KR20150131483A - Method for producing Platycodon grandiflorus fermented broth with enhanced antioxidant activity and inhibition activity of alpha-glucosidase - Google Patents

Abstract

The present invention relates to a method for preparing balloon flower root fermented broth with enhanced antioxidant activity and inhibitory activity of alpha-glucosidase. More specifically, the method for preparing balloon flower root fermented broth comprises the following steps: inoculating a broth including 1-10%(w/v) of balloon flower roots with a Lactobacillus plantarum strain; and fermenting the same at a temperature of 20-40°C for 8-24 hours. The balloon flower root fermented broth prepared by the present invention is useful as a functional food having an antioxidant effect and an inhibitory effect of alpha-glucosidase.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a Platycodon grandiflorus fermented broth with enhanced antioxidant activity and an inhibitory activity against alpha-glucosidase,

The present invention relates to a method for producing a borage seedling broth having enhanced antioxidative activity and alpha-glucosidase inhibitory activity.

Bellflower is a perennial herb that belongs to the lantern family and is native to Korea, China and Japan. It is a medicinal herb that has been used for respiratory diseases such as drainage, germosis, bronchitis and asthma. Bellflower is blended There are 273 prescriptions for Oriental medicine in Oriental medicine and 49 for medicine.

The main pharmacological component of the presently known bellflower is triterpenoid saponin. In animal experiments, this substance has been shown to inhibit the secretion of chondroitin, genomic action, central nervous system (sedative, analgesic, antipyretic effect) Anti-inflammatory action, anti-ulcer and gastric secretion inhibiting action, anticholinergic action to lower blood pressure by expanding blood vessels, hypoglycemic action, and cholesterol metabolism improvement. In addition, the hot water and ethanol extracts of the bellows inhibit the fungal aprotoxin. The inulin fraction has potent antitumor effects against phagocytosis and solid and multiple cancers, while the 40% bellflower extract has been shown to inhibit alcohol absorption . The bellflower is used in the form of eating a bell pepper to eat cough and relieve, or to boil dry bellflower.

In addition, recent studies have disclosed a method for producing fermented Dorridi (Korean Patent Laid-Open Publication No. 2013-0011013), wherein phytotoxin D is increased among the active ingredients of Rhododendron japonica, and the fermented herb including red ginseng extract and Rhododendron japonica Although an antioxidant health food composition (Korean Patent Laid-Open Publication No. 2011-0080470) is known, a method for effectively promoting antioxidative activity and alpha-glucosidase inhibitory activity using bellflower has not yet been known.

DISCLOSURE OF THE INVENTION In order to solve the above problems, an object of the present invention is to provide a method for preparing a brook of fermented broth to produce a health functional food having improved antioxidative activity and alpha-glucosidase inhibitory activity.

In order to solve the above-mentioned problems, the present invention provides a method for producing a bellflower comprising: (1) sterilizing a culture broth containing a bellflower; (2) inoculating the sterilized culture broth of step (1) with the lactic acid bacteria; And (3) fermenting the culture broth inoculated with the lactic acid bacteria in the step (2) at 20 to 40 DEG C for 8 to 24 hours. The antioxidative activity and alpha-glucosidase inhibitory activity And a method for producing the fermentation broth.

The present invention also provides a bellflower broth having improved antioxidative activity and alpha-glucosidase inhibitory activity according to the present invention.

The present invention also provides an antioxidant-functioning food comprising an effective amount of the Dorage fermentation liquid prepared in the present invention.

The present invention relates to a method for producing a borage fermentation broth having enhanced antioxidative activity and alpha-glucosidase inhibitory activity, wherein the fermentation broth of lactic acid bacterium prepared by using the bellflower is a functional food having antioxidative activity and alpha-glucosidase inhibitory activity It is worth using.

FIG. 1 is a graph showing the pH change of a lactic acid bacterial culture medium in a culture medium containing 10% (w / v) platycodon.
Fig. 2 is a graph showing the pH change of the culture solution by the concentration of the balloon contained in the culture solution.
FIG. 3 is a graph showing the pH change of the culture solution by the culture temperature.
4 is a graph showing the pH change of the culture solution according to sugar content.
FIG. 5 is a graph showing the pH change of the culture medium per fermentation time. FIG.
6 is a graph showing DPPH radical scavenging activity before and after fermentation with lactic acid bacteria in a 1% (w / v) broth fermentation broth.
FIG. 7 is a graph showing the pH and total acidity of 10% (w / v) flowering broth before and after lactic acid fermentation.
8 is a graph showing changes in sugar content before and after fermentation with lactic acid bacteria in a 10% (w / v) Doraji fermentation broth.
9 is a graph showing changes in CFU before and after fermentation of lactic acid bacteria in a 10% (w / v) broth fermentation broth.
10 is a graph showing alpha-glucosidase inhibitory activity of the borage broth.

