KR20060021769A - Monascus sp. mutants and cholesterol inhibitors comprising extracts thereof - Google Patents

Abstract

The present invention relates to Monascus mutant strain (KCCM 10586), which is excellent in the ability to produce monacolin K, a cholesterol synthesis inhibitor developed through irradiation, and non-productive fungal toxin (especially citrinin).

The mutant strains of the present invention were irradiated strains of Monascus pilosus with excellent anti-cholesterol activity (monacolin K), and not only excellent in production of monacolin K but also secured safety as non-fungal toxin-producing strains. It is a superior strain with a significant improvement in pigment productivity. Therefore, it is expected to increase the utilization of red yeast rice in the domestic functional food market by developing red yeast fungi that have excellent cholesterol synthesis inhibitory ability and contain a large amount of natural pigments. It can be used as a raw material and it is also possible to develop new food and beverages.

Description

Mutagen Strains and Cholesterol Lowering Agent Using the Same {Monascus sp. mutants and cholesterol inhibitors comprising extracts pretty}

Figure 1 shows the death rate of spore suspension of audited irradiated Monascus sp.

2 shows various Monascus sp. This is a comparative graph of the content of lovastatin from mutant strains.

Figure 3 is Monascus isolate No. HPLC analysis of the extract of 71109.

Conditions: isocratic mobile phase consisted of methanol 0.025 M sodium phosphate (NaH 2 PO 4, pH 4.5, 82:28 v / v), 1 ml / min of flow rate, 20 μl injection, 236 nm detection, and Symmetry C18 5 μm column.

4 is a TLC chromatogram of the metabolite of Monascus isolates under UV light at 254 nm and 365 nm (CHCl 3: MeOH (3: 1, v / v)).

5 shows various Monascus sp. Bioautography of mutant and citrinin standards is shown.

6 is a result of detection by a fluorescence detector (λex = 231, λem = 500) after HPLC analysis for the detection of citrinin.

(A) Culture extract of Monascus isolate No. 71109

(B) Spike test with citrinin 500 ppb

7 is a result of detecting citrinin using RIDASCREEN FAST Citrinin kit.

8 shows cell viability (MTT) test results of citrinin standards for vero cell lines.

(A) citrinin standard

(B) cultured sample from Monascus isolate No. 71109

9 shows cell viability (MTT) test results of citrinin standards for MDCK cell lines.

(A) citrinin standard

(B) cultured sample from Monascus isolate No. 71109

10 is Monascus spp. The amylase activity of tangible cultures and various variants is analyzed.

Figure 11 is a graph showing the concentration of triglyceride and total cholesterol of SD-rat supplied with the erythrocyte strain of the present invention: control, casein & fat diet (Group I), casein & fat diet containing monacolin K 0.1, 0.5, 1.0% (Group II, III, IV) and lovastatin (Group V).

12 is a graph showing the HDL-cholesterol and LDL-cholesterol concentration of SD-rat supplied with the erythrocyte strain of the present invention: control, casein & fat diet (Group I), casein & fat diet containing monacolin K 0.1, 0.5, 1.0% (Group II, III, IV) and lovastatin (Group V).

The present invention relates to a mutant strain of red yeast bacteria and a cholesterol lowering agent using the same, and more particularly, to produce monacoline K (Monacolin K), a cholesterol inhibitor which was developed through irradiation, and a non-productive product of citrinin, which is a fungal toxin. Monascus ) mutant strain (KCCM 10586), its culture method and a cholesterol lowering agent containing the extract.

In recent years, Korea's economic growth has improved living standards and the frequent cultural exchanges with other countries have led to a more westernized diet, which has led to an increase in animal food intake. The development of chronic degenerative diseases, such as obesity, diabetes, hypertension, and cardiovascular disease, which was previously considered a western problem, is gradually increasing in Korea. In particular, if the total Cholesterol or triglyceride in the blood exceeds an appropriate level, it causes heart disease, and obesity, hypertension and arteriosclerosis, hyperlipidemia, etc. cause abnormalities in the blood circulation.

Monascus is classified taxonically as Kingdom Fungi, Phylum Ascomycota, Class Plectomyetes, Order Ascosphaerales, and Family Monascaeae. Monascus have both a formation of cleistohecia with ascospores and a formation of conidia for proliferation. To date, over 20 Monascus strains have been isolated and identified. Monascus red yeast rice (red Koji, Hongqu, Angkak) has been used as a traditional medicine. Monascus species are known for their pigment production, antimicrobial activity, cholesterol synthesis inhibitory activity, and hypotension-lowering activity, and antidiabetic activity has recently been reported, but Monascus species can produce citrinin, a type of mycotoxin.

