KR20180034752A - A Method for Cultivating a Vegetable worms Having a Function of a Natural Preserved Agent and a Freshness Keeping Agent and an Extract of a Vegetable Worms Cordycepin from the Same and a Natural Complex Composition - Google Patents

Abstract

A method of cultivating Cordyceps militaris according to the present invention comprises: a step of inoculating a liquid medium, a solid medium, or an insect with Cordyceps militaris or Cordyceps nutans to obtain mycelia; and a step of culturing the obtained mycelia in a solid medium or an insect to form a fruit body. The solid medium forming a fruit body is composed of corns and a liquid medium, or is composed of rice, silkworm pupae, and a liquid medium; and has a water content of 50-70%, a relative humidity of 80-95%, an ambient temperature of 15-25°C; and has a luminous intensity of 700-1,200 lux. The liquid medium yielding mycelia is composed of MgSO_4·7H_2O, KH_2PO_4, K_2HPO_4, a yeast extract, peptone, and dextrose, or is composed of a yeast extract, dextrose, EBIOS, and Hyponex. The solid medium yielding mycelia is composed of a liquid medium and at least one selected from rice, potato, corn, and silkworm pupae.

Description

Description FIELD OF THE INVENTION The present invention relates to a method of cultivating a Chinese caterpillar fungus having a natural preservative and a preservative agent, Worms Cordycepin from the Same and a Natural Complex Composition}

The present invention relates to a method of cultivating caterpillar fungus having a natural preservative and a primary preservative agent, a method of producing the caterpillar fungus extract and a natural complex thereof, and more particularly, to a method for cultivating caterpillar fungus in a culture medium, Fermented or extracted with a mixture of green tea, garlic, coconut, grape or similar natural products, or with a natural polymer for natural antimicrobial preservation or maintenance.

Physiologically treated by heating, ultrahigh pressure, radiation or light pulse to inhibit the generation of pathogenic microorganisms for a long period of time or stored or stored for a long time with benzoic acid, sorbic acid, chlorine preparation, nitrite or sodium sulfite The same synthetic preservative can be added to the food. However, such physical treatments or addition of synthetic preservatives may cause problems such as destruction of nutrients in food or side effects due to accumulation in the body. As a result, the use of artificial synthetic preservatives is gradually limited, and studies using various natural antimicrobial substances to replace them have been intensively conducted. Materials such as, for example, salt, vinegar, plant extract proteins, nisin, e-polylysine, natamycin, chitosan or organic acids can be used as natural antimicrobial preservatives. However, it has such a taste, smell or irritation, has a weak antimicrobial activity or a narrow antibacterial range.

Japanese Patent Application Laid-Open No. 2004-242507A discloses a carbon source containing rice or corn, at least one selected from the group consisting of peptone, yeast, malt and milk as a nitrogen source and at least one selected from the group consisting of asparagine, aspartic acid and glycine And biotin or vitamin B species, wherein the content of cordycepin is increased.

International Publication No. WO-2012-108557 discloses a method comprising mixing a mixture of ground rice and deep seawater in a sterilized container containing vegetable carbon fibers; A sterilization step at a temperature of 121 ° C for 10 to 30 minutes after the mixing step; A cooling step in which the content temperature is maintained at 20 to 25 ° C by a natural cooling method after the sterilization step; A step of inoculating 20 to 20% (W / v) of the contents of 500 g of Cordyceps mellifera cultured for 10 to 15 days in a PDA medium in an aseptic state after the cooling step; A step of incubating in a dark room for 2 to 8 days under the conditions of a dark room humidity of 70% or more and a temperature of 20 to 25 ° C after the inoculating step; A step of incubating the dark room with shaking light twice a day so as to dilute the contents under an illuminance of 1000 Lux or more using an LED lamp for 10 days under the conditions of a humidity of 70% or more and a temperature of 20 to 25 ° C after the dark room culture step; And a drying step of drying the rice flour at 50 to 70 ° C for 15 to 30 hours after the rice flour culturing step, thereby producing a functional rice containing a large amount of a cordycepin ingredient.

The medium or rice production method disclosed in the prior art has a disadvantage that it is difficult to treat directly with food, fruits or vegetables so as to have the function of a natural antimicrobial preservative or a leading preservative. Therefore, it can be used as a natural preservative added with natural polymer or as a lead preservative, which can be used directly as a natural preservative or as a lead preservative, for extracting green tea, garlic, coconut, grape, It needs to be developed. However, the prior art does not disclose such a method.

The present invention has been made to solve the problems of the prior art and has the following purpose.

