KR101341263B1 - Method for manufacturing fermented turmeric using a probiotic strain, Lactobacills johnsonii IDCC 9203 - Google Patents

Abstract

The present invention relates to a fermented turmeric manufacturing method, characterized in that the fermented turmeric powder can be fermented with Lactobacillus 9203 to produce fermented turmeric with increased active ingredient. The present invention is largely composed of three steps, fermentation process using turmeric powder and probiotic strains, active ingredient extraction process using alcohol, powder production process using starch and lyophilization.
The present invention configured as described above can increase the content of curcuminoid active ingredients, especially solubility of turmeric through the lactobacillus fermentation process of turmeric.
Through the present invention, it is possible to extend the use of turmeric, which has been limited in odor, taste, insolubility, etc., and can provide fermented turmeric excellent in efficacy.

Description

Method for manufacturing fermented turmeric using a probiotic strain, Lactobacills johnsonii IDCC 9203}

The present invention relates to a method for producing fermented turmeric which increases the content and bioavailability of curcuminoid, which is an active ingredient of turmeric, compared to turmeric extract, and removes the peculiar bitter taste and smell of turmeric through fermentation of turmeric using specific lactic acid bacteria. It is convenient to take and maximizes the content and activity of various active substances, including curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), which are known to have anti-inflammatory, anti-allergic and anti-cancer effects. By increasing their bioavailability, there is provided a method for producing fermented turmeric that can effectively inhibit / prevent various allergic diseases and increase its effect even when ingested in the same amount as general turmeric extract.

Turmeric is a perennial herbaceous herbaceous plant belonging to the ginger family Ginger, which grows and grows in Southeast Asia including South China, India and Okinawa. Dried roots of turmeric are called turmeric, which is used as a medicinal herb or as a spice because of its unique taste and aroma. The major components of turmeric are curcumin, which is bactericidal, antibacterial or anti-inflammatory, and has antioxidant and cytoprotective functions. The most common components are DMC and BDMC. In addition, essential oils include tameron, cineol, and alpha crumenmen, and minerals include calcium, potassium, iron, magnesium, phosphorus, and many other flavonoids and vegetable fiber. The pharmacological action of the turmeric is known to promote liver detoxification, bile secretion, anti-ulcer, suppressing blood cholesterol, etc., and is effective for hepatitis, jaundice, cholangitis, menstrual irregularities, high blood pressure, arteriosclerosis, and anti-cancer, anti-diabetic, and antibacterial effects. Effects are also well known. In recent years, it has been reported that Ulgum can prevent dementia (Alzheimer's disease). In fact, it is known that Indians who ingest curry daily have the lowest incidence of dementia worldwide, and the incidence rate is only 1/4 that of Americans . Curcumin is known to be one of the active ingredients in curry because it breaks down toxic proteins accumulated in the brain of Alzheimer's patients. Based on the results of these researches, researches on dementia and brain diseases are being actively conducted in Ulgum in many countries of the world.

Turmeric is hot and hot, improving blood circulation and penetrating the blockage. It has a unique dark color and bitter taste, and has been used as a natural dye since ancient times. Curcumin, widely known as an active ingredient of turmeric, is a very useful substance for the treatment of certain diseases, but it can also be used for indications such as common colds, various allergic diseases, and anti-inflammatory effects. Its use is getting wider. Most of the turmeric products listed to date are tea and beverages containing curcumin extracted from turmeric juice or turmeric powder and curcumin extracted from turmeric. However, when taking turmeric juice or turmeric powder, it can be inconvenient to take due to the bitter taste and aroma of turmeric, and the curcuminoid content in the product is extremely limited, so it cannot be effectively used. There are disadvantages. In addition, curcuminoids, which have various useful functions, are poorly bioavailable due to their insoluble properties in water, and their development into various formulations is limited. Therefore, the amount of curcuminoid that can be actually produced by the body when consumed on the market is very small and the expected effect is also reduced. In order to solve this problem, the effect of increasing the content of curcuminoid, the active ingredient of turmeric, through the fermentation of turmeric and at the same time increasing the bioavailability has more beneficial effects when compared with the same amount of turmeric extract. The effect can be greatly increased.

