore, it was confirmed that the addition amount of the extract did not greatly affect the yield of the fermented product according to the strain.

2.6. Measure the number of lactic acid bacteria

As a result of measuring the number of lactic acid bacteria in the freeze-dried biocide material by mass culture, 1,350,000,000 cfu / g of lactic acid bacteria were confirmed and it was confirmed that it was suitable as a material for producing the fermented probiotic product.

EXPERIMENTAL EXAMPLE 1. Antioxidative Effect Analysis of Wooden Extracts

In order to confirm the antioxidative effect of the extract of the extract of the present invention, (i) the effect of 1,1-diphenol-2-picrylhydrazyl (DPPH) And (ii) nitrite scavenging ability.

1.1. 1,1- Diphenol -2- Picrylhydrazyl (1,1- diphenol -2- picrylhydrazyl, DPPH ) Radical elimination efficacy

The free radical, 1,1-diphenol-2-picrylhydrazyl (DDPH), is purple and shows maximum absorbance at 515 nm, but reacts with a substance with free radical scavenging activity The antioxidant effect of the extract of the present invention was measured using the fact that the absorbance value was lowered when it was yellow.

DPPH was dissolved at the concentration of 5, 10, 50, 100, 300 and 500 ㎍ / ㎖ in order to examine the free radical scavenging effect of DPPH in the extracts of DPPH. The control group, ascorbic acid (vitamin C) showed a scavenging ability of 89% at 100 μg / ml concentration, whereas the probiotics containing Hokutogi extract showed 12% free radical scavenging ability (see FIG. 4). Probiotics containing Huangchil extract showed significantly lower free radical scavenging ability than ascorbic acid, but increased with increasing concentration.

1.2. Nitrite scavenging efficacy

Nitrite is known to react with amines present in meat products, medicines and pesticides to produce nitrosamines, which are known to induce cancer. Thus, nitrite scavenging activity is indirectly measured by measuring the inhibitory effect of nitrosamines Is used as an indicator.

1 ml of the extract of the present invention was added to 1 ml of a 1 mM NaNO ₂ solution and the pH of the mixture was adjusted to 1.2 with 0.1 N HCl (pH 1.2) to make the total volume to 10 ml. The reaction was allowed to stand for 1 hour. 0.4 ml of Griess reagent was added to 1 ml of the reacted solution and mixed well. This was allowed to stand at room temperature for 15 minutes, and the absorbance was measured at 520 nm using a microplate reader to measure the amount of remaining nitrous acid. The blank test was carried out by adding 0.4 ml of distilled water instead of the Greek reagent. Nitrite scavenging activity was expressed as percentage with and without addition of sample.

As a result, the absorbance of L-ascorbic acid as a positive control group was 26% at 1 mg / ml, compared with 9% as a control group (see FIG. 5).

Experimental Example 2. Analysis of inhibitory effect of fat accumulation

2.1. Cell viability measurement

MTT analysis was performed to determine the cytotoxicity of the present invention. Cells were seeded in 96-well plates at a density of 4 × 10 ³ cells / well and cultured for 24 hours. The samples were treated at 0, 5, 10, 50, 100, 150, 300, 500, 1000 μg / Time. The MTT solution is added to each well and reacted for 4 hours. After removing the MTT solution and adding DMSO, the absorbance was measured at 540 nm using an ELISA reader 5 minutes later, and the result was calculated as a ratio to the control value.

As a result, the cell viability was decreased to 85% from 1000 μg / ㎖, although the viable cell count did not show a significant change in cell viability up to 500 μg / ㎖. On the other hand, the survival rate of cellulase extracts containing Hwacheon-gil extract decreased from 500 ㎍ / ㎖ to 79% at 1000 ㎍ / ㎖, although the cell viability did not change significantly up to 300 ㎍ / ㎖. Therefore, treatment was carried out at a concentration of 300 μg / ml or less for the effect of inhibiting fat accumulation and related treatment.

2.2. Induction of differentiation of 3T3-L1 adipose precursor cells

In the 3T3-L1 cell line, when inducers such as DMI (Dexamethasone + IBMX + Insulin) are added to the medium, the growth is stopped and the differentiation into adipocytes begins and the intracellular lipids accumulate. Differentiation was induced.

In order to differentiate 3T3-L1 cells into adipocytes, the cells were divided into 6-well plates at a density of 1 × 10 ⁵ cells per well. When the cells were confluent at 80-90%, 5 μg / ml insulin, 1 μM DEX, 0.5 mM IBMX and 10% FBS to induce differentiation (see Table 6 below). Two days later, the cells were exchanged with DMEM medium containing 5 μg / ml insulin and 10% FBS. After 2 days, the cells were supplemented with DMEM medium supplemented with 10% FBS for 4 days.