In order to achieve the object of the present invention,

(1) sterilizing the culture medium containing the bellflower;

(2) inoculating the sterilized culture broth of step (1) with the lactic acid bacteria; And

(3) fermenting the culture broth inoculated with the lactic acid bacterium at 20 to 40 DEG C for 8 to 24 hours in the step (2); and a step of increasing the antioxidative activity and alpha-glucosidase inhibitory activity A method for producing a bellflower fermentation broth is provided.

In the step (1), the content of the balloon in the culture solution is preferably 1 to 10% (w / v), but is not limited thereto, and most preferably 1 to 5% (w / v) .

The sterilization of step (1) is preferably, but not limited to, sterilization at 121 占 폚 for 15 to 20 minutes.

The step (1) is preferably in the form of powder, but it is not limited thereto, and it is possible to use various forms such as a bellflower extract, if necessary.

The above-mentioned lactic acid bacteria may be any strains which are excellent in acid production ability and harmless to human body, but Lactobacillus plantarum plantarum , and more preferably, Lactobacillus plantarum SRCM100309, SRCM100318 or SRCM100320.

The present invention also provides a bellflower broth having improved antioxidative activity and alpha-glucosidase inhibitory activity according to the present invention.

The present invention also provides an antioxidant-functioning food comprising an effective amount of the Dorage fermentation liquid prepared in the present invention.

When the bellflowy broth of the present invention is used as a food additive, the bellflower broth may be added as it is or may be used together with other food or food components, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the fermentation broth of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. As a specific example, the above-described bellflower fermentation broth can be used to produce a processed food having good shelf-life while modifying the characteristics of agricultural products, livestock products or aquatic products. Such processed foods include, for example, confectionery, drinks, liquor, fermented foods, canned foods, milk processed foods, meat processed foods, noodles and the like. The sweets include biscuits, pies, breads, candies, jellies, gums, cereals (including dinner utensils such as cereal flakes). Drinks include carbonated beverages, functional ionic beverages, juices (such as apples, pears, grapes, aloes, citrus fruits, peaches, carrots, tomato juices, etc.) and sikhye. The mainstream includes sake, whiskey, shochu, beer, liquor, and fruit wine. Fermented foods include soy sauce, miso, and kochujang. Canned products include canned products (for example, tuna, mackerel, sandwiches, canned fish, etc.), canned products (canned beef, pork, chicken and turkey canned products) and canned products (canned products such as corn, peach and canned pineapple). Milk processed foods include cheese, butter, yogurt and the like. Meat processed foods include pork cutlet, beef cutlet, chicken cutlet, sausage, sweet and sour pork, nuggets, nubucki, and the like. And noodles such as sealed packaging raw noodles. In addition, the composition may be used in retort food, soup and the like.

In addition, functional food, health food or health supplement food can be produced by using the above-mentioned Dorage fermentation broth. The functional food, the health food or the health supplement food refers to a food which provides a biocontrol function including a physiologically active ingredient in addition to the nutritional function, and the platycodon fermentation liquid of the present invention has an antioxidative effect and an alpha-glucosidase inhibitory activity, Food, health food or health supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Establishment of optimal fermentation conditions of lactic acid bacteria

1) Production of native resource media and cultivation of lactic acid bacteria

In order to prepare a functional fermentation broth using the bellflower cultivated in the Sunchang area, a culture medium for lactic acid fermentation was prepared by the following method.

The bellflower was diluted with distilled water using 100% powder (powder) and sterilized to prepare a culture medium for lactic acid fermentation.

Three kinds of lactic acid bacteria ( Lactobacillus plantarum SRCM100309, SRCM100318 and SRCM100320 strain) were preincubated and activated in MRS medium, and then inoculated on the prepared broth medium and incubated at 30 DEG C for 24 and 48 hours Lt; RTI ID = 0.0 > 100 rpm. ≪ / RTI > After culturing, the pH of the culture solution was measured to select a strain having a large pH reduction value.

