KR100907296B1 - Rodotorula sp. JSU150-13 with high productivity of coenzyme Q10, and the producing method of Coenzyme Q10 using the strain - Google Patents

Abstract

The present invention relates to a new strain belonging to the genus of Rhototolura producing coenzyme Q10, and more particularly, to a very good new strain JSU150-13, which has a 200 times higher productivity in liquid culture compared to conventional Rhototolura species. And a method for producing coenzyme Q10 therefrom.

Coenzyme Q10, Rhodocola

Description

Coenzyme 90 A new bacterium with excellent productivity, Rodotollula rubra, UCS150-130, and a method for producing coenzyme X110 using the same [Rodotorula sp. JSU150-13 with high productivity of coenzyme Q10, and the producing method of Coenzyme Q10 using the strain}

The present invention relates to a novel strain Rhototoluru lubra JSU150-13 having high coenzyme Q10 productivity and a method for producing coenzyme Q10 using the same, and more particularly, to coenzyme in liquid culture compared to conventional Rhotolululubra species. It relates to very good new strains having a productivity of at least 200 times higher Q10 and a method for producing coenzyme Q10 therefrom.

Coenzyme Q ₁₀ (Coenzyme Q _{10 ,} 2,3-dimethoxy-5methyl-6-decaprenyl-1,4-benzoquinone) is a yellow or orange, fat-soluble quinone compound with properties similar to a odorless or tasteless vitamin. Coenzyme Q10 is universally present in many organisms, including animals and plants, as the Latin word "Ubiquitous" exists universally. Although not well known in Korea, Coenzyme Q10, which has been recognized for its medical value in the United States, Europe, and Japan for 20-30 years, is one of the nutrients that prevents aging. Not only does it act as an antioxidant, it's also essential for cells to generate the energy needed to function normally.

Coenzyme Q10 is a substance found naturally in the human body and is an electron / proton carrier that plays an important role in the function of mitochondria. Therefore, it is involved in the respiratory chain (respiratory chain) to produce energy in the form of ATP (adenosine triphosphate) from oxygen. Coenzyme Q10 is synthesized in the endoplasmic reticulum and has higher concentrations in heart, liver, kidney and pancreas than in other tissues. In addition, coenzyme Q10 has an antioxidant function that removes and neutralizes harmful oxygen free radicals, which are formed in the body during excessive exercise or energy generation and can cause fatal damage to human cells. It also plays the role of -oxidant.

As the important functions of coenzyme Q10 have been revealed, various pharmacological effects such as liver function improvement and hypoglycemic effect by immune enhancing effect have been proved.In addition, anti-aging by oxidative action and increase of energy production in body Various benefits and pharmacological effects of coenzyme Q10 have been confirmed, including enhancement and support for weight loss through metabolic activity. Therefore, coenzyme Q10 is widely used as an additive ingredient in medicines, dietary supplements and cosmetics.

Coenzyme Q10, the most basic metabolism of metabolism, can be synthesized in the body, but the production process of coenzyme Q10 in the body is sufficient by many cofactors such as vitamins B3, B5, B6, B12, C, and folic acid. It is a very complex process that must be present and it is impossible to produce enough coenzyme Q10 if some of them are deficient. And our lifestyle and environment is coenzyme Q10 Affect the level, actually, Deficiency of the cofactor nutrients mentioned above by Karl Folkers (awarded by the American Chemical Society for Coenzyme Research), age (in older, coenzyme Q10 Concentrations), stress and environmental pollution have also been shown to reduce coenzyme Q10 production capacity.

As coenzyme Q10's internal synthesis is complicated and difficult, it is necessary to supplement the coenzyme Q10 lacking in the body through food containing coenzyme Q10. Foods that contain coenzyme Q10 include spinach, broccoli, nuts, meats like guts or beef, fish like sardines or mackerel, or egg yolks. May cause problems, and coenzyme Q10 through food because it can not absorb 100% coenzyme Q10 into the body when consumed as food Ingestion is very inefficient. Therefore, it is desirable to take coenzyme Q10 as a medicine or health food, and in fact, many nutritional supplements sold in pharmacies and health supplements sold at health food stores contain coenzyme Q10. Many products contain coenzyme Q10.