The unique dark color, aroma and bitter taste of turmeric are not suitable for taking turmeric directly or as an additive to health functional foods, and considering the limited content of curcumin, the active ingredient contained in turmeric, and the absorption rate in the body It is difficult to expect various effects expected for turmeric, such as anti-inflammatory activity, anti-allergic, anti-atopic activity, treatment and prevention of various diseases. Therefore, in order to solve the above problems, the fermentation was carried out using a specific lactic acid bacteria. Conventional techniques related to increasing the content of curcumin, an active ingredient of turmeric, by fermenting turmeric using lactic acid bacteria include fermented turmeric compositions using a mixed culture of three kinds of lactobacillus (lactobacillus, leuconostoc, streptococcus) and a method of preparing the same ( Application No. 1-2005-0069028. The fermentation used in the prior art used a method in which three lactic acid strains were mixed and incubated for 7 to 13 days. The above method curcumin increased by fermentation compared to the number of strains used for the culture and a long incubation time. The amount of is increased by 1.29 times. In addition, the conventional technique for the preparation of water-soluble curcumin is a method for producing curcumin-dimannoside with a very high water solubility by connecting curcumin and mannose with alpha-glucosyl bonds using a chemical method (Application No. 10-2005-). 0013587). This is a curcumin solubilization method by synthesis, which requires complex reactions and additionally requires a process for separating / purifying curcumin, resulting in lower production efficiency and higher production cost. In order to solve this problem, in this patent, a single lactic acid strain is cultured for a short time to increase the content of curcumin contained in turmeric by more than three times and increase the content of BDMC and DMC among various types of curcuminoids, and thus the overall bioavailability of curcuminoids. It provides a method of producing fermented turmeric to increase. More specifically, Lactobacillus Johnsoni fermented turmeric for 16-24 hours using a single strain of IDCC 9203 to remove fermented peculiar bitter taste and odor and provide fermented turmeric that maximizes the activity of various active ingredients including curcumin. . Therefore, the main object of the present invention is to provide a fermentation turmeric that increases the bioavailability as the curcuminoid content is increased by fermentation, and another object of the present invention is to produce a fermentation turmeric using a lactobacillus johnsonyi IDCC 9203 To provide. Still another object of the present invention is to add lactobacillus johnsoni IDCC 9203 fermented turmeric thus produced to beverages, foods or cosmetics conveniently and widely spread to increase the effect of treatment and prevention for various diseases.

In order to achieve the above object, the fermentation is performed by inoculating Lactobacillus zonsini IDCC 9203 in a medium containing turmeric powder. In addition, the active ingredient in turmeric culture is extracted by alcohol and freeze-dried the extract.

In more detail, 7 strains of lactic acid bacteria stored in Ildong lactic acid bank were cultured to select strains showing the most optimal culture state in the medium containing turmeric. Lactobacillus cultures using these strains were screened by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot assay using biomarkers related to inflammatory reactions. .

In order to select the medium to be applied to the selected lactic acid strains, the culture medium experiment was conducted to select the optimal medium, and the HPLC analysis confirmed that the content of curcuminoid was increased. Finally, a sample was prepared by culturing the selected medium and lactic acid bacteria to produce fermentation turbulence, and the antiallergic activity in vivo was applied to the atopic dermatitis animal model and passive skin sensitization model using NC / Nga mouse. It was performed to test vivo - in the measuring.

The strain used for the culture in this experiment is Lactobacillus johnsonii IDCC 9203 ( Lactobacillus johnsonii IDCC 9203) was a strain deposited in Korea microbial conservation center (Accession Number: KCTC-10923BP).

