KR101406044B1 - Production of coenzyme Q10 through cultivation of Rhodotorulla sp. in culture medium adding mandarin peels as carbon source - Google Patents

Abstract

The present invention relates to a method for producing coenzyme Q10 using citrus peel, and more particularly, to a culturing technique for producing coenzyme Q10 through cultivation of Rhodotorula using citrus peel as a carbon source. The productivity of coenzyme cetene used was significantly improved compared with the control group. The present invention also provides a novel method of economically utilizing and treating citrus peel as an environmental pollutant by providing a method of producing economic functional foods and pharmaceuticals by using citrus peel, which is difficult to dispose of, as a carbon source .

Coenzyme quenching, citrus peel

Description

[0001] The present invention relates to a method for producing coenzyme Q10 by culturing Rhodotorula in a medium containing citrus peel as a carbon source. in culture medium containing mandarin peels as carbon source}

FIG. 1 is a photograph showing various enzymes of citrus peel according to the present invention (A is a control group and B, C and D are novoferm, citrozym and ultrazym treated photographs, respectively)

      FIG. 2 is a graph showing a comparison of dry cell mass of a cultivar of the genus Torila with the addition of 30 g / L of glucose as a carbon source,

Fig. 3 is a chart comparing the productivity of coenzyme Q10 in Rhodotorula with citrus peel pretreated according to the present invention

The present invention relates to a method for producing coenzyme Q10 using citrus peel (including citrus fruit juice) from Jeju. More specifically, the productivity of coenzyme Q10 in Rhodotorula lucer cultures using citrus peel as a carbon source is improved The present invention relates to a method for reducing environmental pollution by reducing the cost of cultivation by recycling citrus peel which is difficult to dispose of.

Coenzyme Q ₁₀ (coenzyme Q ₁₀ ) It is a kind of quinone that is essential for maintaining the healthy function of living organisms because it plays an important role in generating energy in cells. Coenzyme Q is a substance found naturally in the human body. It is an electron / proton carrier that plays an important role in the function of mitochondria. It is involved in the respiratory chain and is essential for generating energy from oxygen in the form of adenosine triphosphate (ATP). Coenzyme Q10 is synthesized in the endoplasmic reticulum, and its concentration in heart, liver, kidney, and pancreas is higher than other tissues. It also acts as an antioxidant because it has an antioxidant function to remove and neutralize harmful oxygen free radicals, which can be formed in the body during excessive exercise or energy generation and can cause fatal damage to human cells. As the important function of coenzyme Q is revealed, various pharmacological effects such as improvement of liver function by immunopotentiating effect and blood glucose lowering effect and blood cholesterol lowering effect have been proved. In addition, anti-aging by antioxidant activity, Enhancement, support for weight loss through metabolic activity, and various effects and pharmacological effects of coenzyme Q10 have been confirmed. Therefore, coenzyme Q is widely used as a raw material for pharmaceuticals, health supplements and cosmetics.

In the case of coenzyme Q10, which is the most basic and metabolically important substance of human metabolism, it is possible to synthesize in the body, but the coenzyme Q10 in the body is produced by the simultaneous production of many cofactors such as vitamin B3, B5, B6, B12, It is a very complex process that only needs to be present, and it is impossible for sufficient coenzyme QTEN to be produced when some of these components are deficient. And our lifestyle and environment also coenzyme Q It is actually the Dr. The deficiency of the above-mentioned cofactor nutrients by Karl Folkers (awarded by the American Chemical Society as a coenzyme research), age (the older the coenzyme Q in the body Concentration reduction), stress and environmental pollution have also been shown to decrease coenzyme Q production capacity. As such, the synthesis of coenzyme QTEN in the body is complicated and complicated, so the food containing coenzyme QTEN should supplement the deficient parts in the body. Foods containing coenzyme QTEN include spinach, broccoli, nuts, meats such as visceral beef or beef, fish such as sardines or mackerel, or egg yolks, but consuming large quantities of these foods, which are high in cholesterol in their diet, And can not be absorbed into 100% body when consumed as food. Therefore, coenzyme Q10 Ingestion is very inefficient. Therefore, it is preferable to take it as medicines or health foods. In fact, there are many coenzyme QTENs contained in health supplements that are sold in pharmacies, such as nutritional supplements and health supplements. In recent years, cosmetics containing coenzyme QTEN A large amount of products are being sold.

