KR101422078B1 - Manufacturing method of fermented Ophipogon japonicus for improving of blood flow using Cordyceps militaris and Lactic acid bacteria - Google Patents

Abstract

The present invention relates to a method for the production of mycorrhizal fungi, comprising the steps of pretreatment of the gangmongdong to a solid or liquid phase, a step of inoculating the gangbuchocarbons into a special medium to induce mycelial growth by shaking culture, And a step of inoculating a lactic acid bacterium with a lactic acid bacterium in a second step to cultivate the fermented lactic acid bacteria.
The fermented product of the present invention is expected to be an antioxidant, an antithrombotic component (ruscogenin and cordycepin) increase, a thrombolytic enzyme activity, and an ACE inhibitory activity, and is expected to be an agent for the prevention of cardiovascular diseases such as arteriosclerosis and hypertension.

Description

Technical Field [0001] The present invention relates to a method for manufacturing a fermented macromolecule for improving blood circulation using cordyceps and a lactic acid bacterium,

The present invention relates to a method for producing fermented macromolecans for improving blood circulation using cordyceps and lactic acid bacteria. The present invention relates to a method for preparing a microorganism, which comprises the steps of pre-treating ginseng root to a solid or liquid phase, activating ginseng seeds, inoculating the microorganism into a specific medium to induce mycelial growth by shaking culture, And a step of inoculating and culturing the first fermented lactic acid bacteria in a second step.

The present invention is based on the finding that the culture fermented with Lactobacillus after fermentation of McMurdoong with Cordyceps sinensis has increased ruscogenin and cordycepin, increased fibrinolytic effect, and angiotensin The antihypertensive effect and the antioxidant effect are increased by the inhibition of angiotensin converting enzyme activity. Therefore, the fermented product of the present invention can provide a high-performance fermented corn steep product capable of enhancing anti-thrombotic, thrombolytic, antihypertensive and antioxidant effects.

Macmundong (麦 门冬, scientific name Ophiopogon japonicus Ker - Gawler ) is a perennial evergreen herbaceous plant belonging to the family Liliaceae, which is widely grown in the southern part of Korea. McMundong has been known to be effective for strengthening blood sugar and nourishing ginseng as well as nourishing ginseng, Jinhae, gadam and diuretic. It has been used as a prescription for ginseng, liquorice, cheongsimjonja tang, In particular, ruscogenin is one of the most effective antithrombotic agents (Kou JP et al.),

Cordyceps is a genus of more than 200 species of the genus Cordyceps with Clavicipitaceae. At present, about 70 species of Cordyceps sinensis have been reported in Korea and Cordyceps japonica and Cordyceps militaris have been recognized as food. Among them, a species called pupae is a kind of entomopathogenic fungi which mainly forms an amygdaloid type larvae based on a larva or a pupa of Lepidoptera. Artificial cultivation is actively carried out. The percentage of carbohydrates and sugars that are energy sources is low, but vitamin A and minerals are more abundant than other species, making them a health functional food. The pupa of Cordyceps sinensis protects against external substances such as anti-inflammatory, anti-bacterial, antitumor, and immunity enhancement, nourishment strengthening, energetic enhancement, anti-fatigue, exercise capacity improvement, aging prevention, , Cholesterol and neutral lipid lowering, hypoglycemia, hypertension treatment, and so on. Cordycepin, a substance produced by Cordyceps, has been reported to have anti-thrombotic effects (Hyu Jeong Cho et al.).

Lactic acid bacteria has been recognized as a very familiar microorganism by long-term intake of fermented milk, and reports on various health-promoting effects such as effect of dressing, immunity enhancement, anticancer effect and antibacterial effect have been reported. It has been announced that it has a mechanism. Kaizu et al. Reported that Lactobacillus sp . SBT 2028 has antioxidant activity in vivo and decreases the accumulation of active oxygen in the human body. Sanders et al. Reported that lactic acid bacteria have various ability to remove active oxygen. Lin et al. Reported that B. longum and L. acidophilus inhibit lipid peroxidation 28-48 %. H ₂ O ₂ removal capacity of lactic acid bacteria varied by Bifidobacterium infantis, Bifidobacterium breve, Bifidobacter ium the NADH peroxide of aldolesentis and Bifidobacterium longum was decompose _{H 2 O 2 (Knauf et al} ., 1992), Kullisaar , etc. in the field of infant The ability of Lactobacillus fermentum to remove OH- was reported to be high.