The bellflower fermentation broth is 10% (w / v) was prepared so that the contained bellflower, Lactobacillus plan from the subsequent results of the culture of lactic acid bacteria by measuring the pH of the culture, Lactobacillus plan tarum (Lactobacillus plantarum) of the three strains tarum (Lactobacillus plantarum) SRCM100318 and Lactobacillus tarum Plan (Lactobacillus plantarum) the pH value of the strain Lactobacillus SRCM100320 plan tarum (Lactobacillus plantarum SRCM100309, and the pH values after incubation for 24 hours and 48 hours were almost the same, indicating that the difference in incubation time was not large (FIG. 1). Based on the results shown in FIG. 1, the strains to be used for producing the broth fermentation broth were selected from the strains of Lactobacillus plantarum plantarum ) SRCM100320.

2) Establish culture condition

Selected lactobacillus Lactobacillus plantarum SRCM 100320 strain by measuring the pH and incubated with different culture conditions each was to determine the optimal culture conditions.

The selected lactic acid bacterium was inoculated into the broth medium and cultured at the cultivation temperature (20 ° C., 30 ° C., 40 ° C.), 10% (w / v), 5% The optimum pH was determined by measuring pH after culturing with different sugar content (6brix, 10brix).

① Identification of the pH according to the concentration of the bellflower: The medium was prepared by adding the bellflower to the medium in the concentrations of 1% (w / v), 5% (w / v) and 10% (w / v) respectively and then inoculating the strain The pH change of the culture medium was measured while stirring at 30 rpm for 12 hours at 100 rpm. As a result of measuring the pH change, the pH of the culture solution containing 1 to 10% (w / v) bellflower was almost not changed, and it was judged that culturing in a culture solution containing 1% (w / v) (Fig. 2).

② Confirmation of pH according to incubation temperature : The medium containing 5% (w / v) broth was sterilized, and the sugar content was adjusted to 6 brix as in the MRS medium. The strain was inoculated and the pH change of the culture medium was measured while stirring at 100 rpm for 12 hours at 20 ° C, 30 ° C and 40 ° C, respectively. The pH change with incubation temperature showed the lowest pH value when cultured at 30 ° C (FIG. 3).

③ Confirmation of pH according to sugar content : A medium containing 5% (w / v) of ballooning broth was prepared and sterilized. The sugar content was measured and adjusted to sugar-free treatment, 6brix and 10brix, and the strain was inoculated on the sterilized medium The pH change of the culture medium was measured while stirring at 30 rpm for 12 hours at 100 rpm. It was found that the pH difference according to the sugar content was not so large, so that it was possible to culturize sufficiently only with the sugar originally contained in the 10% broth without the brix control (FIG. 4). Therefore, the optimum fermentation conditions were established in a medium containing 1% (w / v) platycodon without any sugar content and cultured at 30 ° C.

3) Analysis of optimal fermentation time

The strains were inoculated with 1% (w / v) broth and sterilized at the optimal fermentation conditions and the pH was changed at 30 ℃ for 0, 12, and 24 hours. The pH was reduced to 3.8 by fermentation for 12 hours and the fermentation for 24 hours showed no significant difference (Fig. 5). Therefore, it was judged that the lactic acid bacteria were sufficiently grown for 12 hours to produce an acid.

Example  2. Preparation of high-capacity functional fermentation broth for industrial application

In order to mass produce the fermented broth by establishing the optimal fermentation conditions, the content was increased and large capacity fermentation was carried out. Fermentation using a fermenter was performed for large - scale fermentation. The broth fermentation broth was fermented at a culture medium concentration of 10% (w / v) at a total volume of 2.5 L for 12 hours at a culture temperature of 30 ° C. and 100 rpm. The fermentation broth was sampled before and after fermentation and used for future functional and quality analysis.

Example  3. Analysis of functional components of fermentation broth

1) Materials

The fermented broth was prepared by using the lactic acid bacteria which were separated and stored in the fermented microorganism industry promotion institute and optimized for the fermentation conditions according to the present invention. At this time, the treatments inoculated with lactic acid bacteria were sampled before and after fermentation and stored in a freezer. The fermentation broth was centrifuged at 10,000 rpm for 20 minutes and then sterilized using a 0.45 μm syringe filter, and then used as a material for functional testing.