The object of the present invention configured as described above is to remove the bitter taste and smell peculiar to turmeric and to produce fermented turmeric with increased bioavailability compared to conventional turmeric extract, such as the active ingredient active, fermented turmeric having the characteristics as described above is effective It is to provide food, health functional foods and cosmetics as ingredients. Foods, health functional foods, and cosmetics containing fermented turmeric produced using Lactobacillus john sony IDCC 9203 as an active ingredient have improved bioavailability and increased in quantity than the active ingredients contained in conventional turmeric extracts. This can be useful for.

Figure 1 shows the results of RT-PCR and inflammation-inducing mediator cycloxygenase-2 for tumor necrosis factor-α (TNF-α) and interleukine-6 (IL-6) as part of the lactic acid bacteria selection experiment Figure 2 shows the results of Western blot assay for COX-2) and inducible nitric oxide synthase (iNOS),
Figure 2 is a view detailing the fermented turmeric manufacturing process,
3 is a view comparing the solubility of fermented turmeric and non-fermented turmeric sample,
4 is a view showing the changes in the content of curcuminoids contained in fermented turmeric and non-fermented turmeric through HPLC analysis,
Figure 5 is divided into the appearance of erythema / hemorrhage, dry / wound, edema, sores / abrasions, thyroiditis over a total of seven times in the atopic dermatitis animal model for the entire duration of the experiment and assigning a score to each item (0-3 points) The symptoms of dermatitis were quantified, and this is a simple graph.
6 is a diagram showing the results of measuring the quantitative change of blood IgE effected by oral administration of each sample using ELISA kit when mite extract induces atopic dermatitis in NC / Nga mouse model.
7 is a diagram showing the inhibitory effect of the sudden hypersensitivity reaction in the manual skin sensitization model of turmeric fermentation product using a PCA animal model.

Preferred embodiments are presented to aid in understanding the invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Lactobacillus Screening Experiment

In the present invention, in order to select the lactic acid bacteria that can be easily cultured in the medium containing turmeric powder, a culture experiment was conducted for 7 weeks of lactic acid bacteria <Table 1>.

No . Strain One B. longum 2 L. gasseri 3 L. johnsonii 4 L. paracasei 5 L. plantarum 6 L. reuteri 7 L. rhamnosus

Specifically, the lactic acid bacteria were precultured in MRS liquid medium for 16 hours, and the preculture was inoculated in the same MRS liquid medium and cultured for 8 hours to perform intermediate culture. GPT medium containing 1% glucose, 0.5% peptone, 1-5% turmeric powder was used as the fermentation medium and incubated at 37 ° C. for 21 hours by inoculating intermediate culture solution. After the fermentation was progressed, the form of lactic acid bacteria was observed through a microscope. Among the 7 strains of lactic acid bacteria stored in Ildong lactic acid bank, the culture status of B. longum and L. gasseri was poor and was excluded from the selection process. In order to select the lactic acid bacteria to be used for the production of fermented turmeric samples, the remaining five lactic acid bacteria cultures were measured to inhibit the inflammatory factor inhibitory activity, one of the functions of curcumin, the active ingredient of turmeric. RT-PCR was performed on TNF-α and IL-6, pro-inflammatory cytokines, and Western blot assay was performed on inflammatory mediators COX-2 and iNOS. Finally, Lactobacillus Johnsoni IDCC 9203 strain was selected. 1>.