Korean Patent Registration No. 449245 (composition for treating neurological diseases including marsupial extract), Korean Patent Registration No. 635440 (composition for protecting brain cells and memory including marshmallow extract), Korean Patent Publication No. 2012 -37201 (an anti-obesity composition containing a mung-mung seed extract), Korean Patent No. 1,093,731 (a composition for treating and preventing inflammatory diseases containing mung-mun-dong extract as an active ingredient), Korean Patent Publication No. 2011-41808 And the like), and the like, but the technical structure is different from that of the present invention.

Among the deaths of modern people, blood circulatory system diseases caused by blood vessel disorders such as blood flow impairment, blood vessel injuries and hypertension are caused by blood clot generated in the blood vessels, and deaths have been shown to account for a large number of deaths. Thrombosis interferes with the circulation of blood in the veins and arteries, blocking the supply of nutrients to the tissues and the supply of oxygen to cause cerebral hemorrhage, cerebral thrombosis, heart failure, myocardial infarction, and arteriosclerosis. As a result, studies have been made actively on a solubilizer that prevents thrombus formation and dissolves the generated thrombus as an important therapeutic agent for adult diseases. When the production of active oxygen exceeds the capacity of the bio-defense system, oxidative stress is caused. This is an important factor causing various diseases such as aging. When an antioxidative substance is taken, it will limit oxidative damage or restrict further progression of diseases . Therefore, antioxidative biofunctional substances capable of eliminating such active oxygen are expected as agents for inhibiting or treating aging and various diseases, and development of therapeutic agents using such antioxidative biofunctional substances is increasingly interested.

The present invention relates to a method for producing a microorganism, which comprises the steps of pretreating gangmongdong to a solid or liquid phase, a pre-culture step in which a mycelial is formed and activated by inoculating a specific gangworm, Followed by fermentation with Cordyceps, followed by inoculation with lactic acid bacteria in a second step.

The thrombolytic activity and the angiotensin converting enzyme (ACE) inhibitory activity of fermented milk were fermented using a jar fermenter to determine the thrombolytic activity and antihypertensive effect on the fermented product, After fermentation, it was increased to 169EU / ml, 2.3 times higher than before fermentation. After fermentation, it was increased to 3.22 times by 272EU / ml after lactic acid fermentation, and it could be used as a thrombolytic agent for food such as nattokinase. ACE enzyme inhibition activity was shown to be 59 ~ 64% inhibition after first fermentation by Cordyceps and 5 ~ 12% by 69 ~ 71% after fermentation of second lactic acid bacteria. It is expected to be a preventive improvement.

The present invention is to provide a blood circulation improving product which converts ophiopogonin component of mukmun dong into ruscogenin, which activates mukmun dong with cordyceps, and increases cordycepin production due to mushroom propagation.