2.3. Oil Red  O dyeing

The triglycerides present in the lipids are produced at the stage of differentiation from 3T3-L1 lipoproteins into adipocytes. The triglyceride produced in the cells continues to form fat globules until the end of differentiation. As time goes by, fatty globules are bound together and their size gradually increases. The amount of fat globules produced was determined by Oil Red O (O) staining method in order to confirm whether the probiotics containing Huangchil extract were effective in inhibiting lipid accumulation.

The formalin solution was added to the 6-well plate, fixed for 30 minutes, and washed once with distilled water. After treatment with 0.3% Oil Red O solution for 1 hour, it was washed once with 60% isopropanol, and the degree of fat accumulation was observed under a microscope using a microscope.

As a result, the probiotics decreased from 300 and 500 μg / ㎖ to 94% and 90%, respectively, and the probiotics containing Hokutogi extract decreased to 98, 89 and 79% from 100, 300 and 500 ㎍ / ㎖, respectively (see FIG. 6) .

2.4. Triglyceride content

The triglyceride content in the cells was measured to determine the decrease of triglyceride constituting the lipid sphere.

Intracellular triglycerides were performed according to the assays as indicated in the commercially available Assay kit. The samples were treated with different concentrations of 3T3-L1 and HepG2 cells, washed twice with PBS, and incubated at room temperature for 20 minutes with 20 μl of AdipoRed reagent, followed by ELISA reader And the results were calculated as a ratio to the control value.

As the results of oil red O, the content of triglycerides decreased with increasing concentration of extract. The contents of triglycerides were decreased to 101, 100, 95 and 89% at 50, 100, 300 and 500 ㎍ / ㎖, respectively, compared to the cells treated with DMI alone. When the probiotics were treated, the contents of triglycerides were reduced to 99, 98, 90 and 82%, respectively (see Fig. 8 (a)).

2.5. Cholesterol content

The cholesterol content was determined according to the quantitative method indicated in the commercially available assay kit. Cell lysate was prepared by washing the cells with the concentration of each sample in a concentration-dependent manner. The cell lysate was prepared according to the kit assay, and then measured using an ELISA reader. The result was calculated as a ratio to the control value.

As a result of measuring the cholesterol content produced after treating the cholesterol accumulated in the adipocyte under the same conditions as the treatment conditions in the oil red 0 staining, the lipid differentiation inducing cells treated with the probiotics showed 50, 100 , 99, 96, and 89% at 300 and 500 μg / ml, respectively, and decreased to 100, 97, 89, and 83%, respectively, when the differentiation-inducing cells were treated with the probiotics containing Huangchil extract (B)).

Experimental Example 3. Analysis of anti-hyperlipidemic effect

3.1. Tributynin plate assay

To investigate the antihyperlipidemic effect of probiotics containing probiotics and yellowtail extracts, the lipid degradation and inhibitory effects of active substances were investigated.

A 1% tributyrin solution was diluted in an appropriate amount to prepare a tributyrin plate with a final volume of 5 ml together with a 2% agar solution. The lipolytic activity of the sample was analyzed by measuring the size of the clear zone by reacting various concentrations of the sample at 37 ° C for 1 to 3 hours by making holes of 3 mm in diameter on the tributynin plates 9).

3.2. Lipolytic activity assay

To investigate the antihyperlipidemic effects of probiotics and Huangchil extract - containing antibiotics, lipid - degradation activity and lipolytic activity were investigated using lipolytic activity assay.

spectrophotometric analysis was performed using a substrate (10 mM) of p-nitrophenyl butyrate (PNPB). (Molecular Devices, Sunnyvale, Calif., USA) was used for analysis of p-nitrophenol (PNP) at 405 nm for 10 minutes, And compared with lipase (lipid-degrading substance) (positive control group).

The efficacy of the group treated with 25 μg of lipase in the positive control group was 1.26 V _max (? Mol / min) and the efficacy of the negative control group was 0 V _max (mU / min). 0, 0, 0.024, 0.0.09, 0.13, 0.25, 0.67 V _max (mU / ml) at 5, 10, 15, 25, 50, 100, 250 and 500 ㎍ of probiotics, respectively. 0, 0, 0.048, 0.12, 0.21, 0.35, 0.48 V _max (mU / min) at 5, 10, 15, 25, 50, 100, ) Were obtained.

3.3. HMG CoA reductase inhibitory effect

Cholesterol synthesis is known to be controlled by feedback from the final product, according to many studies. The most important factors in the regulation of these are LDL receptor and HMG-CoA reductase. When the activity of the MG-CoA reductase is decreased, the activity of the LDL receptor is increased and it is reported that the blood cholesterol concentration is decreased.