The present inventors have studied for several years the screening, screening and characterization of substances such as pigment, antimicrobial activity and mycotoxin productivity of Monascus strains, another bioactive substance by Monascus , high productivity strain of monacolin K, cholesterol synthesis inhibitor Has been trying to develop.

Monacolin K and its derivatives have been commercialized in each country since the United States in 1987, and more than 1 million patients have already been treated. More than half of lovastatin (mevinolin), which is 100% lactone form, lovastatin (Hongguk monacolin K), which is an acid form, is safer for humans. A majority of K is active.

The inventors of the present invention is to increase the productivity by using the synthetic inhibitory ability of cholesterol, if used in the manufacture of health supplements, as well as the substitution effect of imported red yeast, as well as the enormous reduction in medical expenses, national health and It will also make a great contribution to health promotion.

Among the substances that inhibit HMG-CoA reductase, which catalyzes important regulatory reactions in the early stages of cholesterol biosynthesis, Lovastatin, Pravastatin and Simvastatin are the products that inhibit squalene synthetase and include zaragotic acid and squalstatin. Purpacin, epicohliquinone, acaterin, helminthosporol and lacteritin have been reported as cholesterol inhibitors, in particular Acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors.

Monacolin K, a cholesterol inhibitor, was first discovered in M. ruber by Endo in 1979. It is a common secondary metabolite distributed in all species of the genus Monascus , all of which have very low toxicity and HMG-CoA, a regulatory enzyme of cholesterol synthesis. It is characterized by antagonistic inhibition of (3-hydroxy-3-methylglutryl-CoA) reductase.

In spite of these useful aspects, in 1995, research by Blanc et al. Reported that monascidin A, an antimicrobial substance produced by Monascus strain, is a kidney toxin citrinin, a type of mycotoxin. LD ₅₀ of citrinin in mice, rabbits and 4-day-old chicken embryos is known to be 35 mg / kg, 19 mg / kg and 80.5 μg / egg, respectively, and is teratogenic in chicken embryos. Also, LC ₅₀ for Artemia salina larvae is known to be 33.9 μg / ml. Therefore, Monascus fungi used in functional red yeast products must be carried out to review the productivity of fungal toxins, and the commercial red yeast must have such safety. In addition, the production and production of various useful bioactive substances by optimizing the isolation and selection of wild strains and cultivation conditions is limited, so the development of excellent variant strains is essential as a way to overcome this.

In Korea, there are no research reports to produce cholesterol inhibitors using the genus Monascus , and few studies have been found to increase the productivity of metabolites by obtaining mutants by applying irradiation to Monascus or microorganisms. have. An example of the production of cholesterol-inhibiting substances by the genus Monascus is known in Japan, and Korea is currently using it to manufacture functional drinks from expensive red rice soup imported. However, monacolin K activity is low.

The present inventors developed a highly active Monascus mutant strain through irradiation and obtained a significant increase in yield compared to the initial production through the optimization of the culture conditions.

Previously, Monascus focused on the manufacturing process or pigment productivity of foods using Monascus strain, and the development method of mutant strain used UV method, but the sugar strain increased the production ability of monacolin K. The development of mutant strains by irradiation was differentiated from the existing mutant strain development methods and was the first attempted method.

Through the present invention, an appropriate irradiation dose for mutations in the Monascus strain has been suggested, and it is highly safe for use in the production of functional foods because of excellent production of monacolin K and no fungal toxin production by irradiation.

Therefore, the main object of the present invention is to provide a Monascus mutant strain having excellent production ability of Monacolin K and non-productive citrinin, a fungal toxin.

Another object of the present invention is to provide a method for producing Monascus mutant strain of the present invention.

Another object of the present invention is to provide an optimized culture method of the strains of Monascus mutant of the present invention.

Another object of the present invention to provide a cholesterol-lowering functional food and pharmaceutical products containing the extract of Monascus mutant strain of the present invention.

In order to achieve the object of the present invention, the present invention provides a Monascus mutant strain (KCCM 10586) to produce Monacolin K with high efficiency.