Turmeric  Fermentation Condition Experiment

In order to select a medium suitable for fermentation of the Lactobacillus johnsonia IDCC 9203 strain selected in Example 1 and to obtain a fermentation product having the best inflammatory inhibitory activity, fermentation condition experiments were performed. The first step is to increase the cell growth. Inoculate Lactobacillus Johnsoni IDCC 9203 strain into MRS broth containing 1 ~ 4% fish peptone, and proceed with primary culture at 37 ℃ for 16 hours, lactose 2%, yeast Inoculate the primary culture medium in the secondary culture medium consisting of extract powder 2%, casein peptone 1%, fish peptone 2%, sodium acetate 0.5%, sodium citrate 0.2%, CaCl ₂ 0.1%, tween80 0.1% at 37 ℃ The cells were increased by incubating for 8 hours. The second step is to optimize fermentation of turmeric and induce fermentation of turmeric by minimizing nutrients necessary for bacterial growth. Specifically, the fermentation broth containing 1 ~ 5% of turmeric powder, 1% of glucose and 0.5% of peptone was inoculated and fermented at 37 ° C. and 50 rpm while the pH of the fermentation broth was lowered to 4.2 and the pH was 2 ~. Fermentation was terminated when it kept for 3 hours. The time to end of fermentation was usually 20 to 24 hours.

Fermented turmeric  Manufacturing method

After the fermentation was completed, the fermentation broth was recovered by calibrating with sodium hydroxide (10% NaOH) or sodium carbonate (20% Na ₂ CO ₃ ) so that the pH of the fermentation broth was 6.5-7.0. The fermentation filtrate was concentrated under reduced pressure, and the alcohol was extracted by adding 1-3 times the concentration of the concentrate. The alcohol extract was concentrated through reduced pressure, recovered by adding an appropriate amount of water, and a fermentation turmeric was prepared by lyophilization by adding a drying aid (FIG. 2). Fermented turmeric is well hygroscopic after lyophilization and an appropriate protective agent should be selected. As a type of drying aid, lactose, starch, cyclodextrin, maltotextrin may be used in an amount of 1 to 10%, and preferably 1 to 5% of starch compared to the fermentation filtrate. In addition, hot water extract samples were prepared in order to compare component contents and physical properties with the samples that were not fermented (non fermented turmeric). The hot water extract sample was prepared by preparing a GPT medium containing 1 ~ 5% turmeric powder, then bathed at 121 ° C. for 40 minutes, left at 37 ° C. for 21 hours, and then recovering the filtrate by centrifugation. It proceeded similarly to the manufacturing method of a turmeric sample.

Fermentation turbulence samples and non-fermentation turbulence samples were prepared and the solubility was compared. As a sample for comparing solubility, a sample without starch was used. 100 mg of each sample was suspended in 5 ml, and the solubility was observed. As a result, the non-fermented turmeric had many precipitates insoluble in water, but the fermented turmeric had little precipitate (Fig. 3).

Fermented turmeric  Active ingredient ( curcumin Content analysis

 For the fermented turmeric sample and the non-fermented turmeric sample prepared in Example 3, the content analysis of the active ingredient was carried out using HPLC. After extracting the active ingredient from each sample using ethyl acetate, it was dissolved in HPLC grade methanol to a concentration of 1,000 ppm and analyzed by HPLC. The curcumin content in the whole sample made using 1 liter of culture was about 40.7 mg / l in the fermented turmeric sample and 13.1 mg / l in the non-fermented turmeric sample. >. In Figure 4 it can be seen that the fermented turmeric sample significantly increases the amount of curcuminoids as well as other curcuminoids such as BDMC or DMC compared to the non-fermented turmeric sample.

Curcumin of sample 1g
(/ / G) Sample yield
(g / ℓ) Total curcumin
(Mg / l) One Fermentation sample 1375.4 29.6 40.7 2 Non-fermented sample 486.6 27 13.1

Curcumin content in turmeric is the same, and more curcumin can be obtained when fermented turmeric is extracted from alcohol than curcumin obtained by extracting (not fermenting) turmeric into alcohol. This is because fermented strain (Lactobacillus johnsonyi IDCC 9203) ferments turmeric and decomposes macromolecules such as dietary fiber and insoluble protein of turmeric, and organic acids are produced to increase the solubility of curcumin, which can be extracted more as fermentation filtrate. It may be biosynthesized from precursors such as cinnamic acid and p-coumaric acid through fermentation.