; Plasmin 1unit).
도 15는 Renin-Angiotensin system의 항고혈압 메카니즘을 나타낸 것이다.
도 16은 Ophiopogon japonicus K.G.의 발효 중에 안지오텐신 전환 효소 억제 효과를 나타낸 것이다(○ ; 30%　Powder materials, 35% Extract materials).
도 17은 Cordycepin militaris 에 의해 발효된 추출물과 분말의 유산균 배양 후 안지오텐신 전환 효소 억제 효과를 나타낸 것이다(Before(□) ; First fermentation by Cordycepin militaris, After(■) ; Second fermentation by lactic acid bacteria ). Figure 1 shows the antioxidant activity during fermentation of the dried fermented product and the fine granules (○; Dry materials, Chopping materials).
Figure 2 shows the antioxidant activity during the fermentation of the powder and extract (○; 30% powder materials, ■; 35% extract materials).
FIG. 3 is a graph militaris (○: 35% extract materials, ■; 30% powder materials) after fermentation with lactic acid bacteria.
Fig. 4 shows the results of the Ophiopogon japonicus Kg of Cordycepin during fermentation (○; Dry materials, ■; Chopping materials, △; 30% Powder materials, ◆; 35% Extract mater).
Figure 5 is a graph After the fermentation of lactic acid bacteria by extracts and powder fermented by militaris , the content of Codycepin was shown (Before (□); First fermentation by Cordycepin militaris , After (■); Second fermentation by lactic acid bacteria.
6 is Cordyceps militaris . Ophiopogonin by Ruscogenin is a bioconversion process.
FIG. 7 is a graphical representation of Ophiopogon japonicus During the fermentation of KG, the contents of Ophiopogonin D were shown (○; Dry materials, ■; Chopping materials, △; 30% Powder materials, ◆; 35% Extract mater).
Fig. 8 is a graph showing the Ophiopogon japonicus Kg fermentation of Ruscogenin (○; Dry materials, ■; Chopping materials, △; 30% Powder materials, ◆; 35% Extract mater).
Figure 9 is a graphical representation of Cordycepin militaris (D), the content of Ophiopogonin D after the fermentation of lactic acid bacteria with the extract and powder fermented by the fermentation method. (Before (); First fermentation by Cordycepin militaris , After (); Second fermentation by lactic acid bacteria).
Figure 10 shows that Cordycepin militaris (1), and (2), after fermentation of lactic acid bacteria with extracts and powders fermented by fermentation, the content of Ruscogenin was shown (Before (□); First fermentation by Cordycepin militaris , After (■); Second fermentation by lactic acid bacteria.
Figure 11 shows the fibrinolytic activity (fibrin plate) during fermentation of 35% extract.
Figure 12 shows the fibrinolytic activity (fibrin plate) during fermentation of 30% powder.
FIG. 13 is a graph showing the Ophiopogon japonicus Fibrinolytic activity (fibrin solution) during fermentation of KG (35% extract materials, 30% powder materials, Plasmin (1 unit)).
Figure 14 is a graph militaris (2) The fermentation of lactic acid bacteria, which is fermented by Lactobacillus sp., After fermentation with Lactobacillus acidophilus, showed Fibrinolytic activity (Before (□); First fermentation by Cordycepin militaris ,

; Plasmin 1 unit).
Figure 15 shows the antihypertensive mechanism of the Renin-Angiotensin system.
FIG. 16 is a graph showing the Ophiopogon japonicus KG (30% Powder materials, 35% Extract materials) were shown to inhibit angiotensin converting enzyme during fermentation.
Figure 17 shows that Cordycepin militaris And the inhibition of angiotensin converting enzyme by culturing the lactic acid bacteria of the extract and powder fermented by the first fermentation by Cordycepin militaris , After (); Second fermentation by lactic acid bacteria.

The present invention relates to a method of producing a microorganism by inoculating mycelium of Cordyceps sinensis with a special medium to inoculate mycelium of Cordyceps sinensis with a pre-treated cornflower, and cultivating it in a second step to inoculate the lactic acid bacterium in culture to finally increase the content of ruscogenin and cordycepin , An increase in antithrombotic activity, an increase in thrombolysis ability, an increase in angiotensin converting enzyme (ACE) inhibitory activity, and an increase in antioxidant ability.

<Materials>

McMundong used liquorice in the form of dry materials, chopping materials, and powder materials, which were harvested at Geochang, Gyeongsangnam-do in 2012. The extract was prepared directly by the inventor at 4 ℃ It was stored at low temperature and used as needed. Cordyceps militaris was distributed from KCTC to the pupa of Cordyceps militaris . Three strains of Streptococcus thermophilus MG510, Bifidobacterium longum MG723, and Lactobacillus plantarum MG207 were cultured and mixed in the strains belonging to the applicant (MEDIONEN).

The method for obtaining the lactic acid bacteria necessary for the fermentation of the present invention will be described in detail as follows. The following figure is the website of Mediogen Co. (http: // www, mediogen.co.kr).

When 'Product' is selected on the above website, 'Health functional food ingredient' is selected. Through the product information column as shown below, Streptococcus thermophilus MG510, St. thermophilus MG510, (Bifidobacterium longum MG723, B. longum MG723), and Lactobacillus plantarum MG207 (L. plantarum MG207).