In the mutant strain of the present invention, Monascus mutant strain (KCCM 10586) is characterized in that it does not produce citrinin, and is characterized in that it produces red and yellow pigments with high efficiency. Not producing citrinin here means that it is not detected by normal TLC or HPLC.

In order to achieve the other object of the present invention, the present invention comprises the steps of irradiating various doses of radiation to Monascus to build a library of red yeast bacteria, approximately 1000 in the range having a killing rate of 95% to 99.99% High productivity of Monacolin K, including obtaining colonies, and selecting mutant strains having superior activity compared to the parent strain through anti-fungal bioassay. It provides a method for producing Monascus mutant strains low in the productivity of linin (citrinin).

In the production method of the present invention, it is characterized in that the radiation dose of about 2.5KGy to have a killing rate of about 99.99%. At this time, the production of mutant strains Monaolin K (maximum).

In order to achieve the other object of the present invention, the present invention is 2-3% Soytone, 2-3% Glucose, 0.04-0.06g magnesium sulfate (MgSO4), content of barley 75- Provided is a culturing method of Monascus mutant strain (KCCM 10586) of the present invention, characterized by culturing in a medium containing 85%.

In order to achieve the other object of the present invention, the present invention provides a cholesterol lowering agent containing an extract of Monascus mutant strain (KCCM 10586) of the present invention.

In order to achieve another object of the present invention, the present invention provides a functional food for lowering cholesterol containing an extract of Monascus mutant strain (KCCM 10586) of the present invention.

In the functional foods and drugs of the present invention, the extract is characterized in that it contains Monacolin K (Monacolin K). Monacolin K antagonically inhibits HMG-CoA (3-hydroxy-3-methylglutryl-CoA) reductase, a regulator of cholesterol synthesis.

In the functional foods and pharmaceuticals of the present invention, the extract is added to an extraction device and an extraction solvent commonly used in the art, that is, water or an organic solvent or a mixed solution thereof, and any device at room temperature or below the boiling point of the solvent. It can be obtained by extracting using. In addition, in the present invention, the extract of hongguk bacteria is understood to include hongguk (red rice) itself and the extract containing the honggukyun.

Examples of the organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; C2-C5 polyhydric alcohols, such as 1, 3- butylene glycol, a propylene glycol, and glycerin, etc. are mentioned, The mixed solution of these organic solvents and water, etc. can be used.

Cholesterol lowering agents of the present invention may be prepared by methods known in the pharmaceutical art, and may be mixed with itself or with a pharmaceutically acceptable carrier, excipient, diluent or the like to form a powder, granule, tablet, capsule, or injection. It can be prepared and used as. They can also be administered orally or parenterally.

The dosage of the extract of Monascus mutant strain (KCCM 10586) according to the present invention as an active ingredient may be appropriately selected depending on the age, sex, condition, and symptoms of the disease, preferably 500 mg per day ( Adults who have insomnia are administered 250 mg powder capsules in the morning, 2 times a day before bedtime).

The inventors of the present invention have selected high-quality strains with high activity of monacolin K, whose physical, chemical, and biological safety have been examined, and established conditions for optimizing monacolin K productivity and safety. As a sample, the improvement of hyperlipidemia of red yeast rice using SD-rat which finally induced safety and hyperlipidemia was verified.

Therefore, it is necessary to secure the production of cholesterol-inhibiting substance and blood pressure-lowering substance through the development of excellent mutant strains, and to secure the competitiveness of foreign products only by securing strains through the search for fungal toxin (citrinin). It is expected to bring about the creation of added value.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

(Experimental material)

1. Microorganisms

Monascus isolates isolated from Angkak, a red yeast rice, were used for irradiation. Aspergillus nidurans KCTC 6910 was used as a test organism for testing antifungal bioassay. These fungal microorganisms were incubated at 28 ° C. in potato dextrose agar slant (PDA, Difco Lab. Detroit, USA). Bacillus subtilis KCTC 1021 was used as a test organism for bioautography test. The microorganism was incubated for 8 h in nutrient broth and the culture was plated on plates. To detect antimicrobial activity, the following strains were used: Bacillus cereus KCTC 1012, Yersinia enterocolitica ATCC 23715, Pseudomonas aeruginosa ATCC 1750, Shigella dysenteriae ATCC 13313, E. Coli O157; H7 KCTC 1039, Salmonella enterotidis ATCC 11376 , Listeria mono All these microorganisms were purchased from the American Type culture collection (ATCC) and the Korean Collection of Type Culture (KCTC).