Fermented turmeric curcuminoid  Content ratio analysis

When the turmeric fermented with Lactobacillus johnsoni IDCC 9203 strain, each component was analyzed by HPLC analysis to determine the change in the content ratio of curcuminoid. Curcumin analogs (curcuminoids) contained in turmeric include curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). Curcumin accounts for more than 50% of curcuminoids. The curcumin content ratio of the fermented turmeric sample prepared in Example 4 was 52%, which was reduced from 58% of the non fermented turmeric, but BDMC showed a tendency to increase from 19% to 23%. It is estimated that curcumin's methoxy group is converted to hydrogen and converted to DMC and BDMC during fermentation. Fermentation turmeric produced through this fermentation process contains more BDMC and DMC in content ratio or absolute amount than non fermentation turmeric. BDMC and DMC with hydroxyl groups have been reported not only to have more potent antioxidant activity than curcumin, but also to exhibit anti-allergic activity in rats (Sun-Mee Lee, 2007, Hepatoprotective activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin, Kor. J. Pharmacogn. L38). (2): 139 ~ 147) It has been reported that BDMC, among the curcuminoids, the main ingredient of curry, activates the immune system to remove beta amyloid, a feature of senile dementia (Milan Fiala, 2007, Innate immunity and transcription of MGAT-III). and toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin, PNAS, vol. 104, 12849-12854). As such, there are many reports that BDMC and DMC are superior in various physiological activities than curcumin. By the fermentation turmeric manufacturing method proposed in the present invention can not only increase the BDMC and DMC content, including curcumin but also fermentation turmeric with an increased content ratio of BDMC excellent in physiological activity.

Raw material BDMC (%) DMC (%) Curcumin (%) One Fermentation sample 21.729 25.615 52.656 2 Non-fermented sample 19.672 22.545 57.783

Fermented turmeric  Efficacy Evaluation

In this example, animal experiments were conducted on two experimental models to determine how the content and compositional differences of fermented turmeric and non-fermented water turmeric affect the efficacy.

<6-1> Atopic Animal Experiment Using NC / Nga Mouse Model

NC / Nga mice were used as experimental animal models for the induction of atopic dermatitis. The present inventors epilated the back of the NC / Nga mice, and then applied the SDS solution to the cotton wool and evenly applied to the epilated back. Mite extract ointment was then applied to cause atopic dermatitis. Dexamethasone (5 mg / kg) was used as a drug control group, phosphate buffered saline (PBS) was used as a control group, and the test group was orally fermented turmeric (FT340) at a concentration of 340 mg / kg. The symptoms of dermatitis were observed and recorded seven times in total.

4, the group orally administered FT340 showed that atopic dermatitis symptoms gradually worsened without significant ups and downs compared to the control group administered orally PBS, which may occur when sudden exposure to a large amount of external antigen occurs. Fermentation turbulence may be thought to have alleviated physical skin damage and various diseases caused by sudden hypersensitivity. In addition, orbital blood collection was performed twice to measure blood IgE levels related to atopic dermatitis and analyzed using IgE ELISA kit. In Figure 5, comparing the quantitative changes in IgE in the blood during the administration period of the sample it can be seen that the group of the FT340 administration was reduced in the amount of IgE in the blood than the group receiving the dexamethasone drug control group and the number It can be seen that the low level is almost equivalent to the non-induced group. Blood collected at the end of both atopic induction and oral administration increased overall IgE concentrations compared to blood collected during administration, but the FT340 oral group showed similar IgE levels compared to the control group. It was reduced by 24%. Administration of fermented turmeric can be thought to reduce the IgE concentration in the blood and alleviate atopic dermatitis. Passive skin hypersensitivity test (PCA) was conducted to confirm that fermented turmeric inhibits the production of allergy-inducing factor IgE and to compare the allergic effects with non-fermented turmeric.