Prior Art 1: Japanese Patent Publication No. JP 2004-242507A (OUBIKEN: KK, published on Sep. 02, 2004) Prior Art 2: International Publication No. WO-2012-108557 (FARMS KOREA AGRICULTURAL ASSOCIATION, published on Aug. 16, 2012) Functional rice containing a large amount of cordycepin ingredient and method for producing the same

It is an object of the present invention to provide a method for cultivating Cordyceps sinensis cultured artificially as a mycelial or fruiting body in the form of a natural preservative and a leading preservative agent, and to thereby provide Cordyceps sinensis extract.

It is another object of the present invention to provide a method for the production of an antimicrobial and antioxidant activity, a hypoglycemic action and an immune cell proliferation enhancing action, an anticancer and stress In order to increase the expression level of cordycepin, which has various functions such as suppression, fatigue and liver protection, asthma action and radiation protection effect, the extracts of Cordyceps sinensis extract And at the same time, the freshness maintenance ability and the preservation ability are improved, and a natural complex thereof.

According to one embodiment of the present invention, there is provided a method for cultivating Cordyceps sinensis, comprising the steps of: obtaining a mycelium by inoculating Codiceceps militaris or Codisse Nutanscule in a liquid medium, a solid medium or a saline; And a step of culturing the obtained mycelium in a solid medium or a medium to form a fruiting body, wherein the solid medium for forming the fruiting body comprises corn and a liquid medium or comprises rice, silkworm cocoons and liquid medium, Wherein the liquid medium for obtaining the mycelium is at least one selected from the group consisting of MgSO4H2O, KH2PO4, K2HPO4, yeast extract, peptone, and dextrose Wherein the solid medium for obtaining the mycelium is at least one selected from the group consisting of rice, potato, corn, and cocoon pupa, and a liquid culture medium containing at least one selected from the group consisting of rice, potato, maize, .

According to another embodiment of the present invention, the extract obtained from the mycelium or the fruiting body is fermented by the lactic acid bacteria.

According to another embodiment of the present invention, the natural Chinese cordycem complex is extracted from a mycelium or fruiting body cultured in a liquid medium, a solid medium or a feces and contains 2.0 to 8.0 wt% of water, 2.0 to 3.5 wt% of crude fat, wt% of crude protein, 5.0 to 8.0 wt% of extract components such as minced minerals; And an additive comprising cellulose, pectin, carrageenan, starch, protein, enzyme or chitosan, and is spread on fruit vegetables or added to foods for preservation or for natural antimicrobial preservation.

According to another embodiment of the present invention, the Chinese Cordyceps native compound is extracted from Cordyceps mellifera and comprises 2.0 to 8.0 wt% water, 2.0 to 3.5 wt% crude fat, 30 to 50 wt% crude protein, 5.0 to 8.0 wt% A base material comprising min. And additives such as green tea, garlic, coconut, grape or herbal medicine to enhance the antibacterial effect.

The mycorrhizal fungus cultivated in accordance with the present invention cultivated according to the present invention has a large antibacterial activity, and when the above-mentioned Cordyceps sinensis extract is mixed with green tea, garlic, coconut and grape, It can be used as a natural antimicrobial preservative. The extract of Codispein can also be used as a preservative for inhibition of browning and decay in storage and distribution of fruit and vegetables. In addition, the addition of auxiliary substances such as cellulose, pectin, cariguanan, starch, protein, enzyme and chitosan as a natural polymer can further maintain the lead retention time. This makes it possible to replace physically processed or artificial synthetic preservatives used for preservation or maintenance of the known art. Codisepin extracts extracted by fermentation methods can be applied to foods such as hamburgers, fish cakes or chilled meats, and do not affect the pH of the food itself and are not toxic. The extract of Codispein according to the present invention has high stability and sensory characteristics.

The culture method according to the present invention is a method for cultivating Cordyceps sinensis by cultivating a vegetable brown rice based on an existing animal larva or a pupa, and is characterized by its antimicrobial and antioxidant activity, hypoglycemic action, Such as suppression, fatigue and liver protection, asthma action, radiation protection effect, etc., which are known to improve the expression level of cordycepin. Such an effect is achieved by fermentation of Lactobacillus acidophilus, and the extract or complex according to the present invention enhances the freshness maintenance and storage ability of the food through the combination of herbal medicines and food. In addition, the extract or complex according to the present invention exhibits excellent antimicrobial effects by the addition of natural products such as green tea, garlic, coconut or grape, thereby allowing the lactic acid fermented caterpillar fungus complex extract to be substituted for sulfur treatment. In addition, it was found that more than 80% of the participants in the sensory evaluation of lactic acid fermented caterpillar fungus extract did not feel any difference from the untreated control, or did not feel it in the case of single ingestion, It is effective to maintain.