2. Cell line and medium

Cell lines (MDCK and Vero) were purchased from Korean cell line bank (KCLB, No. 10034 and No. 10081). DMEM medium, RPMI 1640 medium, horse serum donor herd, fetal bovine serum and antibiotic-antimycotic were purchased from Gibco-BRLTM (Gaithersburg, MD, USA).

3. Chemicals

All microbial media were purchased from Difco Lab (Detroit, USA) and citrinin was obtained from Sigma chemical co. (St. Louis, MO, USA). Silica gels and TLC plates are available from Merck Co. (Darmstadt, Germany). All other chemicals used analytical grade.

Example 1: Isolation of Mutantism

The production of mutant strains induced mutations by irradiating ⁶⁰ Co γ-ray in various doses (Korea Atomic Energy Research Institute). Radiation irradiation Monascus library was constructed by applying various doses suitable for Monascus , and approximately 1000 colonies were obtained in the range of 95% to 99.99% mortality, and the parent strain at 2.5KGy with 99.99% mortality. Mutant strains with superior activity compared to Monacus isolate 711 were obtained through anti-fungal bioassay.

To study the effect of irradiation on Monascus isolates, spores were harvested from PDA agar plates after incubation at 27 ° C for 7 days. 110 mm Whatman filter paper No. After filtration with 1 (England), spores were diluted in dilute solution with ca. Suspension was at a concentration of 1.5 × 10 6 spores / ml. About 2 ml of spore suspension was irradiated with gamma () rays at room temperature.

Irradiation was carried out at Co ± 60 gamma irradiator (point sourcem AECL, IR-79, Nordion International Co., Ltd, Ontario, Canada). Dosimetry was performed using 5 mm diameter alanine dosimeters (Bruker Instruments, Rjeomstettem, Germany). In this experiment, the radiation dose was used as a target dose of 0-3 KGy.

Irradiation with a dose of 0-3 Kgy resulted in a death rate of spores of 95-100%. To select high productivity strains of monacolin K, spores with a killing rate of 95-99.99% were used for continuous selection. Specific mortality according to the radiation dose is shown in FIG. 1.

After culturing the mutant strains, the agar plug method was performed (MS Kumar, et al., J. Microbiological Methods 40 99-104 (200)). The agar floc was punched out with a 6 mm stainless steel cork borer. Spores were picked to inoculate the agar flocs. Inoculated agar flocs were incubated at 28 ° C. for 10 days. Test organisms, A. nidulans , were grown on PDA plates for 10 days. Spores were harvested in diluent. 100 μl of a spore suspension containing 1.3 × 10 7 spores per ml were plated on a PDA plate. 20 μl of the alcohol extract from the agar plug was transferred onto a 6 mm diameter paper disk and dried and placed on the surface of the agar plate smeared on spores of A. nidulans . Zones of inhibition (in mm) were recorded after the plates were incubated at 28 ° C. for 27-32 hours.

To find active strains, a number of strains obtained by audit irradiation were screened by the agar plug method and anti-fungal bioassay. Most strains obtained by 2.5 KGy irradiation showed strong fungal inhibitory activity. Among thousands of Monascus mutants, only 9 strains exhibited an inhibition zone of at least 7 mm. Table 1 shows the inhibitory regions for A. nidurans .

TABLE 1

The anti-fungal activity of various Monascus sp. Mutants

Example 2: Quantitative Analysis of Nomacholine K by HPLC

For the quantitative confirmation of Monacolin K, the strains 126 and 71128 showed the highest activity in the strains identified by anti-fungal bioassay and found to be larger than the parent strain. High activity was found in order.

HPLC analysis was performed with a Waters 2690 apparatus (USA). Symmetric C18 5 μm column (reverse phase, 3.9150 mm, Waters) was used. An isocratic mobile phase consisting of methanol-0.025 M sodium phosphate (NaH 2 PO 4, pH 4.5, 82:28 v / v) was flowed at a flow rate of 1 ml / min in a 20 μl injection. Detection was maximized at 236 nm using a photodiode array detector (Waters 996). The sample was dissolved in methanol at a concentration of 5 mg / ml and the injection volume was 10 μl. Data collection was done using Waters' Millennium software.