&Lt; Example 1 > Culture of Cordyceps

The seed culture of P. japonicus was inoculated into PDA (Potato Dextrose Agar), then cultured at 24-27 ° C for 3-5 days, activated, and inoculated into the preculture medium of Table 1 and incubated at 24-27 ° C for 3-5 days at 100-150 rpm. When mycelium was sufficiently produced by shaking culture for 5 days, it was used for fermentation of each McMurdo-Dong sample.

 Composition of medium for cultivation of Cordyceps badge Furtherance Concentration (%, w / v) PDA
( Potato Dextrose Agar ) Potato Dextrose Agar
(Diffico) 3.9

Cordyceps militaris
Media Glucose 4 Yeast extract One Proteose pepton 0.2 KH ₂ PO ₄ 0.2 MgSO4 ₄ 0.02 pH 6.5

&Lt; Example 2 > Cultivation of lactic acid bacteria

Streptococcus thermophilus MG510 and Lactobacillus plantarum MG207 were cultured in MRS media at 35-38 ° C for 12-15 hours, and Bifidobacterium longum MG723 was cultivated in the following Table 2 After incubation at 35 ~ 38 ℃ under anaerobic conditions for 24 ~ 48 hours using Modified MRS media, 1% of each of them was used for fermentation.

 Medium for culture of lactic acid bacteria badge Furtherance Concentration (%, w / v)
Lactobacilli MRS Broth Lactobacilli MRS Broth
(Diffico)
5.5
Modified MRS Broth
Lactobacilli MRS Broth 5.5 L-cysteine HCl 0.05 Na ₂ CO ₂ 0.02

&Lt; Example 3 > Primary fermentation of mungum-dong using caterpillar fungus

Fermentation of Cordyceps sinensis in the form of solid fermentation and liquid fermentation was performed. Solid materials and chopping materials were used for solid fermentation, and powder materials and extract materials were used for liquid fermentation.

&Lt; Example 3-1 > Dry godmongdong

1 kg of the raw material was thoroughly washed, immersed in 3 times of water, and then heated at 105 ° C for 40 minutes. After cooling, the pH was adjusted to 6.5 with NaOH, and the mixture was placed in a fermentation bottle and sterilized at 121 ° C for 20 minutes. After sterilization, the cells were inoculated with 10% of total amount of Cordyceps, which had been pre-incubated, and then fermented at 25 ° C for 10 days.

<Example 3-2> Chopping materials

Fermentation was carried out by adding 3 times of water to 1 kg of raw material and immersing it at 105 ° C for 40 minutes. After cooling, the pH was adjusted to 6.5 with NaOH, and the mixture was sterilized at 121 ° C for 20 minutes in a fermentation bottle. After sterilization, the cells were inoculated with 10% of total amount of Cordyceps, which had been pre-incubated, and then fermented at 25 ° C for 10 days.

<Example 3-3> Powder materials

In the case of powdery manganese, the preliminary experiment showed that 30% was a proper solution concentration. Therefore, 30% of McMundong powder was prepared, and the pH was adjusted to 6.5 with NaOH. Then, the mixture was placed in a shaking flask and sterilized at 121 ° C for 20 minutes. After sterilization, the cells were inoculated with 10% of total amount of Cordyceps, which had been pre-cultured, and then fermented at 25 ° C for 10 days with shaking at 100 to 150 rpm.

<Example 3-4> Extract extract materials

In the case of the extract, the result of the preliminary experiment showed that 35% was a proper concentration. Therefore, it was immersed in water with 35% water content, and then it was heated at 105 ℃ for 60 minutes. Then, solids were sieved, and only the extract was used. The extract was adjusted to pH 6.5 with NaOH, placed in a shaking flask and sterilized at 121 ° C for 20 minutes. After sterilization, the cells were inoculated with 10% of total amount of Cordyceps, which had been pre-cultured, and then fermented at 25 ° C for 10 days with shaking at 100 to 150 rpm.

&Lt; Example 4 > Fermentation of Lactobacillus Lactobacillus fermented with Cordyceps

The mushroom extract and powder were fermented in the liquid fermentation with Cordyceps, pH adjusted to 6.5 with NaOH, and sterilized at 80 ℃ for 40 min. After cooling, three kinds of activated lactic acid bacteria ( Streptococcus thermophilus MG510, Bifidobacterium longum MG723, and Lactobacillus plantarum MG207) were inoculated at 1% each, and then subjected to secondary fermentation at 37 ° C for 24 to 48 hours.