Figs. 1A to 1I show the results of an efficacy test of an antibacterial preservative of an extract according to the present invention. In FIGS. 1A to 1I, the control (control) is an antimicrobial preservative prepared from naturally-produced Cordyceps Extract, the storage time is in days, A, B and C are hamburgers, Or a refrigerated surface.
Figs. 2A to 2C show the results of the pH effect according to the treatment of the extract according to the present invention. 2A to 2C, the control (control) is an antimicrobial preservative prepared from naturally-produced Cordyceps sinensis extract, the storage time is in days, A, B and C are hamburgers, Or a refrigerated surface.
3A to 3C show the results of acidity change according to the treatment of the extract according to the present invention. In FIGS. 3A to 3C, the control (control) is an antimicrobial preservative prepared from a naturally occurring Chinese cabbage extract, storage time is in days, A, B and C are hamburgers, Or a refrigerated surface.
4A to 4C show progress of the sensory test according to the treatment of the extract according to the present invention. In FIG. 4A to FIG. 4C, the control group is an antimicrobial preservative prepared from a naturally-produced Cordyceps mellifera extract. The storage time is one day, and the storage period is 0, 1, 2, 3 and 4 days and at a temperature of < RTI ID = 0.0 > 12 C. < / RTI >
FIGS. 5A to 5D show the toxicity test results of the extract according to the present invention, and FIGS. 5A and 5B show the results of treatment with A375 cells and HaCat cells at a concentration of 1%, respectively.
Figs. 6A to 6C and Figs. 7A to 7E show the results of the efficacy test of the lead retention agent of the extract according to the present invention. FIG. 6A shows the evaluation results of moisture evaporation inhibiting power for 0, 24 and 72 hours, showing untreated, commercially available coating materials and treatment results according to the present invention. Figures 7a and 7c show the results after 5 days, 10 days and 20 days after inoculation of the isolates, respectively, and Figures 7d and 7e show the results after 1 to 6 days and 16 to 22 days after inoculation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the embodiments shown in the accompanying drawings, but the present invention is not limited thereto. In the following description, components having the same reference numerals in different drawings have similar functions, so that they will not be described repeatedly unless necessary for an understanding of the invention, and the known components will be briefly described or omitted. However, It should not be understood as being excluded from the embodiment of Fig.

The method for cultivating Cordyceps sinensis according to the present invention comprises the steps of: obtaining a mycelium by inoculating a liquid medium, a solid medium or a solid medium with Coryseps milliliter or Corysepsu nutsan gum; And a step of culturing the obtained mycelium in a solid medium or a medium to form a fruiting body, wherein the solid medium for forming the fruiting body comprises corn and a liquid medium or comprises rice, silkworm cocoons and liquid medium, A content of 50 to 70%, a relative humidity of 80 to 95%, an ambient temperature of 15 to 25 캜, and a light intensity of 700 to 1200 lux.

The caterpillar fungus cultivated in the medium is in the form of a mycelium or fruiting body, and the mycelium or fruiting body can be separated from the culture medium. And the active ingredient can be extracted from the mycelium or fruiting body of the separated cordyceps. As described above, the mycelium or fruit body extract obtained through the fermentation process can be added to foods, medicines, cosmetics, fibers or daily necessities so as to exhibit antimicrobial, preservative, leading-preserving or antioxidant functions. The mycelium or fruiting body of the caterpillar fungus cultivated in the medium contains the same components as the natural caterpillar fungus and has been found to contain the necessary codistin components for antimicrobial preservation or maintenance. According to these results, the fermented Cordyceps sinensis extracts of the fermented Cordyceps sinensis extracts were found to contain the same or more effective ingredients as the Cordycepsin extract of the natural cordyceps and could replace synthetic preservatives.

This is explained in detail below.

1. Culture of mycelium

The mycelium can be obtained by culturing in a liquid medium, a solid medium, or a medium, and a medium for liquid medium, solid medium, or humor can be produced in various ways.

end. Mycelial culture in liquid medium

Cordyceps militaris or Cordyceps nutans were inoculated into 20 ml of seven liquid media and cultured at 24 to 25 ° C, aeration of 1.0 ± 0.5 vvm, pH of 5 ± 0.5 vvm , And cultured for 7 days. The mycelia were filtered from the culture solution and dried at 80 ° C for 2 hours to obtain dried mycelia.

badge Badge 1 Badge 2 Badge 3 Badge 4 Badge 5 Badge 6 Badge 7 Sugar 2 One 2 Malt extract 2 0.3 potato 20 20 Yeast extract 0.2 0.3 peptone 0.2 0.5 MgSO4.7H2O 0.05 0.015 0.05 KH₂PO₄ 0.046 0.015 0.1 K2HPO4 0.1 Dextrose 2 One 2 CaCl₂ 0.005 NaCl 0.00025 FeCl3 (1%) (ml) 0.12 Thiamine-HCl 0.000001 Ebios 0.5 0.5 Hyponex 0.3 water 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml

≪ Liquid medium composition for culturing mycelium (unit: g) >

The amount of dried mycelium cultured in each medium is as follows.

badge Codiceceps militaris
(C. militaris) Cody Sens Nutans
(C. nutans) Badge 1 0.196 0.198 Badge 2 0.134 0.128 Badge 3 0.262 0.262 Badge 4 0.092 0.102 Badge 5 0.164 0.172 Badge 6 0.560 0.342 Badge 7 0.120 0.230

≪ Liquid medium The dried mycelium (g / 20 ml)

I. Culture of fermented mycelium in solid medium

In the solid medium, the mycelium can be obtained from the mycelium cultured in the liquid culture medium, for example, the mycelium cultured in the medium such as the liquid culture medium 3 can be inoculated into the solid culture medium to obtain the mycelium. The solid medium may be maintained at 70 to 90% humidity, at a temperature of 20 to 30 占 폚, and may be cultured for 25 to 40 days.

Solid medium composition Rice 5 g + liquid medium 50 ml Potato 5g + liquid medium 50ml Corn 5 g + liquid medium 50 ml Rice 5g + corn 5g + liquid medium 50ml Rice 50g + cocoon cocoon pupa 5g + liquid medium 75ml

≪ Solid medium composition for culturing mycelium >

All. Mycelial cultures from the trunk

Mycelial cultivation in the trunk can be carried out under the same or similar conditions as mycelial cultures in solid medium.

Composition of trophic culture medium Cocoon cocoon pupa 20g + sterilized water 100ml Cocoon Pupa 20g Cocoon pupa 20g + water 20ml

≪ Composition of donor medium for culturing mycelium >

The culture conditions of the mycelium in the liquid medium, the solid medium and the saline medium were confirmed, and the result of mycelial culture in the solid medium was difficult to separate completely.

Solid medium Mycelial growth Rice 5 g + liquid medium 50 ml + Potato 5g + liquid medium 50ml ++ Corn 5 g + liquid medium 50 ml +++ Rice 5g + corn 5g + liquid medium 50ml +++ Rice 50g + cocoon cocoon pupa 5g + liquid medium 75ml +++

<Mycelium of Cordyceps caught in solid medium>

Bulbous medium Mycelial growth Cocoon cocoon pupa 20g + sterilized water 100ml +++ Cocoon Pupa 20g ++ Cocoon pupa 20g + water 20ml +++

<Mycelium of caterpillar fungus cultivated in the trunk>

+: 10-30% mycelial formation

++: 30 to 70% mycelial formation

+++: more than 70% mycelial formation

As can be seen from Tables 3 to 6, the mycelium was best cultured in solid medium containing 50 ml of liquid medium in 5 g of corn and showed good growth in solid medium made of 5 g of rice + 5 g of corn + 50 ml of liquid medium . In addition, the growth of the mycelium in the larva cultured medium was found to be best when 20 g of the silkworm cocoons and 100 ml of sterilized water were added.

2. Cultivation of fruit bodies by fermentation

end. Fruit body formation in solid medium

Fruiting bodies were cultured from mycelia cultured in solid medium and fruiting bodies were formed in solid medium. For the fruiting body, the solid medium was maintained at a water content of 50-70%, a relative humidity of 80-95% and an ambient temperature of 15-25 ° C, and mycelia was cultured for 60-90 days at a light intensity of 800-1500 lux, It was made.

Solid medium Mycelium formation Rice 5 g + liquid medium 50 ml + Potato 5g + liquid medium 50ml ++ Corn 5 g + liquid medium 50 ml +++ Rice 5g + corn 5g + liquid medium 50ml +++ Rice 50g + cocoon cocoon pupa 5g + liquid medium 75ml +++

+: 10-30% fruiting formation

++: 30 to 70% fruiting body formation

+++: more than 70% fruiting

The fruiting bodies cultured on the solid medium were difficult to separate from the solid medium, and the fruiting bodies were visually observed, and the formation area ratio of fruiting body per area of the fruiting bodies was measured to measure the formation level of fruiting body.

I. Fruiting body formation in the trunk

Fruit bodies were formed by inoculation of mycelia cultured in solid media and maintained in the same condition as fruiting body in solid medium with culture condition of fruiting bodies in the rumen.