HPLC effectively separates monacolin K with a delay of 4.5 0.5 min. This delay was found in crude samples incubated and extracted from agar pieces. First, it was found that 9 selected strains produced monacolin K of at least 1 μg / agar piece. Among them, 4 strains produced more than 7 μg / agar piece of monacolin K (FIG. 2). Among these, Monascus isolate No. The HPLC analysis of the extract of 71109 is shown in FIG. 3.

Example 3: Identification of Mycotoxin Non-producing Strains by Physical, Chemical or Biological Methods

In order to identify fractions similar to the Rf value of citrinin on TLC, strain 71109 was finally selected as a highly productive and safe strain, except for 71126 and 71128 strains, which were found to be citrinin-produced by comparative analysis using high sensitivity HPLC. Finally, the safety of the culture was verified through cytotoxicity and mutagenicity experiments.

200 ml of culture were grown in REB medium for 10 days and the culture was grown to 110 mm Whatman filter paper No. Mycelium was removed by filtration with 1 (England). The filtrate was extracted five times with chloroform of eastern blood. The extract was separated with a separatory funnel and concentrated at 30 ° C. with a vacuum rotary evaporator (EYELA; Japan).

 In order to perform thin layer chromatography (TLC), a silica gel TLC plate (Kieselgel 60, F254, 0.2 mm, Merck, Germany) was used. The cultured and concentrated samples were dissolved in methanol, 0.5 μl of the sample was spotted on the plate, and the spotted plate was developed in chloroform and methanol (3: 1 v / v). The plate was taken out and dried, and then visualized in visible and ultraviolet light (360 nm and 254 nm) to determine the Rf value.

For bioautography, 20 ml of soft agar mixed with test organisms ( B. subtilis KCTC 1021) and maintained at 45 ° C. were poured into a Petri dish containing the developed TLC plate. Petri dishes were dried and incubated at 37 ° C. for 12 h. After 12 h, staining with 0.02% 2, 3, 5-triphenyltetrazolium chloride (Sigma) stained the inhibitory region.

Extracts and citrinin standards of various cultures were developed. The Rf value of the Citrinin standard was 0.45 0.5 and the spot of the citrinin standard was clearly observed under UV light at 254 and 360 nm (FIG. 4). Inhibition of B. subtilis at an Rf value of 0.45 was observed only in the citrinin standard and not in the other samples (FIG. 5).

For HPLC quantitation of citrinin, both gradient and isocratic elution were used. Both analyzes used XTerra MS C18 5 μm column (reverse phase, 4.6150 mm, Waters). Waters 2690 apparatus was used for isocratic elution. The mobile phase had methanol 0.25 M H 3 PO 4 (80:20 v / v) and injected 20 μl at a flow rate of 1 ml / min. Fluorescence detection was performed at excitation wavelength 331 nm and emission wavelength 500 nm. Gradient elution was performed for 40 min with (20:80 v / v) to (100: 0) methanol water elunt composition. Chromatograms were recorded with a ProStar 320 UV / Vis detector at 250 and 275 nm wavelengths. Detection was performed with a fluorescence detector. Almost no citrinin was detected in the crude sample. To determine the presence of citrinin, the spike test was performed with 0.5-1 ppm of commercial citrinin. 6 is a comparative chromatogram. As shown in FIG. 6, M. The extract of 71109 (FIG. 6A) did not have a peak matching the spike chromatogram of citrinin (FIG. 6B). As a result, M of the present invention. 71109 produced high monacolin K but produced little citrinin. Thus, M. Mutant strain 71109 was deposited with the Korean National Center for Microbiological Conservation (KCCM) under accession no. KCCM-10586 dated August 5, 2004.

In order to reconfirm detection of citrinin, enzyme immunoassay was performed using RIDASCREEN FAST Citrinin kit. Even with this experiment, Monascus. Mutant 71109 did not detect any citrinin.

In order to verify the stability of the culture medium of 71109, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reduction assay was performed. MDCK and Vero cells were cultured in 100 mm tissue culture dishes and cells were seeded in 96 well plates containing 100 μl medium per well (106-107 cells / mL). The samples were incubated overnight before the samples were processed. Sample treated cells were preincubated for 48 hours before MTT testing. MTT stock (2.5 mg / mL) was prepared in PBS and MTT reduction was started by adding 10 μl MTT stock per well. The plate was incubated at 37 ° C. for 3 hours, then 150 μl DMSO was added to stop the reaction. The absorbance was measured at 570 nm and the reference wave-length was 630 nm, which was determined using a microplate reader (Model 550, BIO-RAD Laboratories, CA, USA). 8 shows the effect of citrinin and cultured samples on vero cells. Treatment of vero cells with citrinin reduced their survival. Dilution of citrinin increased survival. In contrast, cultured samples did not reduce viability at various concentrations, demonstrating that they are not toxic to vero cells. In addition, MDCK cells were exposed to various concentrations of citrinin and cultured samples. The toxic effect of citrinin was shown to decrease cell viability, which shows the concentration-dependent toxicity of citrinin. In contrast, the cultured samples did not change viability in all experimental groups (FIG. 9).