Despite its large market size, coenzyme QTEN is extremely difficult and difficult to select and develop a highly productive strain due to its extremely low concentration in the cell and its complicated regulatory mechanism. It also makes it difficult to develop a production strain of coenzyme QTEN that is synthesized by complicated biosynthetic process However, systematic research through microbial fermentation has not progressed in Korea because genetic engineering and metabolic engineering approaches are required.

About 20% of Jeju Island's total citrus production is used as mandarin orange. The citrus fruit produced in the citrus processing industry is estimated to be 100,000 tons per year. Currently, it is pointed out that it is one of the most important causes of environmental pollution in Cheju Island, which is the largest tourist spot in Korea, because it is difficult to completely treat citrus peel. Studies on the use of citrus peel for feed and fertilizer have been continuously carried out, but there is a great difficulty in industrialization due to economical efficiency and inefficient production thereof. Currently, most of the citrus peel is marine dumping, but since July 07 when the London Treaty comes into force, the ban on marine dumping of citrus peel is completely banned. Therefore, it is not an exaggeration to say that the establishment of the treatment method of citrus peel is the most influential factor in the development of the citrus processing industry.

The moisture content of citrus fruit juice, which is a waste of citrus juice, is maintained as much as Td or 82% depending on the time of harvesting, which is the biggest constraint factor for the treatment of by - products and useful resources. Citrus juice leaves contain 10-15% of pectin and 1-3% of flavonoids in dry weight. Pectin is a complex polysaccharide mainly present in the middle lamella of a plant, and refers to a substance that binds and binds cells. It is widely used as a gelling agent and has effects such as prevention of hypertension, arteriosclerosis, angina pectoris, removal of heavy metals, anti-radiation, lowering of cholesterol in blood, proliferation of intestinal useful microorganisms, and strengthening of immune system. There are about 60 kinds of citrus flavonoids, common flavonoids such as rutin and theosmin, and flavonoids specific to citrus such as neranine and hesperidin, novirretin and benzeretin. It has been found that there are many excellent natural materials with excellent physiological activity among flavonoids. In particular, antioxidant activity, prevention of circulatory diseases, anti-inflammatory, antibacterial, antiviral. Immune enhancement, liver disease improvement effect, anti-cancer function has been revealed various biocontrol functions. Using these characteristics, it is possible to develop functional foods and medicines for the prevention and treatment of diseases of adult diseases such as hypertension, arteriosclerosis, angina pectoris, and diabetes.

The present invention provides a method for producing coenzyme cetenes by efficiently decomposing and treating the cultured medium using rhodotorula lubra cultures using citrus peel (containing citrus fruit juice), which is difficult to dispose of, as described above .

The present invention relates to a method for producing coenzyme Q10 by using citrus peel as a culture medium for cultivation of Rhodotorula rubra, and is characterized by comprising the following steps.

1) Acid treatment step of citrus peel

Mix citric acid and distilled water at a ratio of 1: 2, adjust the pH to 3.0 using sulfuric acid, and sterilize at 121 ° C.

2) Enzyme treatment of citrus peel

After the acid treatment step, the pH is adjusted to 4.5 with NaOH, the enzyme is added at an appropriate concentration, and the reaction is carried out at 45 ° C for 12 hours.

3) Culture step

An appropriate amount of the treated citrus peel is added as a carbon source, and other additional ingredients are added thereto to prepare a coenzyme Q10 production medium, which is then sterilized at 121 DEG C for 15 minutes. Then, the production strain Rhodotorula rubra is inoculated and incubated for about 4 days at 28 DEG C and 220 rpm with shaking.

