KR20090090855A - Large-scale production of b-glucan through semi-continuous fermentation performed with sparassis crispa mycelia - Google Patents

Abstract

A method for culturing a Sparassis crispa mycelia is provided to massively produce Sparassis crispa mycelia and shorten culture time through semi-continuous culture of liquid phase. A medium for culturing Sparassis crispa mycelia in liquid phase comprises 0.01~3.0% of glucose, 0.01~0.5% of yeast extract, 0.01~0.2% malt extract, 0.01~1% of potassium monophosphate, and 0.01~0.04% of iron sulfate. A method for culturing the 0.01~0.04% in liquid phase comprises: a step of germinating a spore of Sparassis crispa mycelia; a step of culturing in liquid phase medium at 23‹C, 200 rpm of shaking speed for seven days; and a step of collecting beta-glucan from Sparassis crispa mycelia.

Description

Large-scale production of b-glucan through semi-continuous fermentation performed with Sparassis crispa mycelia}

The present invention relates to a method for culturing mushroom mycelium, and to a liquid culture method of the mycelium mushroom mycelium.

Commonly blossomed mushrooms ( Spanrassis crispa Wulf. ex Fr.) is a fungus with two species of genus 1 and 1 genus. In English, cauliflower mushroom or crested sparassis, the Japanese name Hanabiratake, and the Chinese name water plum.

The blossom mushroom is when the fruiting body matures, the whole is 95-225x110-285mm large, somewhat round, small petal-shaped shades gather to form a cabbage-seaweed. The stems are 25-55x 28-46mm, short, blunt, hard, and repeatedly cracked upward to form numerous short branches, and the branches become flat, thin, wavy petals. The top of gat is smooth and white-pale yellow but yellow after growth. The fruiting layer is on the back or bottom of each small lampshade, and is smooth, initially pale yellow, yellow when grown, and brown when homeless. The tissue is thin, elastic, flexible, fleshy, and white.

As a result of analyzing the composition of the blossom mushroom in March 2001, the blossom mushroom was found to contain 43.6% (43.6 g of 100 g) of beta-glucan in the dried blossom mushroom. Such a flower mushroom may be a beta-glucan lump, and the beta-glucan has been found to correct the body's immune system as a key component for enhancing the body's immunity, and to control cancer, high blood pressure, diabetes, and the like.

In addition, the blossom mushroom is known to not only lower blood pressure or return blood sugar to normal in addition to excellent anti-cancer effect, but also has a hematopoietic effect, but since the amount that can be captured in the natural state is extremely low, efforts to artificially cultivate it continue. come.

On the other hand, Japanese Patent Laid-Open No. 11-56098 discloses a method of cultivating zinnia mushrooms using a medium containing nutrients after extracting hot water from larch and removing soluble ingredients, and Korean Patent Publication No. 2002-48337 In the artificial culture method of mushroom, activated carbon and activated calcium were added to the medium mixed with larch and broadleaf tree, and the temperature change when cultivating the mushroom mushroom mycelium after sterilizing the medium containing nitrogen source commonly used at high temperature and high pressure. Artificial cultivation method of the blossom mushroom has been disclosed.

In addition, the method of cultivating the artificial mushrooms of the blossom mushroom as well as the Korean Patent Publication No. 2004-0071926 'culture method of the mushroom mushroom mycelium' is not an artificial cultivation for producing the fruiting body of the blossom mushroom, but simply using a mycelium of the mushroom mushroom Liquid phase culture methods have been disclosed.

However, the artificial cultivation method of the flower mushroom as described above has a problem that a large number of mycelium does not develop into fruiting body or fruiting body during the cultivation period, the fruiting body produced is small because it does not meet the proper conditions during fruiting hypertrophy growth, blossoming Simple liquid culture method using a mycelium of mushrooms has a problem that the final productivity of the flower mushroom mycelium that can be obtained in one culture because the culture period from growth culture to production culture is long.

In order to solve the above problems, an object of the present invention is to increase the productivity of the blossom mushroom mycelium while significantly reducing the total incubation period compared to the conventional cultivation of the blossom mushroom mushroom and batch culture of the blossom mushroom mycelium in order to mass-produce mycelium of the blossom mushroom This is to provide a semi-continuous culture process more than three times increased.

As described above, the artificial cultivation method of the flower mushroom has a problem that a large number of mycelium does not develop into fruiting body or fruiting body during the incubation period, and the fruiting body is small because the fruiting body does not achieve proper conditions during fruiting growth. Simple liquid culture method using a mycelium of mushrooms has a problem that the final productivity of the flower mushroom mycelium that can be obtained in one culture because the culture period from growth culture to production culture is long.

The present invention relates to a liquid culture method of mushroom mycelium, and more particularly, to a method for mass production of mycelium through semi-continuous liquid culture using beta-glucan high content of agar mushroom. The semi-continuous liquid culture resulted in a productivity increase of about 235% compared to the conventional method.