100 μl of 0.5 uM phosphate buffer solution (pH 7.0), 100 μl of 2 mM DTT, 100 μl of 0.5 mM β-NADPH, and 10 μl of HMG-CoA (pH 7.0) were added to 10 μl of each sample dissolved in distilled water 10 μl of reductase (liver of Sirian hamster, 10 mg protein / ml) was added, and the mixture was preheated at 37 ° C. for 5 minutes. Then, HMG CoA was added thereto, and the absorbance at 340 nm was measured while reacting for 3 minutes. Using these values, the inhibitory activity was calculated as follows. A control experiment (blank) was also carried out by adding distilled water instead of HMG-CoA:

As a result, the activity of the probiotics containing probiotics and Huangchil extract was found to be 24.01% and 13.03%, respectively, as measured by HMG-CoA reductase inhibitory activity.

Experimental Example  4. Anti-thrombosis  Effect analysis

4.1. Fibrin block analysis

In order to form a fibrin clot, the inhibitory activity against fibrin formation was analyzed by simultaneously reacting the mixture with all the materials and various concentrations of the sample or reacting with each material in advance at various times. After adding 90 μl of 1% human fibrinogen (prepared in 20 mM Tris-HCl (pH 7.4) containing 100 mM NaCl) to 10 μl of 0.5 U / ml thrombin and various concentrations of sample simultaneously or in advance , And the resulting mixture was reacted for a period of 1 hour with a few times of pyrotechnicity to measure the inhibitory effect by measuring the weight of the formed fibrin mass. Controls were prepared by reacting mixtures without added samples under the same conditions. For the analysis of the results, a negative control (a negative control with no treatment of fibrinogen, and a negative control with fibrinogen and thrombin alone formed a negative control), a positive control (a control group treated with u-PA under the same conditions as the experimental group) (A group treated with various concentrations when forming fibrin masses).

As a result of the experiment, it was confirmed that the fibrin block was strongly formed through the negative control group and that the inhibitory effect of the negative control group in which only human fibrinogen and human thrombin were reacted was 0%. In the positive control group, 20U t-PA, And inhibited the formation of granules. In the group treated with the probiotics, the effect of inhibiting fibrinogen formation was observed at about 0% at 10 μg, about 9% at 20 μg, and about 15% at 50 μg. In the group treated with the extract, the inhibitory effect on the formation of fibrinogen was observed at about 10%, about 20%, and about 50%, respectively.

4.2. Thrombin analysis

Thrombin assay is a method using human thrombin, which is a major coagulation factor for forming fibrin block, which is a major constituent of thrombus. It is used to evaluate the thrombin enzyme activity inhibition effect of samples by simultaneously treating or pretreating samples with thrombin .

In order to form fibrinogen into fibrin and analyze the inhibitory effect on thrombin, which is an important factor for the coagulation reaction, enzyme activity was measured using thrombin-synthesized substrate (S-2238) after pretreating the sample with thrombin. Samples of various concentrations and 0.25 U of thrombin were titrated to a final volume of 30 μl. After pretreatment for 10 minutes, 10 μl (4 mM) of thrombin synthesized substrate (S-2238) was reacted for 5 minutes and measured at 405 nm. The results were analyzed by positive control (positive control treated with thrombin only) and experimental group (treated with various concentrations of thrombin).

As a result, the OD ₄₀₅ value of thrombin-thrombin-specific substrate reacted with the thrombin-only positive control was 100% for thrombin enzyme activity. , 100% at 100%, 89% at 100 ㎍, 100% at 100 ㎍, 98% at 20 ㎍, 90% at 50 ㎍ and 81% at 100 ㎍, respectively.

4.3. Turbidity analysis

Turbidity assay is also an analytical method using human fibrinogen and human trombone, which are major factors for forming fibrin block, the main constituent of thrombus.

After the simultaneous treatment of the sample and the main factors of the fibrin formation, the inhibition activity of the sample was analyzed by real - time tracking of the density of the fibrin mass at 405 nm. 10 μl of 10 U / ml thrombin and various concentrations of sample were simultaneously added to 90 μl of 1% human fibrinogen (prepared in 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCl) Loaded onto a 96-well microplate and measured at 405 nm for 1 hour. Controls were prepared by reacting mixtures without added samples under the same conditions.

As a result, in the negative control group in which only human fibrinogen and human trombone were reacted, the fibrin mass density was continuously increased for 60 minutes, and in the positive control group in which 20 U t-PA and 4 mU plasmin were added to human fibrinogen and human thrombin After the start of the reaction, the density of the fibrin aggregates was increased, but it was confirmed that the subsequent formation was strongly inhibited. Compared with the fibrin layer density of the negative control group, the inhibitory effect was 16% at 50 μg in the probiotics and 29% at 50 μg in the probiotics containing the extract.