Bulbous medium Mycelial growth Cocoon cocoon pupa 20g + sterilized water 100ml +++ Cocoon Pupa 20g ++ Cocoon pupa 20g + water 20ml +++

&Lt; Cordyceps sinensis cultivated in the trichomes >

+: 10-30% fruiting formation

++: 30 to 70% fruiting body formation

+++: more than 70% fruiting

As can be seen from Tables 7 and 8, the fruiting body was best in a solid medium containing 50 g of rice + 5 g of silkworm cocoon pupa and 75 ml of liquid medium. Solid medium containing 5 g of rice, 5 g of corn and 50 ml of liquid medium, The culture was found to be good in the order of solid medium supplemented with 5 g rice + 50 ml liquid medium, solid medium supplemented with 5 g potato and 50 ml liquid medium. When a fruiting body is formed by using a fertilization medium, it can be seen that the best culture condition is obtained in a solid medium supplemented with 20 g of silkworm cocoon pupa and 20 ml of agar.

3. Analysis of components of mycelium and fruiting bodies

The components of mycelia or fruiting bodies fermented in a liquid medium, a solid medium or a saliva were analyzed. The mycelia cultured in the liquid medium were filtered and lyophilized, and fruiting bodies were obtained and lyophilized. The collected mycelium and fruiting bodies were analyzed for mycelia and fruiting bodies according to crude-protein measurement using Micro-Kjeldhl, crude fat measurement using Soxhlet extraction method, and inquiry method using heat and dry weight measurement, respectively, The components were analyzed. In addition, the components of the randomly selected wild cordyceps were analyzed and compared according to the same method. In the case of wild cordyceps, it was difficult to separate mycelium and fruiting body, and the total components of mycelium and fruiting body were analyzed together.

Cordyceps sinensis component mycelium Fruit body Additive-free moisture 2.8 6.0 10.84 Crude fat 3.07 2.08 8.4 Crude protein 40.36 30.93 25.32 Views min 7.51 5.32 4.1

<Cultivation Compared with the composition of mycelium and fruiting body of Cordyceps mellifera and the wild caterpillar fungus (Unit:%)>

From the results shown in Table 9, it was found that the components of the wild-type Cordyceps, the mycelium fermented according to the present invention, and the components of fruiting bodies were similar to each other. Therefore, it can be seen that the mycelium and the fruiting body according to the present invention are artificial fermentation culture forms of Cordyceps sinensis. Accordingly, it can be seen that the mycelia and fruiting bodies according to the present invention can be used as an antimicrobial preservative or a leading preservative based on the cordycepin component. Furthermore, if natural fermentation is added to enhance the efficacy of Codispein in cultured media and cultures and to incorporate fermentation technology and to extract green tea, garlic, coconut, grape, or natural antimicrobial preservation or maintenance, It was found that the retention ability can be improved.

In accordance with the present invention, further tests have been conducted for the efficacy of mycelia and fruiting bodies as antibacterial preservatives or lead preservatives.

4. Efficacy Test

Antimicrobial preservatives and starch preservatives were prepared from the mycelia and fruiting bodies extracted for efficacy tests and classified into BHC-G3 without acetic acid and BHC-F3 with acetic acid. Antimicrobial activity, antioxidant activity (DPPH), MIC and physiological activity, sensory evaluation or preservative effect were tested for each.

end. Antimicrobial preservative efficacy test

Three food groups were selected to test the efficacy of the cultured mycelium and fruiting body as an antimicrobial preservative. An accelerated storage test at 12 ° C for selection of the type and concentration of the antimicrobial preservative was used to determine storage stability (microbial and sensory Quality) test was conducted.

A. Preservative efficacy

The criteria for the hamburger patty, fish cake and cold side were set according to the results of MIC (Minimum Inhibitory Concentration) test, and tests for 1/2 × MIC and 2 × MIC concentration were added.

- hamburger patty: 0.200, 0.400 and 0.800 wt%

- Fish cake: 0.225, 0.450 and 0.900 wt%

- Refrigerated side: 0.100, 0.200 and 0.400 wt%

BHC-G3 and BHC-F3 were mixed in 10 ml of water to the concentrations specified above, and processed into hamburger patties, fish cake and cold surfaces to make 10 g each, placed in a zipper bag and mixed at 0, 2, 4 and 6 The results were observed while being stored for days. The results were analyzed using the ANOVA procedure of the SAS statistical program (Version 9.1, SAS Institute, Cary, North Carolina, USA). The results were analyzed by microbiological characterization, pH measurement, titratable acidity and sensory evaluation . The mean value was calculated using Duncan's multiple range test when each treatment showed a statistically significant difference (p ≤ 0.05).