The present invention is directed to the development of a semi-continuous mycelium liquid culture method for producing a high concentration of beta-glucan zinnia mushroom. The content of the invention will be described in more detail by way of examples. However, these examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Liquid Culture Method of Blossom Mushroom Mycelium

(1) germinating spores of the flower mushroom to obtain mycelium;

The mycelium of zinnia mushroom was used as a mycelium obtained by germinating spores obtained from the fruiting body of zinnia mushroom collected in the wild. Obtained by yeast-potato starch-glucose agar medium (3g yeast extract, 10g potato starch 10g glucose, 15g agar, 1L distilled water) and incubated at 23˚C for 15 days. On the other hand, the mycelium is inclined in a test tube containing yeast-potato starch-glucose agar medium and stored at 4˚C, subcultured every month to use.

(2) culturing the mycelium in a liquid medium;

The mycelia grown on the slope medium are aseptically collected and then inoculated in a liquid medium at 10% (v / v). As a liquid medium, 3% glucose, 0.5% yeast extract, 0.2% malt extract, 1% potassium monophosphate, and 0.04% iron sulfate have good mycelial productivity when using media adjusted to 4.5. Liquid culture of the mycelium is incubated for 7 days under the conditions of 23 ° C., agitation speed 200 rpm, aeration rate 1vvm in a fermenter.

(3) beta-glucan recovery step from the flower mushroom mycelium;

Centrifuge the culture medium in which the mycelium mushroom mycelium was incubated at 10min, 8000rpm to collect only the cells and then dried for 90˚C, 12hr to obtain the cells from which moisture is removed. Thereafter, the cells were extracted with hot water for 150 ° C. for 2 hr, and the beta-glucan was concentrated using an ultrafiltration membrane having a fraction molecular weight of 30 K. The final 3-fold volume of ethanol was added to recover the beta-glucan. The recovered beta-glucan is hot-air dried to volatilize the ethanol, and then frozen at -70 ° C and dried using a lyophilizer. The dried beta-glucan is pulverized with a grinder to prepare a powder sample.

Example 2 Determination of Optimal Production Conditions of Blossom Mushroom Mycelium by Simple Batch Cultivation and New Medium Exchange Time in Semi-Continuous Culture

In general, fungi grow in the form of mycelia and pellets during liquid culture, and when grown in pellets, the viscosity of the culture solution decreases to facilitate delivery of oxygen and nutrients to the mycelia. Therefore, in order to grow mycelium smoothly, the growth of pellets should be made. Inoculation, one of the factors affecting the growth of pellets, is an important factor for increasing the initial growth rate of cells and increasing the yield of the target product, and affects the size of the pellets to change the morphological characteristics. In addition, the initial pH of the medium affects the function of the cell membrane, the morphology and structure of the cells, the solubility of various salts in the medium, and the nutrient intake rate in the medium. In general, mycelial growth and production of the target product occur within a specific pH range, and there are cases where the optimum pH for mycelial growth and the optimum pH for the production of the target product are different. As such, pH is an important factor in mycelial growth and production of target products. Therefore, in this example, the inoculum was changed to 2.5%, 5%, 10%, 15%, and 20% to determine the optimal condition of the inoculum and the initial pH of the culture through simple batch culture using a 5L top fermenter. The initial pH of the medium was changed from 3 to 8 to investigate the effect on mycelial growth. As a result, as shown in FIG. 1 and FIG. 2, the inoculation amount showed the smoothest mycelial growth at 10%, and the mycelial production increased with increasing pH, showing the highest yield at initial pH 5. Therefore, the optimum production conditions through batch culture of the mycelium mushroom mycelium were determined as the inoculum 10%, the initial pH of the medium, respectively. In addition, in order to determine the feeding time of the new feed medium in the semi-continuous culture using the blossom mushroom mycelium under the optimum conditions, the growth curve and carbon source consumption of the blossom mushroom mycelium were cultured using a 5L top fermenter. It investigated by doing. As shown in FIG. 3, the mycelial growth was vigorous until the 5th day of culture, and after that, the concentration of the carbon source was sharply reduced, which acted as a stress on the mycelium, indicating that the mycelium growth was slowed. Therefore, the replacement time of the new feed medium for the semi-continuous cultivation of zinnia mushroom was determined on the fifth day of culture.