4) Stage of Coenzyme Q10

After the culture is completed, the optimum culture medium is recovered and pulverized to confirm the productivity of coenzyme Q10.

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

≪ Example 1 > Measurement of degree of glycation in the acid treatment step of citrus peel

In order to utilize citrus peel, which is mainly composed of cellulose and pectin, as a carbon source for cultivation of Torula rubra, first saccharification is required to decompose them into minimum units. In this embodiment, the citrus peel was disassembled using an acid. The citrus peel solution (citrus peel: water = 2: 1) was adjusted to pH 3.0 using sulfuric acid (H ₂ SO ₄ ) and sterilized at 121 ° C for 0, 5, 10, 15, One). In the control group without addition of sulfuric acid, about 9% of reducing sugar was present. Sulfuric acid was added and reducing sugar was measured at different sterilization times. When sterilized for 20 minutes, about 0.36 g of reducing sugar / g of dried citrus peel reducing sugar was produced.

<Table 1> Comparison of saccharification degree of citrus peel according to sterilization time

Sterilization time Reducing sugar concentration (g Reducing sugar per gram dried citrus) Conversion Rate (%) 0 (no addition of sulfuric acid) 0.9 9 5 0.21 21 10 0.27 27 15 0.32 32 20 0.36 36

&Lt; Example 2 > Measurement of saccharification degree in the enzyme treatment step of citrus peel

Various enzymes were added to the citrus peel solution which had been sterilized for 20 minutes in Example 1 and the degree of saccharification was measured at pH 3, 4, and 7 for 12 hours. The results are shown in Table 2. Overall, the glycosylation level decreased at pH 4 over pH and peaked at pH 4. The highest enzyme activity of novoferm was observed at pH 4 and 0.68 g of reducing sugars / g of citrus peel at pH 4. A photograph of the degree of glycation of each condition is shown in FIG.

<Table 2> Comparison of saccharification degree of citrus peel according to enzyme type and pH

(g reduction sugar per gram of citrus peel)

pectinex viscozyme novoferm citrozym ultrazym pH 3 0.4 0.5 0.6 0.48 0.5 pH 4 0.45 0.55 0.68 0.52 0.45 pH 7 0.42 0.45 0.5 0.56 0.44

<Example 3> Rhodotorula lubra cultured with citrus peel as a carbon source

Rhodotorula rubra cultures were performed with different concentrations of citrus peel treated with sulfuric acid and hydrolytic enzymes, with 30 g / L of glucose as a carbon source. Table 3 shows the composition of the basic synthetic liquid medium. The culture method is as follows. The citrus peel solution treated under the respective conditions is centrifuged at 2000 rpm for 10 minutes to take only the supernatant. Then, prepare the basic synthetic liquid medium as shown in Table 3 and add the citric acid supernatant. The medium is sterilized in 30 ml portions in a 250 ml Erlenmeyer flask and inoculated with 5% (v / v) Rhodotorula rubra cultured in liquid YPD medium for approximately 24 hours. The cells were incubated at 30 ° C for about 4 days to confirm the amount of dried cells (Fig. 1).

<Table 3> Composition of basic synthetic liquid medium

ingredient Concentration (g / L) Ammonium sulfate
Dipotassium hydrogen phosphate
Magnesium sulfate
Calcium chloride
Fine element 7
0.5
0.35
0.2
1 ml

_{* Fine Elements: Zinc sulfate  3.5 g of manganese chloride, 0.1 g of manganese chloride, Sodium molybdate  0.01 g, copper sulfate 0.02 g / 100 ml}

As compared with the culture with 30 g / L of glucose as a control, the amount of dried cells increased with incubation with high citrus concentration. In the culture with 30 g / L of citrus peel, the dried cell mass was 83%. As a result, it was confirmed that the citrus peel after the stepwise treatment was available as a carbon source to replace glucose.