a. Analysis of microbiological quality characteristics

Rojas-Graua et al. (Bagmixer® 400, Interscience®, USA) were mixed with 20 mL of sterile 0.2% peptone water (PW, Difco, Detroit, MI, USA) after taking 10 g of the sample aseptically in a sterile bag (3M) , France) for 90 seconds. 0.1 mL of each sample solution was taken and stepwise diluted into 9.9 mL of diluent. Step 1 Dilute 1 mL of the solution with 3M ™ aerobic counting Petrifilm (3M, St Paul, MN, USA) and incubate at 37 ° C for 24-48 hours. The number of colonies was measured and expressed as log CFU / g. The yeast / mold counts are prepared by diluting the yeast / mold samples in the same manner as the total number of microorganisms, and gradually diluting them in 1 mL each of 3M ™ Yeast and mold Petrifilm, which is a dry medium, at 25 ° C. for 3-5 days, Respectively. The samples were pretreated in the same manner as the Escherichia coli / Escherichia coli total bacterial counts, and then diluted in a stepwise manner. 1 mL of each was aseptically dispensed into 3M ™ E. coli / Coliform Petrifilm, a dry medium film, And the number of colonies was measured.

The measurement results are shown in Figs. 1A to 1I.

Figs. 1A to 1C show the total number of bacteria, E. coli / E. coli, yeast / fungus counts when an antibacterial preservative is added to a hamburger patty. G3 and F3 were added at concentrations of 0.200, 0.400 and 0.800 wt% and stored at 12 ℃ for 0, 2, 4, and 6 days. When 0.83 wt% of G3 was added, total bacterial counts were 0.83-1.89 log CFU / g, which is lower than that of the control. In particular, it can be seen that the addition of 0.800 wt% of G3 inhibited the yeast / fungal count to the detection limit (≤ 0.48 log CFU / g) during 6 days of storage. When F3 was added at 0.800 wt%, total bacterial count was inhibited by 6.54 log CFU / g on the 6th day of storage. However, when 0.83 wt% of F3 was added, the yeast / mold number was effective until the 2nd day of storage, but it was abruptly increased from the 4th day of storage and showed an insufficient effect on the yeast / mold number. Therefore, the addition of 0.83 wt% of G3 to hamburger patty is the most effective microbiological safety until 4 days storage.

Figs. 1D to 1F show total bacterial counts, Escherichia coli / Escherichia coli counts, and yeast / fungal counts when an antibacterial preservative was added to fish paste. G3 and F3 were added at concentrations of 0.225, 0.450 and 0.900 wt% and stored at 12 ℃ for 0, 2, 4 and 6 days. The total bacterial count was inhibited by 7.42 log CFU / g on the 4th day of storage compared to the control, and the total bacterial count was rapidly increased on the 6th day of storage, but the total bacterial count was lower than that of the control by 2.78 log CFU / g . The addition of 0.900 wt% F3 showed that the total bacterial count was effectively reduced to the detection limit (≤ 0.48 log CFU / g) at 6 days of storage. Escherichia coli / coliform strain was lower than that of the control, but there was no significant difference (p> 0.05). Yeast / fungal counts were found to be effective when G3 and F3 were added at concentrations of 0.450 and 0.900 wt%, which was 3-4 log CFU / g lower than that of the control at 6 days of storage. Therefore, adding 0.900 wt% of G3 or F3 to fish cake is the most effective for microbiological safety.

FIGS. 1G to 1I show the total number of bacteria, E. coli / E. coli, and yeast / mold counts when an antimicrobial preservative is added to a refrigerated surface. G3 and F3 were added at concentrations of 0.100, 0.200 and 0.400 wt% and stored at 12 ℃ for 0, 1, 2 and 3 days. On the 3rd day of storage, it was found that F3-1 was treated at 0.400%, and E. coli / E. coli was effective at a level of 5.50 and 1.99 log CFU / g, respectively, lower than the control. However, the number of E. coli / Escherichia coli and yeast / mold in the G3 treatment was lower than that of the control, but the effect of total bacteria was insufficient. Therefore, the addition of F3 0.400 wt% in the case of refrigerated surfaces proved to be most effective in terms of microbiological safety.

b. pH measurement

The pH was measured by using a digital pH meter (Beckman Counter, model pH, USA) after 10 g of sample and 20 mL of distilled water were homogenized by a stomacher for 90 seconds according to the method of Kwak et al. And the results are shown in Figs. 2A to 2C.