<Example 3> Determination of the exchange amount of the culture medium and fresh feed medium for the semi-continuous cultivation of blossom mushroom mycelium

 A semi-continuous culture method was developed to maximize the productivity of the zinnia mushroom mycelium. Semi-continuous culture is a production culture system that repeats the process of removing part of the culture medium (medium + bacteria) from the bioreactor and injecting fresh medium again during the culture period. Such a semi-continuous culture can be characterized as an advantage and the ability to obtain a continuous useful metabolite through long-term production culture without going through the growth culture step before the production culture. Therefore, the success of the semi-continuous culture can yield high metabolite productivity during the short incubation period, and furthermore, it can be expected to reduce the cost due to the reduction of the number of days of culture. In this example, 5L fermenter (operating volume: 3L) was used for semi-continuous culture. The first batch culture was carried out daily, and after 5 days, a certain percentage (70%, 80%, 90%) of the culture solution was removed as soon as possible with a metering pump, and a fresh medium was injected as much as the ratio of removing the medium (medium batch exchange). Semi-continuous culture). The secondary, tertiary and quaternary batch cultures were all carried out for 5 days for a total of 20 days of production culture, and samples were collected at 24 hour intervals. At the time of replacing the medium, each sample was collected and compared before and after the exchange. The experimental results of the semi-continuous culture are shown in FIG. 2. As a result, the yield of the flower mycelium showed the same productivity as the first batch culture after the completion of the fourth batch culture under the condition that the exchange amount between the culture medium and the new feed medium was 70%. On the other hand, at 80% and 90%, the mycelial production of the cultivated mycelium was the same in the first and second batch cultures, but the mycelial production decreased gradually in the third and fourth batch cultures. Therefore, the exchange amount of the culture medium and the new feed medium for the semi-continuous cultivation of the final blossom mushroom mycelium was determined to be 70%.

Example 4 Determination of Feed Rate of Fresh Medium for Semi-Continuous Culture of Blossom Mushroom Mycelium

Semi-continuous culture is a culture method in which the recovery of culture medium and cells and the introduction of a new medium are repeated repeatedly for a long period of time. In the case of strains in which beta glucan is unstable in productivity or easy to change in cell activity, the higher production strains become closer to the latter stage of culture. Low-growth, faster-growing strains or less-active strains or reverse mutations occupy most of the cells, resulting in reduced productivity at the end of the culture. Therefore, in the present embodiment, in order to slow down the chemical environment change of the culture medium generated when the new medium is injected, an intermittent semi-continuous culture in which a small amount of the medium is removed intermittently and then a small amount of the medium is injected daily. pulse feeding (PF)) and medium continuous injection (CF) to continuously inject a small amount of medium. The final volume injected was adjusted to the same amount. Fermenter conditions and the number of batches were the same as the semi-continuous culture conditions of Example 3. The experimental results for this are shown in FIG. 3. As a result, both of the above injection methods increased the mycelial production of zinnia mushroom every time the medium was exchanged, compared with the conventional semi-continuous culture method, especially when the medium continuous feeding (CF) was used. Mycelial concentration was up to 20g / L. As a result, the medium continuous injection and semi-continuous production culture developed by the researchers were able to significantly reduce the incubation time compared to the conventional batch culture. If the growth culture period is 10 days and the production culture period is 5 days, the total production from the semi-continuous production culture for 30 days will be 60 days. As a result, the same result can be obtained by reducing the incubation time of 30 days. In addition, the results of comparing the production of the mushroom mushroom mycelium obtained through the simple batch culture and the medium continuous injection semicontinuous culture in Table 1 showed that the medium continuous injection semi-continuous culture was more than twice as high as the batch culture.

TABLE 1

<500L standard> Monthly mycelial production (kg) Productivity increase rate (%) Simple batch culture 15 kg 100% Medium continuous injection, semi continuous culture 35.3 kg 235%

1 is a graph showing the production concentration of zinnia mushroom mycelium according to the inoculation amount through a simple batch culture,

2 is a graph showing the production concentration of zinnia mushroom mycelium according to the initial pH of the medium through a simple batch culture,

3 is a graph showing the concentration of the flower mycelium and the consumption of the carbon source by a simple batch culture method,

4 is a graph showing the production concentration of zinnia mushroom mycelium in the exchange amount of the culture medium and the fresh medium during the semi-continuous culture,

5 is a graph showing the production concentration of the mycelium mushroom mycelium according to the feed rate of the new medium when the culture medium and the new medium exchange during the semi-continuous culture,

Table 1 is a table comparing the monthly productivity of the mycelium mushroom mycelium according to the simple batch and semi-continuous culture method.

Claims (4)

In the liquid culture of the mushroom mycelium, Glucose 0.01 ~ 3.0%, yeast extract 0.01 ~ 0.5%, malt extract 0.01 ~ 0.2%, potassium monophosphate 0.01 ~ 1%, iron sulfate 0.01 ~ 0.04% pH is adjusted to 3.5 ~ 7.5 culture of mushroom mycelium Way The method of claim 1, A method for cultivating mycelium mushroom mycelium through a medium single-stage semi-continuous culture for exchanging medium of 0.1% to 99.9% (v / v) of the culture volume. The method of claim 1, A method for cultivating mycelium mushroom mycelium through medium intermittent injection and semi-continuous culture for exchanging medium of 0.1% to 99.9% (v / v) of the culture volume. The method of claim 1, A method for cultivating mycelium mushroom mycelium through the medium continuous injection semi-continuous culture to exchange the medium of 0.1% ~ 99.9% (v / v) of the culture volume.

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