Example 4 Production of Coenzyme Q10 by cultivating Rhodotorula Lubra using citrus peel as a carbon source

Coenzyme Q10 of HPLC  Analysis method

HPLC (High Pressure Liquid Chromatography) was used to measure the content of coenzyme Q10. For pretreatment of the sample, saponification-solution for hydrolysis is added to 20 ml of the cultured medium, and then the cells are hydrolyzed at 90 ° C. N-Hexane was added to the hydrolyzed cells, and after shaking for a predetermined time, only the n-hexane layer in which coenzyme Q10 was dissolved was collected to evaporate n-hexane to obtain coenzyme Q10 crystals. The coenzyme Q10 crystals were again suspended in ethanol, Mu] m microfilter to prepare a sample for HPLC analysis. The HPLC analysis conditions are shown in Table 7 below.

<Table 5> HPLC analysis conditions

column Mightysil RP-18 GP 250-4.6 (5 占 퐉)
(Kanto chemical co., INC.) Mobile phase Methanol: Ethanol = 13: 7 Column temperature 35 ℃ by temperature controller Detectors & Conditions HP1100series G1313A ALS (Hewlett packard co.) UV wavelength  275 nm Flow rate 1 ml / min Sample injection  5 μl

Citrus peel As a carbon source  Culture method used

An experiment was conducted to confirm the productivity of coenzyme Q10 in Rhodolurala bra culture using citrus peel as a carbon source. The composition of the production medium is shown in Table 4.

<Table 4> Coenzyme Q10 production basic medium

Furtherance Concentration Beef extract
Skim milk
Malt extract
NaNO ₃
FeSO ₄
CaCO ₃ 2.5
4
4
4
0.4
5

Coenzyme Q10 production was carried out through Rhodotorula sp. Culturing using the composition of Table 4. The experiment was carried out in the same manner as in Example 3 except that the control group was added with glycerol 60 g / L as a carbon source, and citric acid treated with sulfuric acid and novoferm, which is a lytic enzyme, was added to the test group and cultured at 30 ° C for about 4 days. The results of this incubation are shown in Fig. When the concentration of coenzyme Q10 in a culture supplemented with 60 g / L of glycerol as a carbon source was taken as 100%, the coenzyme Q10 of the culture treated with novoferm increased about 2.5 times to 250%. In addition, 120, 200% of the cultures treated with other enzymes showed significantly higher coenzyme Q10 productivity than the control. This phenomenon is thought to be caused by the fact that the saccharified citrus peel is used for cell growth and the carbon source decomposed slowly by the microorganism is continuously supplied to the cell metabolism. The results show that when citrus peel is used as a carbon source to cultivate Rhodotorula genus, not only the citrus industry wastes are effectively used but also Coenzyme Q107, which is a useful substance, can also be produced in high yield.

The present invention relates to a method for producing coenzyme Q10 using citrus peel from Jeju. More specifically, the productivity of coenzyme Q10 in Rhodotorula genus culture using citrus peel as a carbon source is improved compared to the control, It provides a method of recycling citrus peels to lower the cost of producing materials and reduce environmental pollution.

Claims (6)

I) Mixing the citrus peel and distilled water at a ratio of 1: 2, adjusting the pH to 3.0 with acid, and sterilizing at 121 占 폚 to prepare an acid decomposition product of citrus peel; And Ii) adjusting the acid decomposition product of the citrus peel to pH 4.5 with NaOH, followed by enzymatic degradation by adding the enzyme and reacting at 45 ° C for 12 hours to obtain a carbon source of the citrus peel- &Lt; / RTI &gt; The method according to claim 1, wherein the acid is sulfuric acid. 3. The method according to claim 1, wherein the enzyme is any one of novopearl, viscose, and citrozem. A method for producing coenzyme Q10, comprising inoculating a culture medium containing a citrus peel-derived carbon source produced by the method according to claim 1 into a culture medium containing Rhodotorola sp. 5. The process according to claim 4, wherein the Rhodotorola sp. Strain is Rhodotorula rubrene. 5. The method according to claim 4, wherein the culture is cultivated at 28 DEG C to 30 DEG C for 4 days.

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