2A to 2C, it can be seen that the initial pH of the hamburger patty, fish paste and chilled surface are 5.98, 6.92, and 5.84, respectively, and G3 does not cause pH change at all concentrations. When the F3 2x MIC concentration was treated at 0 day of storage, the pH of the hamburger patty, fish cake and cold side decreased to 0.07, 0.78, and 0.90, respectively, but there was no substantial change. Therefore, no substantial change in pH is observed during storage as a whole, and it is considered that G3 and F3 do not affect pH on food.

c. Titratable acidity measurement

The titratable acidity was determined according to the AOAC Standard Test Method. For this purpose, 10 g of sample and 20 mL of distilled water were placed in a sterilized bag and homogenized for 90 seconds with a Stomacher. Then, 1 ~ 2 drops of 1% phenolphthalein (77-09-8, Duksan pure chemicals, Gyeonggi-do, Korea) was added to 10 mL of the homogenized sample and 0.1 N NaOH (S0610, Samchun pure chemical, Gyeonggi-do , Korea). The state in which the red color was maintained for 30 seconds in the colorless state was regarded as the end point, and the amount of consumed 0.1 N NaOH was measured to calculate the content (%) of the organic acid.

In the above formula

- Vol. of NaOH (ml) = Total volume of NaOH used for titration

- N = Normality of titration (NaOH)

- Acid meq factor = miliequivalent weight of acid

Respectively.

The measurement results are shown in Figs. 3A to 3C. 3A to 3C, the initial acidity was found to be 0.10, 0.11, and 0.02% when the antimicrobial preservative was added to the hamburger patty, fish cake, and cold storage, and the change in pH was not substantially observed in all treatments during the storage period . Therefore, the addition of G3 and F3 to the hamburger patty, fish cake, and chilled surface showed no change in acidity.

d. Sensory test

The test was conducted on 100 hamburger patties, fish cake, and cold noodles, and the sensory evaluation was performed with a 5-point categorization divided into appearance, color, flavor and overall preference. The higher the score, the better the taste Was added. The sensory test results are shown in Figs. 4A to 4C.

FIG. 4a shows the result of sensory evaluation when the antibacterial preservative was added to the hamburger patty. In the case of storage at 0 day, G3 and F3 showed a negative effect on taste by 1.75 and 2.50 points at 0.800 wt% treatment, respectively. On the contrary, the appearance, color, flavor and overall acceptability were found to be acceptable at more than 3.00 points in all treatments. In case of storage 6 days, F3 (0.800 wt%) showed higher score than control. Also, in case of 0.800 wt% treatment of G3, all the evaluation items showed acceptable level of 3.00 or more.

Fig. 4b shows the result of sensory evaluation when the antimicrobial preservative was added to the fish paste. In the case of 0 day of storage, G3 0.900 wt% F3 was 2.00, 2.67 and 2.50 points at 0.450 and 0.800 wt%, respectively, appear. However, appearance, color, and aroma were found to be acceptable at more than 3.00 points in all treatments. However, overall acceptability was lowered to 2.86 points for F3 0.450 and 0.900 wt% treatment, respectively. G3 showed higher score than other treatments during storage at 0.900 wt%, and it was found that 6 days of storage had a similar or higher score to the control than the control.

FIG. 4c shows the result of sensory evaluation by adding an antimicrobial preservative to the refrigerated surface. In the case of storage 0 days, the taste was found to be at an acceptable level of 3.00 or more in all treatments. Appearance and color were 3.43 ~ 4.29 which were similar or slightly lower than the control, and all were acceptable. However, on the 0th day of storage, the odor and overall acceptability were not acceptable at 2.00 and 2.86 points for F3 0.400 wt% treatment, respectively. In the case of 6 days storage, G3 0.100 wt% treatment showed acceptable values of over 3.00 points in all evaluation items. The appearance and color of F3 at 0.200 and 0.400 wt% were higher than those of control at 3.00 or more, indicating that they had good effect on sensory properties.

According to the above results, it can be seen that addition of G3-1 0.800% and 0.900% for hamburger patty and fish cake does not affect the sensory properties except for taste, and for the refrigerated surface, G3 0.100 wt% or F3 0.200 wt % Is added, it can be understood that no influence is exerted on the sensory properties.

B. Toxicity test

The ability of mitochondria to reduce MTT tetrazolium insolubles by aqueous dehydrating enzymes by dehydrating enzymes was tested by MTT assay. The absorbance of the untreated sample was measured, and then each sample was treated and tested for toxicity by measuring the absorbance at 540 nm with a microplate reader, and the test results were shown in FIGS. 5A to 5D.

Referring to FIGS. 5A to 5D, when treated with 1 wt% of G3, the keratinocyte HaCaT cell showed an increase in cell growth and no toxicity. In case of treatment with 1 wt% of F3, keratinocytes, which are normal cells, and cancer cells that produce melanin, did not affect cell growth.

I. Lead preservative

To test the leading preservative function of G3 and F3, a test was conducted on the fruits and vegetables, and the results are shown in Figures 6a to 6c.

Referring to FIGS. 6A to 6C, it can be seen that the treatment of cereals with G3 or F3 and subsequent treatment with natural food preservatives is advantageous for germ control. Specifically, it can be seen that the effect of the natural food preservative is improved by treating and processing the agricultural products for the function of the lead preservative, G3 or F3.