Example 3 Production of Product Using Probiotic Powder

3.1. Manufacture of yogurt candy

The recipes were selected to prepare lactic acid bacterium candies by mixing freeze-dried powder containing the extract of Hwangchujang extract and the ingredients (see Tables 7 to 9, unit weight%). Yogurt candy was prepared with three flavors including plain, blueberry, and strawberry flavor. The lactic acid bacteria content of lactic acid bacteria candy was set to be 100 million per gram of probiotic lactic acid bacteria.

Plain-1 Plain-2 Plain-3 Freeze-dried powder 23 Freeze-dried powder 27 Freeze-dried powder 30 Tapioca powder 4 Tapioca powder 4 Tapioca powder 4 Skim milk powder 3 Skim milk powder 3 Skim milk powder 3 Vitamin C 6 Vitamin C 8 Vitamin C 5 glucose 50 glucose 46 glucose 45 Xylitol 5 Xylitol 5 Xylitol 5 Whey protein 2 Whey protein 2 Whey protein 2 Yogurt flavor powder 7 Yogurt flavor powder 5 Yogurt flavor powder 6 Sum 100 Sum 100 Sum 100

 Lactobacillus candy 1 Lactobacillus Theory Lactobacillus content: 2.8 billion / 1 tablet = 20% lactic acid bacteria reduction at 2 years and 6 months of distribution = 1.1 million after 2 years / 1 party = Therefore, it is set to contain 100 million lactic acid bacteria per 1 Lactobacillus candy.

Strawberry-1 Strawberry-2 Strawberry-3 Freeze-dried powder 23 Freeze-dried powder 27 Freeze-dried powder 30 Tapioca powder 4 Tapioca powder 4 Tapioca powder 4 Skim milk powder 3 Skim milk powder 3 Skim milk powder 3 Vitamin C 6 Vitamin C 8 Vitamin C 5 glucose 50 glucose 46 glucose 45 Xylitol 5 Xylitol 5 Xylitol 5 Whey protein 2 Whey protein 2 Whey protein 2 Strawberry flavor powder 7 Strawberry flavor powder 5 Strawberry flavor powder 6 Sum 100 Sum 100 Sum 100

 Lactobacillus Candy 1 Lactobacillus Theory: Lactobacillus acid content: 2.8 billion / 1 tablet = 20% lactic acid bacteria reduction at 2 years and 6 months of distribution = 1.1 million after 2 years / 1 party = Therefore, it is set to contain 100 million lactic acid bacteria per 1 Lactobacillus candy.

Blueberry-1 Blueberry-2 Blueberry-3 Freeze-dried powder 23 Freeze-dried powder 27 Freeze-dried powder 30 Tapioca powder 4 Tapioca powder 4 Tapioca powder 4 Skim milk powder 3 Skim milk powder 3 Skim milk powder 3 Vitamin C 6 Vitamin C 8 Vitamin C 5 glucose 50 glucose 46 glucose 45 Xylitol 5 Xylitol 5 Xylitol 5 Whey protein 2 Whey protein 2 Whey protein 2 Blueberries
Fruit juice powder 7 Blueberries
Fruit juice powder 5 Blueberries
Fruit juice powder 6 Sum 100 Sum 100 Sum 100

 Lactobacillus candy 1 Lactobacillus Theory Lactobacillus content: 2.8 billion / 1 tablet = 20% lactic acid bacteria reduction at 2 years and 6 months of distribution = 1.1 million after 2 years / 1 party = Therefore, it is set to contain 100 million lactic acid bacteria per 1 Lactobacillus candy.

3.2. Measure hardness of prototype

The results of measuring the hardness of the prototype are shown in the table. The hardness of the prototype was 12,913.72 (± 1,247.25) g.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

Claims (6)

delete As a method for producing a probiotic agent,
Preparing a mixture by mixing milk, Hwangryeok tree extract, glucose and skim milk powder;
Sterilizing the mixture and ice-cooling the mixture;
Inoculating the ice-cooled mixture with lactic acid bacterium and culturing to produce a culture; And
Lyophilizing the culture
Lt; / RTI >
Wherein the Hwigyeolmu tree extract is prepared by mixing Hwangwolmyeo and water at a weight ratio of 1: 1 to 1:10 and extracting by hot water extraction method at a temperature of 80 to 120 DEG C for 5 to 13 hours.
3. The method of claim 2,
Wherein the concentration of the extract is 1 to 5% by weight.
3. The method of claim 2,
Wherein the lactic acid bacterium is any one selected from the group consisting of Lactobacillus brevis , Lactobacillus fermentum , Lactobacillus plantarum , and combinations thereof.
The probiotic agent produced by the method for producing a probiotic agent according to any one of claims 2 to 4.
A lactic acid bacterial product comprising the probiotic agent according to claim 5.

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