2) Analysis method

① Total polyphenol ( polyphenol ) content

The total phenolic content was determined by appropriately diluting the extracted samples according to the Folin-Denis transformation method. 12.5 μl of Folin-Ciocalteu reagent was mixed with 200 μl of each concentration, and the mixture was allowed to react at room temperature for 120 minutes. The absorbance of the reaction solution was analyzed at 750 nm using a UV-Vis spectrophotometer (DU 800, Beckman coulter, Fullerton, CA, USA). The total phenolic content of gallic acid was calculated by the same method as the analytical sample and the total phenol content of the sample extract was calculated from the standard calibration curve. The total polyphenol content was expressed as mg gallic acid equivalent in 1 liter.

The polyphenol content of the fermented broth inoculated with 1% (w / v) bellflower was measured. As shown in Table 1, the broth fermentation broth was not treated with the control (broth without lactic acid bacteria) The amount of polyphenol was slightly increased.

Table 1. Polyphenol content of 1% (w / v) Doraji fermented broth

The polyphenol content of the 10% (w / v) Doraji fermentation broth was measured and the results are shown in Table 2. The fermented broth of Staphylococcus aureus showed a tendency to increase to 142.38mg / L in the fermented broth inoculated with the strain.

Table 2. Polyphenol content of 10% (w / v) Doraji fermentation broth

② DPPH Radical Scatters  Measure

DPPH is a measure of the degree of discoloration of deep purple in yellow by reducing with ascorbic acid, tocophenol, polyhydroxy aromatic compounds, aromatic amines and measuring the electron donating ability of antioxidants. When used. That is, DPPH contains a stable radical located in the molecule, but when it is contacted with a substance having antioxidative activity and alpha-glucosidase inhibitory activity, radicals are eliminated, and the behavior of DPPH at this time is similar to the hydroxyl group (-OH). The degree of decrease of the DPPH radicals is widely used as a method for measuring the antioxidant activity and alpha-glucosidase inhibitory activity of a sample by measuring the degree of reduction with a spectrophotometer.

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability was modified by Blois (1958) and the reducing power of each sample was measured by the effect on electron donation abilities (EDA). That is, 150 μl of a 0.15 mM DPPH solution (dissolved in 99% (v / v) MeOH) was added to 50 μl of a sample, and the mixture was left at room temperature for 30 minutes. Then, the absorbance at 517 nm was measured using a spectrophotometer . The radical scavenging ability of each sample was expressed as a percentage (%) by the following equation (1) as the difference in absorbance between the sample additive and the non-additive.

Equation (1)

Electron donating ability (%) = [1- (absorbance of sample added / absorbance of no added)] × 100

As shown in Table 3 and FIG. 6, the DPPH radical scavenging activity of the 1% (w / v) Doraji fermentation broth was lower than that of the positive control tocopherol (1% (v / v) Which was higher than that of control.

Table 3. DPPH radical scavenging activity of 1% (w / v) Doraji fermentation broth

③ α-G lucosidase Inhibitory activity screening

Each of the strains was subjected to stationary culture at 30 ° C for 2 to 5 days in MRS (Lactobacilli MRS broth media, Difco). After heat treatment of the culture solution for 100 minutes, the supernatant obtained by centrifugation (9,200 g, 10 minutes) - > glucosidase inhibitory activity. AGI (α-glucosidase inhibitory) activity was measured by the following method. That is, 50 μl of the sample was mixed with 50 μl of 0.5 U / ml α-glucosidase enzyme solution (0.1 M PBS, pH 6.8) and preincubated at 37 ° C. for 10 minutes. 3 mM ρ-NPG (ρ- nitro-phenyl-α-glucopyranoside, in 0.1M PBS, pH 6.8) was added to 100㎕ After 10 min at 37 ℃ 0.1M Na ₂ CO ₃ Was added to stop the reaction. The amount of ρ-nitrophenol produced at this time was measured at 405 nm by using a spectrophotometer (elisa reader, Infinite 200 TECAN). In this case, 0.1 M PBS (pH 6.8) was used instead of the culture medium, and 0.1 M PBS (pH 6.8) was used instead of the culture medium and enzyme solution as the blank.

Equation (2)

Enzyme inhibitory activity (%) = [1 - (p-nitrophenol production amount in reaction vessel / p-nitrophenol production amount in untreated treatment)] × 100

The results of measuring the AGI (α-glucosidase inhibitory) activity of each strain are shown in Table 4 below.