Example 4 Biological and Biochemical Properties of Selected Superior Mutants

4-1) Identification of good mutant stocks

Hawksworth and Pitt estimate (1983), how an excellent mutant 71 109 by to each CYA, MEA and PDA was inoculated to the media and 7 days incubation at 25 ℃ relatively weak ability morphology, culture characteristics observed results Citrinin producing Monascus pilosus It became.

The morphological observations of Monascus mutant 71109 showed various types of Monascus sp. And parent strain Monascus isolate No. It was performed with 711. According to Hawsworth and Pitt ( Aust. J. Biot . 31 81-91 (1983)), colonies of Monascus pilosus showed orange and red color when incubated at 25 ° C. for 7 days on MEA medium. M. mutant 71109 also showed similar color to M. pilosus .

4-2) Pigment, Antimicrobial Activity and Enzymatic Characteristics of Highly Modified Wines

71109 showed excellent properties in red pigment and yellow pigment production compared to other mutants and showed no antimicrobial activity. Amylase activity was confirmed to be improved than the parent strain.

Park et al . According to the method ( Kor. J. Appl. Microbiol. Biotechnol. 27 (2) 172-178 (1999)), the pigment production activity was tested, and the parent strain M. 711 showed very low pigment production activity, but several mutants showed strong pigment production activity of red and yellow. Among them, M. 71109 showed the strongest red and yellow pigment formation activity.

Monascus isolate No. Anti-microbial activity was tested in several target organisms with chloroform extract of 711 and its variants. As a result, Monascus 481 used as a positive control inhibited Bacillus cereus (Gram positive) like Monascus strain, but only some of the mutant strains showed anti-microbial activity and also targeted several Gram negative strains, especially Pseudomonas aeruginosa and Shigella dysenteriae . It was made.

The sample was added to the starch solution and a method of reducing sugar is then incubated discharge DNS (N. Saroja, et. Al , Proc. Biochem. 36 119-125 (2000)) was measured, the other Monascus spp 71109 mutant strain of the present invention Similarly, it had amylase activity (FIG. 10).

Example 5: Optimization of Culture Conditions Using Response Surface Methodology (RSM)

5-1) Response surface methodolog

First, the A factor at a time method, which was used in the conventional multifactor design, was used. This is an experimental method that fixes all other factors and changes only one factor under certain cell conditions. From these experiments, four factors, Soytone, Glucose, MgSO4 and barley, which were most influential on monacolin K production, were selected and continuously optimized for RSM. For experimental design of response surface methodology, treatment combinations in this experiment were assigned using Interiorly-Augmented Central Composite Design (Table 2). In this experiment, the response, ie M. The production of monacolin K (μg / g) from mutant 71109 was assumed to be under the influence of the above 4 factors. Data was analyzed using SAS system for statistical analysis.

To model the response, generalized linear modeling was performed. As a result, the response can be expressed as the following model.

y = log (estimated response mean) = 5.0607)