<6-2> Animal Experiments Using Passive Cutaneous Anaphylaxis Models

Antimicrobial injection of 20 μl of anti-denitrophenyl IgE (anti-DNP IgE) into the ear of an ICR mouse administered orally with fermented turmeric, and 24 hours later, an antigen solution containing human serum albumin (HSA) and evans blue (sigma) in the same volume Was administered through the microvein to produce a manual skin sensitized animal model. The concentration of evans blue solution was used as an indicator of skin hypersensitivity. Thirty minutes after the antigen was administered through the microvein, each group of mice was lethal and the ears were extracted, and evans blue was leached from the extracted ear tissue. The solution was centrifuged to obtain a supernatant and the absorbance was measured at 620 nm. . The quantification of the pigment was performed using the evans blue standard curve. Figure 6 shows that when dexamethasone, fermented turmeric, and fermented turmeric (FT340) were orally administered, the group treated with dexamethasone showed a 77% decrease in hypersensitivity compared to the PCA-positive group, and the group that was orally administered nonfermented turmeric At 25%, the oral administration of fermented turmeric showed a reduction of about 50%, indicating a 30% lower sensitivity than the non-fermented group. As a result, it was confirmed that fermented product fermented with lactobacillus johnsoni IDCC 9203 contained a substance capable of significantly suppressing sudden hypersensitivity from foreign antigens, and its activity was further increased by fermentation of turmeric. It could be confirmed.

Hereinafter, a formulation example combining the fermented products of the present invention will be described, but the present invention is not intended to limit the present invention, but is intended to be described in detail.

Preparation Example 1 Preparation of Health Food

Fermented turmeric 500 mg of the present invention

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is a composition that is relatively suitable for health foods, the composition ratio may be arbitrarily modified, and the granules are prepared by mixing the above components according to a conventional health food production method. And it can be used for manufacturing a health food composition according to a conventional method.

&Lt; Formulation Example 2 > Preparation of health drink

Fermented turmeric 100 mg of the present invention

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantity

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Preparation Example 3 Preparation of Cosmetics

Fermented turmeric 1% of the present invention

Stearic acid 2%

Stearyl Alcohol 3%

1,3-butylene glycol 5%

4% glycol monostearate

Potassium Hydroxide 0.1%

Purified water 78.9%

Lecithin 5%

Cholesterol 1%

The oil and aqueous components in the composition were heated to 75 ° C., respectively, and then emulsified using a homogenizing mixer in a cream manufacturing mixer, followed by degassing, filtration and cooling to prepare a cream.

Preparation Example 4 Preparation of Yogurt

Fermented turmeric 5% of the present invention

10% skim milk powder

Glucose 2%

2% sucrose

Purified Water 81%

Mix and homogenize with the same composition as above, heat treatment at 90 ℃ for 10 minutes, and then treat 2% of mixed lactic acid bacterium in the cooled sample to incubate at 40 ℃ for 8 hours to proceed with fermentation and homogenize the curd Used to prepare the yoghurt composition of the present invention.

Claims (5)

delete delete (a) Neutralizing the turmeric fermentation solution produced by fermenting turmeric with Lactobacillus johnsonii IDCC 9203 to 10% (w / v) sodium hydroxide or 20% (w / v) sodium carbonate to pH 6.5-7.0 Making;
(b) extracting the active ingredient by adding alcohol 1 to 3 times the fermentation concentrate;
(c) Method of producing fermented turmeric with increased content of curcumin, demethoxycurcumin and bisdemethoxycurcumin, the active ingredient comprising the step of lyophilizing by adding 1-5% (w / v) starch as a freeze-drying aid.
A food composition comprising the fermented turmeric produced by claim 3 as an active ingredient.
Cosmetic composition comprising a fermented turmeric produced by claim 3 as an active ingredient.

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