  Despite having a large market size, coenzyme Q10 is extremely difficult and difficult to select and develop high-productivity strains due to its extremely low concentration and complex regulatory mechanisms, and also to develop coenzyme Q10 produced by complex biosynthetic processes. Since genetic and metabolic engineering approaches are required, systematic studies through microbial fermentation are not properly conducted in Korea.

  In the case of microorganisms, the physiological and morphological characteristics of cultures are significantly different depending on the media components. Therefore, in order to improve productivity, it is necessary to supply an appropriate amount of medium, as well as to optimize the composition and composition of each nutrient source.

The present invention has been made to overcome the limitations of the prior art, an object of the present invention is to provide a very good new strain of coenzyme Q10 in the liquid culture more than 200 times the productivity of liquid culture compared to the rodotolurubrabra, the parent strain. There is.

It is another object of the present invention to provide a method of culturing the Rhodotolurubrabra JSU150-13 to produce coenzyme Q10 from the culture.

The above-mentioned problem can be solved by the inventors of the Rhodo Tolu Rubra JSU150-13 (Accession No. KACC 93056P). By culturing such a new strain can be obtained coenzyme Q10 from the culture with high efficiency. And at this time, the culture can be liquid culture.

The new strain according to the present invention is a very good strain having a productivity of coenzyme Q10 more than 200 times higher in liquid culture compared to the rodotolurubrabra, which is a parent strain. Therefore, according to the present invention, it is possible to supply a large amount of coenzyme Q10, which is widely used as an additive raw material for medicines, health supplements, and cosmetics by various effects and pharmacological effects.

The present invention provides RODOTOLURA RUBRA JSU150-13 with high coenzyme Q10 productivity. And it shows a method of producing coenzyme Q10 from the culture by culturing the Rhototolula rubra JSU150-13.

Hereinafter, the content of the present invention will be described in more detail.

Rhototolula rubra JSU150-13 (hereinafter referred to as 'new strain') of the present invention is a parental strain of Rhototolula rubra which is a production strain of coenzyme Q10 having an inhibitory effect on cholesterol biosynthesis (hereinafter referred to as 'friendly strain') The strain was confirmed to produce the most coenzyme Q10 among those screened as resistant strains after a predetermined mutation treatment.

Coenzyme Q10 produced by the new strain or the strain produced according to the present invention may be provided as a medical, cosmetic or functional food use. When provided as a drug or food additive, coenzyme Q10 cultures may be prepared and provided in the form of hydrothermal, alcoholic or organic solvent extracts obtained therefrom. When administered orally, the isolated coenzyme Q10 may be mixed with a pharmaceutically acceptable excipient or the like, and in some cases, it may also be formulated into gelatin capsules, tablets and the like.

After inoculation into the agar medium which is commonly used for the storage of new strains and pre-colonized strains according to the present invention, the cells are collected by distilled water containing 20% of glycerol and filtration. After removing the residue through the liquid stock was made to 1.0 × 10 ⁹ cells / ㎖. Liquid stock was stored in -80 ℃ deep freezer containing 20% glycerol (glycerol). During the cultivation, the stock cells were collected by distilled water containing 20% glycerol after incubation in YPD (Yeast extract Peptone Dextrose) agar. The growth medium was inoculated at a volume of 10% (v / v) and incubated at 28 ° C. and 220 rpm in a shaker incubator. Production culture was inoculated in a volume of 10% (v / v) cells obtained in the growth culture and then incubated at 28 ℃, 220 rpm.

Hereinafter, the present invention will be described in more detail with reference to 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 Isolation of Mycobacteria

Mutant strain screening

In this example, Rhodotorula rubura , which was distributed in order to obtain a resistant mutant resistant to a mutagen for coenzyme Q10 production, was used as a parent strain. In order to obtain the mutant strains from the parent strains, the strains were grown by spreading the strains by spreading the strains on agar plate YPD plates and using only the pure cells through filtration. The obtained cells were washed by centrifugation, counted using a hemocytometer, and diluted to 1 × 10 ⁷ cells / ml. The diluted cells were placed in a glass plate and placed on a rocker, and then mixed with gentle UV irradiation. The spore suspension was treated with UV, the mutagen, to induce mutations.