   + (-0.6582) * x1

   + (-1.0291) * x2

   + (-0.9482) * x3

   + (-1.0726) * x4

   + (-0.8106) * x1 * x2

   + (-0.8023) * x1 * x3

   + (-0.7036) * x1 * x4

   + (-0.8833) * x2 * x3

   + (-0.8024) * x2 * x4

   + (-0.7216) * x3 * x4

   + (-0.8531) * x1 * x1

   + (-1.1454) * x2 * x2

   + (1.4086) * x3 * x3

   + (0.0730) * x4 * x4

   + (-0.8200) * x1 * x2 * x3

   + (-0.6464) * x1 * x2 * x4

   + (-0.7333) * x1 * x3 * x4

   + (-0.8478) * x2 * x3 * x4

   + (0.2614) * x2 * x2 * x2

   + (0.2075) * x3 * x3 * x3

   + (0.2702) * x4 * x4 * x4

   + (-0.9473) * x1 * x2 * x3 * x4

   + (0.2972) * x2 * x2 * x2 * x2

   + (-0.3468) * x3 * x3 * x3 * x3

The response surface model was significant as r 2 = 0.993. This means that this model is suitable for this experiment. To find the point that maximizes the estimated response mean, we searched the area in the spherical design. As a result, the optimized maximum points (x1, x2, x3, x4) = (0.48, 0.41, -1.47, 1.20) were found. Convert it to the actual level: (Soytone, Glucose, MgSO4, content of barley) = (2.48%, 2.41%, 0.053g, 80%)

M from the effect of the four factors listed in Table 2. The yield of monacolin k was calculated from mutant 71109 and the optimized maximum point was (x1, x2, x3, x4) (0.48, 0.41, -1.47, 1.20) and the results showed that 71109 monacolin K optimized culture conditions (Soytone, Glucose, MgSO 4, content of barley) = (2.48%, 2.41%, 0.053 g, 80%).

TABLE 2

Actual factor levels correspongings to coded factor levels

TABLE 3

Experimental Design and Responses

Example 6: In vivo test of Monacolin K

Animal experiments were conducted on high-fat diet 71109 using high-fat diets and their effects on blood cholesterol were examined. Monascus mut using SD-rat. The diet was added to the casein & fat containing diet 71109 was fed to 60 days after the blood was collected and examined. Fig. 11 is a graph showing the triglyceride and total cholesterol concentration of SD-rat supplied with the mutant strain of the present invention Are: control, casein & fat diet (Group I), casein & fat diet containing monacolin K 0.1, 0.5, 1.0% (Group II, III, IV) and lovastatin (Group V). 12 is a graph showing the HDL-cholesterol and LDL-cholesterol concentration of SD-rat supplied with the erythrocyte strain of the present invention: control, casein & fat diet (Group I), casein & fat diet containing monacolin K 0.1, 0.5, 1.0 % (Groups II, III, IV) and lovastatin (Group V).

In the case of red yeast rice, the triglycerides and total cholesterol levels of blood were low and LDL-cholesterol concentration was low. In the case of red yeast rice content of more than 0.5%, triglycerides, total cholesterol and LDL-cholesterol concentrations are lower than those of the normal feed feeding control. It is thought to play a role in preventing and atherosclerosis.

As described above, the mutant strain of the present invention as a radiation mutant strain of Monascus pilosus having excellent anti-cholesterol activity (monacolin K), as well as excellent production of monacolin K, and safety as a non-fungal toxin-producing strain, Compared with the parent strain, it is a superior strain which has greatly improved the productivity of red pigment. Therefore, the development of hongguk bacteria, which have excellent cholesterol inhibitory ability and contain a large amount of natural pigments, is expected to increase the utilization of hongguk in the functional food market in Korea. It can be used as a raw material, and new food and beverage can be developed through this.

Claims (9)

Monascus mutant strain (KCCM 10586) which produces Monacolin K with high efficiency. The method of claim 1, wherein the sheet rinin honggukgyun (Monascus) mutant strain (KCCM 10586), characterized in that (citrinin) did not produce. According to claim 1, Monascus mutant strain (KCCM 10586) characterized in that the production of red and yellow pigments with high efficiency. Irradiating Monascus with various doses of radiation to build a Hong Kong bacteria library, obtaining approximately 1000 colonies in the range of 95% to 99.99% mortality, and compared with the parent strain Monascus mutants with high productivity and low productivity of citrinin, including mutant strains having good activity, are screened through anti-fungal bioassay. Manufacturing method. The method of claim 4 wherein the method for manufacturing a honggukgyun (Monascus) variant, characterized in that by irradiating a radiation dose of about 2.5KGy to have a mortality rate of about 99%. Cultured in a medium containing 2-3% Soytone, 2-3% Glucose, 0.04-0.06g MgSO4, 75-85% Content of barley Cultivation method of Monascus mutant strain (KCCM 10586) of claim 1. Cholesterol lowering agent containing the extract of Monascus mutant strain (KCCM 10586) of claim 1. Functional food for lowering cholesterol containing the extract of Monascus mutant strain (KCCM 10586) of claim 1. The cholesterol lowering agent or functional food according to claim 7 or 8, wherein the extract contains Monacolin K.

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