&Lt; Example 5 > Jar-fermentor culture

Culture experiments were carried out on jar fermentor for mass culture. For the liquid culture of jar-fermentor, 30% of the powder and 35% of the extract were suitable, and they were fermented by sterilization at 121 ° C for 40 minutes. The fermentation conditions of the first caterpillar fungus were maintained at 250 ~ 300rpm, pH 6.5, temperature 25 ℃, aeration 5vvm, and defoamer (Dow Corning) was added at any time. The fermentation of the second lactic acid bacteria was carried out at 37 ° C for 48 hours under anaerobic conditions in a non-confluent condition.

As a result of liquid fermentation of 35% of extract and 30% of powder by using jar-fermentor, the first fermentation of mulmun-dong was shortened from 10 days to 7 days in the liquid culture, And the culture time was shortened, and the number of bacteria was increased about 2 times. In addition, antioxidant activity was maintained at 52%, and thrombolytic enzyme activity was increased to 135EU / ml and 169EU / ml, respectively, 2.0 ~ 2.3 times higher than before fermentation and ACE enzyme inhibitory activity was increased by 44 ~ 48%. Cordycepin content was increased to 1195mg / L and 1122mg / L, and ruscogenin contents were increased to 685mg / L and 565mg / L. The antioxidant activity of the extracts was increased by 14 ~ 15%, the activity of thrombolytic enzyme was increased by 103 ~ 115EU / ml, and the activity of ACE Enzyme inhibition activity increased to 69 ~ 71%. In addition, ruscogenin content was increased to 50 mg / L and 44 mg / L, respectively, but cordycepin content decreased to 138 mg / L and 22 mg / L, respectively, after the first fermentation (Table 3 to Table 4).

Comparison of efficacy and composition of 1st and 2nd fermented products of 35% extract of McMundong Items Before fermentation First fermentation Second fermentation pH 6.50 6.41 4.15 ° Brix 25 15 12 Antioxidant activity (%) 26 52 66 Codycepin ( mg / L) 0 1,195 1,057 Ophiopogonin ( mg / L) 322 32 11 Ruscognein ( mg / L) 1,522 2207 2257 Fibrinolytic  activity ( EU / ml ) 59 135 250 Angiotensin converting
enzyme inhibition (%)
16
64
69
Viable cell counts
( cfu / ml )

ND

PDA 7.7 x 10 ⁸
BCP 6.7 x 10 ⁷ BS 4.5 × 10 ⁷ BL 9.9 × 10 ⁷

 Comparison of efficacy and composition of 20% powder material of secondary fermentation Items Before fermentation First fermentation Second fermentation pH 6.50 6.49 4.04 ° Brix 20 17 10 Antioxidant activity (%) 21 52 67 Codycepin ( mg / L) 0 1,122 1,100 Ophiopogonin ( mg / L) 276 36 19 Ruscognein ( mg / L) 2,053 2618 2,662 Fibrinolytic  activity ( EU / ml ) 84 169 272 Angiotensin converting
enzyme inhibition (%)
15
59
71
Viable cell counts
( cfu / ml )

ND

PDA 9.8 x 10 ⁸
BCP 8.8 x 10 ⁷ BS 6.4 x 10 ⁷ BL 2.2 × 10 ⁸

The quality evaluation of the present invention was carried out on cultured fermented lactic acid bacterium after fermentation with mushmundong as Cordyceps sinensis, and the antioxidant activity, changes in contents of ophiopogonin, ruscogenin and codycepin, thrombolytic enzyme titer, and angiotensin converting enzyme inhibitory activity Respectively. The statistical processing of all the experiments of the present invention was repeated three times and the average and standard deviation of the values were expressed as Microsoft office Excel 2007, Microsoft, USA.