In the UV treatment, two 254 nm UV lamps were irradiated in the dark for 0 to 120 seconds and plated on agar plates in 0.1 ml increments. Surviving colonies (coony) were counted to obtain a percentage of the untreated UV treatment, and the UV treatment time of approximately 70-80% mortality was used as the variation condition (see FIG. 1). As a result, UV mutation experiment was performed at 120 seconds, the minimum inhibitory concentration (MIC) of the R. rubra strain of UV. After UV irradiation, a strain was strained on agar plate YPD plate to screen for resistant strains. After culturing the smeared for about 4-6 days in the cow, mutant strains that formed colonies were selected and subcultured in fresh agar medium.

Coenzyme Q10  High Productivity Strain Selection

In this example, a large number of mutant strains having excellent coenzyme Q10 production capacity were selected, and after culturing liquids, the strains having the highest productivity were selected by measuring the content of coenzyme Q10 by HPLC. A simple screening experiment was carried out again to ensure a more stable strain producing coenzyme Q10. In order to select more coenzyme Q10 high productivity strains with improved production capacity, the UV irradiation time was gradually increased, and the screening process was repeated.

Experiment result

As a result, in the first mutation compared to the parent strain, the strains were resistant to coenzyme Q10, and the productivity of coenzyme Q10 was three times higher than that of the parent strain, and the strains were selected (see FIG. 2). As a result, it was possible to select the strain having the highest coenzyme Q10 productivity among the strains resistant to UV at 150 seconds, which was 200 times more productive than the parent strain (see FIG. 3), and the strain was identified as' Rhodotorula rubra JSU150-13. '(See Table 1). As such, the finally selected coenzyme Q10 high productivity strain was deposited on May 31, 2007 with accession number KACC 93056P to the National Institute of Agricultural Biotechnology (NIAB) (KACC, 441-707, Suwon, Korea).

TABLE 1 Coenzyme Q10 Productivity of Parents and Mutants of the Invention

Example 2 Establishment of Culture Conditions for Coenzyme Q10 Production

Carbon source  Coenzyme according to the concentration of nitrogen source Q10  Productivity Experiment

CaCO ₃ , FeSO ₄ , NaNO ₃ , and KCl, which are common components of R9 medium determined through the media experiments, were added with the existing concentrations (see Table 2). Types and concentrations of the carbon and nitrogen sources that showed the difference were added as shown in Table 3 below. Glycerol was added to each medium at intervals of 10 g / L in the range of 30 to 80 g / L to confirm the optimal concentration of the carbon source. Fixed to. In addition, the concentration of beef extract, which is a nitrogen source, was added at intervals of 0.5 g / L in the range of 2.5 to 5 g / L. At this time, the concentration of carbon source was fixed and malt extract and scheme milk, which are other nitrogen sources except beef extract, were adjusted to the existing concentrations. The experiment was performed by making a combination as shown in Table 3 according to the presence or absence of the addition.

<Table 2> Medium Contents of YP and R9

<Table 3> Coenzyme Q10 productivity test according to carbon source (glycerol) and nitrogen source (beef extract, skim milk, malt extract)

Experiment result

First, as a result of the coenzyme Q10 productivity test according to the carbon source concentration based on the YP production medium, it was confirmed that as the amount of glycerol added increased, it had a positive effect on the production of coenzyme Q10. Coenzyme Q10 production appears to be relatively good in the range of g / L (see Table 4).