Table 4. AGI (α-glucosidase inhibitory) activity of isolated strains

④ α-glucosidase inhibitory activity

AGI (α-glucosidase inhibitory) activity was measured by the following method. That is, 50 μl of 0.5 U / ml α-glucosidase enzyme solution (50 μl in 0.1 M PBS, pH 6.8) was pre-incubated at 37 ° C. for 10 minutes. After adding 100 μl of 3 mM ρ-NPG (ρ-nitro-phenyl-α-glucopyranoside, in 0.1 M PBS, pH 6.8), the reaction was carried out at 37 ° C. for 10 minutes and 0.1 M Na ₂ CO ₃ Was added to stop the reaction. The amount of ρ-nitrophenol produced at this time was measured at 405 nm by using a spectrophotometer (elisa reader, Infinite 200 TECAN). In this case, 0.1M PBS (pH 6.8) was used instead of the culture medium, and 0.1 M PBS (pH 6.8) was used instead of the culture medium and enzyme solution as a blank.

The α-glucosidase inhibitory activity of the final selected fermentation broth is shown in FIG. 10 and Table 5. The fermentation broth in which the strains were inoculated exhibited higher activity than the control inoculated with the strains.

Table 5. α-glucosidase inhibitory activity of lactic acid fermentation broth (containing 1% (w / v) bellflower)

Example  4. Analysis of Quality Characteristics of Platycodon sp.

The quality characteristics and microbiological characteristics of the Doraji fermentation broth prepared in Example 2 were analyzed. The bacterial counts and the number of fungi (fungi, yeast) were analyzed by pH, total acidity, sugar content and microbial growth rate. 10 g of the sample was stirred in sterilized saline, diluted, ^{Petrifilm TM aerobic count, yeast and mold} count; was measured using a 3M, USA), food poisoning microorganism analysis was performed in accordance with the Food Code method.

The acid production ability of the selected strains was confirmed from the total acidity (%) measurement through neutralization titration. The neutralized isolate was inoculated into GYE medium (1% yeast extract, 5% (w / v) glucose, 10% (v / v) ethanol) adjusted to 10% (v / v) And shake-cultured at 27 DEG C and 170 rpm for 3 days. After diluting the culture with distilled water 20 times, 40 ㎕ of 1% phenolphthalein was added, and then 50 0.1 of 0.1 N NaOH was added until red color was observed for 30 seconds. The total acidity (%) was calculated by the following formula (3) using the amount of 0.1N NaOH added to the neutralization titration.

Equation (3)

Total acidity (%) = [(0.1 N NaOH titration (ml) x coefficient (0.0063) x dilution multiple (20) / sample amount (ml)

After fermentation, the pH of the fermentation broth was 3.0, the total acidity (%) was 0.7%, the sugar content was 6 brix, and the cell mass was 10.84 log ₁₀ CFU / ml (Figs. 7 to 9).

The results of analysis of the microbiological characteristics of the final fermentation broth were as shown in Table 6, and the total number of bacteria in the fermentation broth was detected to be 10 ¹¹ CFU / g. No fungi and food poisoning microorganisms were detected in all fermentations.

Table 6. Microbiological characteristics of the final fermentation broth

Claims (7)

(1) sterilizing the culture medium containing the bellflower;
(2) inoculating the sterilized culture broth of step (1) with the lactic acid bacteria; And
(3) fermenting the culture broth inoculated with the lactic acid bacterium at 20 to 40 DEG C for 8 to 24 hours in the step (2); and a step of increasing the antioxidative activity and alpha-glucosidase inhibitory activity A method for producing a bellflower fermentation broth. The method according to claim 1, wherein the lactic acid bacterium is a Lactobacillus plantarum strain, wherein the antioxidative activity and alpha-glucosidase inhibitory activity are enhanced. 3. The method according to claim 2, wherein the Lactobacillus plantarum ) is a strain of Lactobacillus plantarum SRCM100309, SRCM100318 or SRCM100320, wherein the antioxidative activity and alpha-glucosidase inhibitory activity are enhanced. [2] The method according to claim 1, wherein the culture medium of step (1) is a culture medium containing 1 to 10% (w / v) Gt; [2] The method according to claim 1, wherein the step (1) is in the form of a powder, wherein the antioxidative activity and the activity of inhibiting alpha-glucosidase are enhanced. A bellflower fermentation broth having enhanced antioxidative activity and alpha-glucosidase inhibitory activity produced by the method according to any one of claims 1 to 5. An antioxidant functional food comprising the bellflowy fermentation broth of claim 6 as an active ingredient.

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