&Lt; Test Example 1 > Measurement of antioxidant activity change by fermentation

DPPH (1,1-diphenyl-2-pricrylhydrazyl) radical scavenging assay, which is widely used, was used for the assay of free radical scavenging samples. Add 200 μl of the centrifuged sample to a reaction vessel containing 2 ml of MeOH, add 400 μl of DPPH solution (0.8 μl), vortex for 10 seconds, and leave at room temperature for 10 minutes in a dark room. The absorbance at 517 nm was measured using a spectrophotometer (Biochrom Lilra S12), and the difference in absorbance between the sample and the control was expressed as a percentage (%), and the electron donating ability was calculated as follows. Control blank was replaced with 200 μl of MeOH in place of the sample.

Inhibition = [(Absorbance of control) / (Absorbance of control)] 100

100 ppm of ascorbic acid was used as an antioxidant standard, and ascorbic acid showed a suppression rate of 95.54%. Each of the DPPH inhibition rates showed a double antioxidative effect after fermentation at a inhibition rate of 44 ~ 50% after the first ciclosporal fermentation at 20 ~ 25% inhibition rate before fermentation (Figs. 1 and 2). In the four test groups, the initial DPPH inhibition rate was 2 ~ 4% lower than that of the other groups, but there was no significant difference among the test groups. The DPPH inhibition rate in the second fermentation with Lactobacillus japonica increased from 44 ~ 50% to 55 ~ 60% at the end of the fermentation, and the antioxidative effect was increased by the fermentation of the second lactic acid bacteria (Fig. 3).

&Lt; Test Example 2 > Measurement of cordycepin and ophiopogonin and ruscogenin by fermentation

(1) Cordycepin analysis

The fermented extract and powdered fermented product were centrifuged at 12,000 rpm for 20 minutes. In the case of the dried product and the fine granules, the fermented product was pulverized with a hand mixer and subjected to centrifugation by hot water extraction at 105 ° C for 1 hour. Ethanol (99.9%) was added to the filtrate four times and allowed to stand at 4 ° C for 24 hours. The mixture was centrifuged at 6,000 rpm for 15 minutes and concentrated using a rotary vacuum evaporator (BUCHl F-200W) to evaporate the alcohol. After filtration with a 0.45 μm membrane filter, it was analyzed by HPLC (Agilent 1200 seris RRLC). Cordycepin crystalline (Sigma-Aldrich) was used as a standard for the experiment. The final concentration was 1000 ppm, 500 ppm, and 250 ppm.

Cordycepin (3'-deoxyadenosine) is a derivative of nucleoside that has been reported to have various physiological activities such as immunoenhancing activity, anticancer activity, antiviral effect, anti-inflammatory effect and antithrombotic effect. Cordycepin was not detected at all before fermentation, (850mg / L), and then in the fermentation of chopping materials and dry materials. In addition, the fermentation of chopping materials and dry materials 716 mg / L and 692 mg / L, respectively, and the lowest content of cordycepin was 541 mg / L for 30% powder materials (FIG. 4). After fermentation with mungumdong extract and mungumdong powder by fermentation with Cordyceps sinensis, fermentation with lactic acid bacterium secondarily resulted in decrease of cordycepin contents to about 28 mg / L and 40 mg / L, respectively (Fig. 5). Lactic acid bacteria It is presumed that this cordycepin is magnetized.