Table 4 Comparison of Coenzyme Q10 Productivity According to Carbon Source and Nitrogen Source in YP Medium

Secondly, the carbon source of R9 production medium showed the best coenzyme Q10 productivity when glycerol was added at 60 g / L. Therefore, it would be better to increase the amount of glycerol added from 40 g / L to 60 g / L, and the results of the investigation of the concentrations of beef extract, skim milk and malt extract, which are nitrogen sources of R9 medium (skim milk, malt extract and Concentration of Beef Extract, Irradiation Range: 2.5 ～ 5 g / L), Coenzyme Q10 Productivity could not be confirmed by the addition of Scheme Milk and Malt Extract. When added, the addition of the scheme milk and malt extract at the same time was confirmed to have a positive effect on the increase in cell concentration. Therefore, in the case of R9 medium, it would be desirable to add the skim milk and malt extracts at the same concentration as the existing concentration, and the results of coenzyme Q10 productivity according to the concentration of beef extract were the highest at 3 g / L, which was slightly higher than the existing concentration. If good productivity was seen, at higher concentrations, the productivity decreased (see FIG. 4). Therefore, the concentration of beef extract in R9 medium was determined to be 3 g / L (see Table 5).

Table 5 Comparison of Coenzyme Q10 Productivity According to Carbon Source and Nitrogen Source in R9 Medium

Finally, the productivity in YP media and R9 media was compared and finally the results of the addition concentration and addition concentration determination experiments of the carbon source and the nitrogen source were summarized (see FIG. 4). Compared with YP media, R9 media is considered to be more productive and stable. As a result of the concentration test of carbon and nitrogen sources, coenzyme Q10 production medium was composed of glycerol 60 g / L, beef extract 2.5 g / L, skim milk 4g / L, malt extract. 4 g / L, NaNO ₃ 4 g / L, FeSO ₄ 0.4 g / L and CaCO ₃ 5 g / L.

Example 3 Culture of New Strains and Optimization of Coenzyme Q10 Production in Optimized Production Medium

Liquid culture method

In order to confirm the production of coenzyme Q10 was carried out liquid culture of each strain. Each strain was incubated for 49 hours in a YPD plate and collected with 20% glycerol solution, and then inoculated into growth medium. 30ml growth medium was added to a 250ml flask and incubated at 220 rpm for 40 hours at 28 ° C. Coenzyme Q10 production was analyzed by HPLC after inoculating the optimized production medium at 10% (v / v) for 72 hours. The growth medium and optimized production medium used in this experiment are shown in Table 6 below.

TABLE 6 Badge Composition

Coenzyme Q10 of HPLC  Analysis method

High Pressure Liquid Chromatography (HPLC) was used to determine the content of coenzyme Q10. For pretreatment of the sample, add saponification-solution for hydrolysis to 20 ml of the culture medium, and hydrolyze the cells at 90 ℃. The hydrolyzed cells were added with n-Hexane and shaken for a certain time to recover only the n-Hexane layer in which coenzyme Q10 was dissolved. Then, n-hexane was evaporated to obtain coenzyme Q10 crystals, which were then suspended in ethanol. Samples for HPLC analysis were prepared through a micron microfilter. HPLC analysis conditions are shown in Table 7 below.

TABLE 7 HPLC Analysis Conditions

Figure 1 shows the result of confirming the minimum inhibitory concentration (irradiation time) according to the UV treatment of codonzyme Q10 production strain Rhodotolura according to the present invention (resulting in step by step because the survival rate was 20.93% when UV irradiation for 120 seconds) Coenzyme Q10 gradually increasing UV irradiation time Select mutant strains with improved production capacity).

Figure 2 is a comparison of the productivity of coenzyme Q10 of the primary strain and mutant strains showed resistance at 120 seconds UV in accordance with the present invention.

Figure 3 is a comparison of the productivity of coenzyme Q10 production strain showing resistance at 150 seconds UV in accordance with the present invention.

Figure 4 is a comparison of productivity according to the concentration of the carbon source and nitrogen source of coenzyme Q10 production medium components according to the present invention.

Claims (3)

Coenzyme Q10 RODOTOLURA RUBRA JSU 150-13 (Accession No. KACC 93056P) with high productivity. It is characterized by culturing Rhodotolurubrabra JSU150-13 (KACC 93056P) to produce coenzyme Q10 from the culture, Process for preparing coenzyme Q10. The method of claim 2, Stomach culture is characterized in that the liquid culture, Process for preparing coenzyme Q10.

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