For the lead preservative zero efficacy test, the receptacles were prepared so that the concentration of G3 or F3 was 3 to 6%, the perspex was immersed for 50 to 80 seconds and then dried. On the 2nd day, spores were obtained by adding sterilized water to a plate culture medium in which two kinds of fungi isolated from the persimmon germ and one strain of the strains were cultured. Then, the obtained fungus spores were diluted so that the concentration was controlled by confirming the number of bacteria through a microscope or a smear, and then inoculated into the persimmons by several times. After the inoculation, they were placed in a container while keeping the humidification condition, and the results were shown in Figs. 7A to 7E.

FIGS. 7A to 7C show the results of the 5th, 10th, and 20th day observation, respectively, and FIGS. 7D and 7D show the results of Day 1 to 6th day and 16th to 22nd day of the measurement.

7A to 7E, it can be seen that fungi are generated before the fifth day in the case of no treatment in severe conditions such as fungal inoculation and humidifying conditions, and that fungi are hardly generated in case of sulfur treatment, BHC-F3 and BHC-G3 Able to know. Specifically, in the case of no treatment

The number of onset individuals: 8/10 and the incidence rate of fungi: 40% in total, and the number of onset cases in BHC-G3: 1/10. On the 20th day of the fungus inoculation, it can be seen that the number of onset individuals: 10/10, the incidence area rate: 20%, the number of onset individuals: 8/10, and the incidence area rate of BHC-F3 are 40%. Therefore, it can be seen that under severe conditions, BHC-F3 can replace sulfur treatment. In the case of BHC-F3, more than 80% of the participants in the sensory evaluation did not feel any difference from the control group or did not feel it in the case of single intake.

The mycelium or cordierite extract of Cordyceps sinensis cultured according to the present invention can be used as a natural antimicrobial preservative having a large antimicrobial activity and a wide antimicrobial range. In addition, the fermentation technology and the fermentation solution and the fermentation which increase the efficiency by adding the natural polymer to the natural antimicrobial preservation or the maintenance of the leading, by combining the fermentation technology and the green tea, garlic, coconut, Codisepin extracts from water can be used as a preservative for inhibition of browning and decay in storage and distribution of fruit and vegetables. This makes it possible to replace physically processed or artificial synthetic preservatives used for preservation or maintenance of the known art. Also, the extracted Codispein extract can be applied to foods such as hamburger, fish cake or chilled surface, and does not affect the pH of the food itself and is not toxic. The extract of Codispein according to the present invention has high stability and sensory characteristics.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention . The invention is not limited by these variations and modifications, but is limited only by the claims appended hereto.

BHC-G3: non-acetic acid extract
BHC-F3: Acetic acid-added extract

Claims (4)

Obtaining a mycelium by inoculating a liquid medium, a solid medium or a saliva with Coryseps milliliter or Coryseps naturus; And
Culturing the obtained mycelium in a solid medium or a medium to form a fruiting body,
The solid medium for forming the fruit bodies is composed of corn and a liquid medium or comprises rice, silkworm cocoons and liquid medium and has a moisture content of 50 to 70%, a relative humidity of 80 to 95%, an ambient temperature of 15 to 25 캜, 700 to 1200 lux,
The liquid medium for obtaining the mycelium comprises MgSO4 占 H2O, KH₂PO₄, K₂HPO₄, yeast extract, peptone and dextrose, or a yeast extract, dextrose, ebiose and hyponex. The solid medium Wherein the culture medium comprises at least one selected from the group consisting of rice, potato, corn and silkworm pupa, and a liquid medium. [Claim 4] The method according to claim 1, further comprising the step of fermenting the extract obtained from the mycelium or fruiting body with lactic acid bacteria. Such as from 2.0 to 8.0 wt% of water, from 2.0 to 3.5 wt% of crude fat, from 30 to 50 wt% of crude protein, from 5.0 to 8.0 wt% of crude protein extracted from mycelia or fruiting bodies cultured in liquid medium, solid medium or embryo Extracting component; And
Cellulose, pectin, carrageenan, starch, protein, enzyme or chitosan,
Natural compound of caterpillar fungus that is sprayed on fruit vegetables or added to food for preservation of leading or natural antimicrobial preservation. A base material extracted from Cordyceps militaris and comprising 2.0 to 8.0 wt% moisture, 2.0 to 3.5 wt% crude fat, 30 to 50 wt% crude protein, 5.0 to 8.0 wt% ash; And
A natural compound of caterpillar fungus including caterpillar fungus including additives such as green tea, garlic, coconut, grape or medicinal herb to enhance antimicrobial activity.

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