(2) Analysis of Ruscogenin and Ophiopogonin D content by fermentation

The extracted fermented product and the powdery fermented product were centrifuged at 12,000 rpm for 20 minutes for analysis. In the case of the dried product and the fine granules, the fermented product was pulverized with a hand mixer and subjected to centrifugation by hot water extraction at 105 ° C for 1 hour. The extracted filtrate (200 ml) was extracted three times with water-saturated n-butanol and concentrated under reduced pressure using a rotary vacuum evaporator (BUCHl F-200W). As a final injection solvent, 10 ml of methanol (99.9%) was added to the solution, which was then filtered through 0.45 mm membrane filter and analyzed by HPLC (Agilent 1200 seris RRLC). Standard concentrations of ruscogenin (European Pharmacopoeia) and ophiopogonin D (Hangzhou Dayangchem CO., LT) were dissolved in methanol (99.9%) and diluted to 1000 ppm, 500 ppm and 250 ppm, And ophiopogonin D were analyzed by HPLC (Agilent 1200 seris RRLC). Ruscogenin and ophiopogonin are the most effective antithrombotic agents in the marshmallow, and ruscogenin is known to be superior to ophiopogonin in antithrombotic effect. Ophiopogonin exists in the form of A to D, and ruscogenin is the parent nucleus and has a different glycoside bond (Fig. 6). Therefore, in order to measure the conversion of ophiopogonin - conjugated glycosides to ruscogenin by fermenting Macmundan with the use of Cordyceps, samples were taken at 2 - day intervals to measure rucogenin and ophiopogonin D contents. Ophiopogonin D showed a tendency to decrease as the first fermentation of the four fermented water samples proceeded, and ruscogenin tended to increase overall, indicating that the ophiopogonin substance was converted to ruscogenin by the fermentation of Cordyceps. In the case of extractive fermentation, ophiopogonin D decreased 288 mg / L to about 34 mg / L after 10 days fermentation from about 322 mg / L before fermentation, whereas ruscogenin decreased to 2733 mg / And ophiopogonin A, B, and C, which are contained in the marshmallow, are also converted by fermentation (Figs. 7 to 8). The content of ophiopogonin D decreased by about 20 mg / L and 15 mg / L in the fermented extract and powdered fermented product, while the content of ruscogenin decreased by 50 mg / L and 36 mg / L, indicating that ophiopogonin, which was not degraded by Cordyceps, was degraded again (Figs. 9-10).

&Lt; Test Example 3 > Measurement of thrombolytic enzyme activity following fermentation

Activity of fibrinolytic activity was measured by two methods of activity measurement using fibrin plate (Astrup deformation method) and fibrin solution (Ehrlich deformation method).

(1) Results of measurement of thrombolytic activity using fibrin plate

In the case of the primary fermentation of Cordyceps sinensis, the thrombolytic activity of fermented macromuscularum was high in measuring the thrombolytic ability, and the activity of thrombolytic activity was measured by diluting the sample 10 times. As the fermentation proceeded, the fibrinolytic capacity gradually increased and showed the highest value on the 8th day of fermentation. Particularly, in the case of powdered fermented product, the size of the dissolved transparent ring was about 50 times higher than that of the control plasmin (1 unit). On the 10th day of fermentation, the thrombus dissolving ability tended to decrease rather than on the 8th day, and the dissolution activity showed the highest titer after 8 days of fermentation (Figs. 11 to 12).

(2) Results of measurement of thrombolytic activity using fibrin solution

The amount of soluble low molecular weight degradation products increased by the degradation of peptide bond when fibrin is acting on the thrombolytic enzyme was measured by measuring the absorbance absorbed in the region of 275 nm. The amount of enzyme increased by 0.01 was increased to 1 unit ( EU). As a result, it was found that the activity of thrombolytic enzyme gradually increased with the progress of fermentation. The activity of thrombolytic enzyme was about twice as high as that of the extract, and showed the highest thrombolytic activity at the 8th day of fermentation. The initial enzyme activity was about 50 ~ 100 EU / ml. On the 8th day of fermentation, the enzyme activity was about 250 ~ 200 EU / ml and about 5 times higher enzyme activity. On the 10th day of fermentation, the fibrinolytic capacity was decreased rather than on the 8th day. Therefore, in order to maximize the activity of the enzyme, it is considered to be most appropriate to make the fermentation of the mungum-dong (Chinese cabbage) to 8 days (Fig. 13). The activity of thrombolytic enzyme after fermentation of Lactobacillus acidus was increased by 2 ~ 2.5 times as compared with that of the first fermentation. The activity of fibrinolytic enzyme was slightly higher in the extract than in the case of the fermentation of Cordyceps, and the activity of fibrinolytic enzyme after fermentation was about 1.5 times higher than that of the control plasmin 1 unit (FIG. 14).

Test Example 4: Angiotencin converting enzyme inhibitory activity upon fermentation

Angiotensin converting enzyme (ACE) inhibitory activity was measured by the method of Cushman and Cheung. Samples of ACE inhibitory activity were centrifuged at 10,000 × g for 20 min and used as a supernatant. 150 μl (2.8 units) of ACE and 100 μl of 100 mM sodium borate buffer (pH 8.3) were added to 50 μl of the sample and reacted at 37 ° C for 10 minutes. Then, 50 μl of Hip-His-Leu solution was added and reacted at 37 ° C for 10 minutes. After the reaction, 250 μl of 1N HCl was added to stop the reaction. Ethyl acetate (1 ml) was added, and the mixture was stirred for 30 seconds, centrifuged at 3,000 × g for 15 minutes, and the supernatant was dried in a dry oven. After drying, 1 ml of 100 mM sodium borate buffer (pH 8.3) was added and dissolved. The absorbance at 228 nm was measured and calculated according to the following equation.

B: Absorbance at the time of adding sample, B: Absorbance at the time of adding distilled water, and C: Absorbance at the time of addition of sample after stopping the reaction)

Angiotensin converting enzyme (angiotencin converting enzyme) converts angiotencin I into physiologically active angiotencin II to contract blood vessels and thus raises blood pressure. Therefore, angiotensin converting enzyme inhibition inhibits angiotencin II production and decomposition of bradykinin, (Fig. 15). Based on the mechanism of action, angiotensin converting enzyme inhibitory activity was measured in the fermented mushroom fermented product. As the first fermentation of Cordyceps sinensis was progressed, the fermentation of the mungmundong powder was increased to about 3 times at the initial inhibition level of 15 ~ 20%, to 55 ~ 60% at the 8th day of fermentation, and about 5% Respectively. As the fermentation progressed, the angiotencin converting enzyme (ACE) inhibitory activity was gradually increased, which was similar to the activity of thrombolytic enzyme, (Fig. 16). After the second lactic acid fermentation, the ACE inhibitory activity was about 60% to 65% (Fig. 17), which was about 10% higher than that of the first fermentation.

These results suggest that the antimicrobial activity, thrombolytic activity, angiotensin converting enzyme (ACE) inhibitory activity and antioxidative activity of ruscogenin and cordycepin are increased by the fermentation of McDonald's In the future, it is expected to improve arteriosclerosis, hypertension, and prevention of cardiovascular disease.

The fermented product of McMundum Dong is anticipated as an antioxidant, antithrombogenic agent (ruscogenin and cordycepin) increase, thrombolytic enzyme activity, and ACE inhibitory activity, and is expected to be a preventive agent for cardiovascular diseases such as arteriosclerosis and hypertension. Therefore, the fermented product of the present invention can be industrially used.

Claims (9)

A step of pre-treating ginseng root to a solid or liquid phase, a step of culturing the ginseng root by inoculating the ginseng root into a special medium to form a mycelium, a step of inoculating and culturing the pre-treated ginseng root to inoculate with a cordyceps, A step of culturing lactic acid bacterium in a second step and culturing the fermented lactic acid bacterium; The fermented product of claim 1 or 2, wherein the fermented product is fermented by inoculating a lactic acid bacterium after sterilization, followed by fermenting the fermented product. How to make McMundong The method according to claim 1, wherein the extract obtained by hot-water extraction from McMundum is cultured by inoculation with Cordyceps, followed by sterilization, followed by inoculation with lactic acid bacteria. The method according to claim 1, wherein the Cordyceps is either one or two selected from the group consisting of pupae or pine needles. 2. The method according to claim 1, wherein the primary fermentation is pre-treated with a primary fermentation, the seedling is inoculated in a special medium, cultured at 24 to 26 DEG C at 100 to 150 rpm for 3 to 5 days, A method for producing fermented macromolecule for improving blood circulation by using Cordyceps mellifera and lactic acid bacteria The method according to claim 1, wherein the secondary fermentation is fermented by inoculation with lactic acid bacteria which have been sterilized and then activated by fermenting the fermented mungbean root with cordyceps, The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is cultivated in a fermented mushroom broth of Streptococcus thermophilus MG510, Bifidobacterium longum MG723 and Lactobacillus plantarum MG207, respectively, and then fermented at a temperature of 35 to 38 ° C for 15 to 24 hours under anaerobic conditions And a method for producing fermented macromolecule for improving blood circulation using a lactic acid bacterium A method for improving blood circulation obtained by mixing a fermented macromolecule for improving blood circulation obtained by the method of any one of claims 1 to 7 with a food acceptable excipient

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