JP3001826B2 - Method for increasing productivity of Monascus pigment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for increasing the productivity of Monascus pigments, and more particularly, the present invention relates to a method of cultivating Monascus, a fungus producing edible red pigment and yellow pigment, in a mixed culture with yeast or mold. , Yeast or fungal culture filtrate, amylase, protease (prote)
a), cellulase, or chitinase to dramatically increase the production of Monascus red pigment and yellow pigment on liquid and solid media.

[0002]

2. Description of the Related Art Synthetic coloring agents have been widely used in place of natural coloring agents derived from animals, plants and microorganisms from the viewpoints of stable color tone, inexpensive price and ease of use. However, since the safety of the synthetic colorant for humans has begun to be doubted, for example, in the 1960s, a component that induces cancer was detected from tar-based dyes, interest in natural colorants has been higher than ever before [see: Appl. E
nviron. Microbiol. , 43 (3): 6
71 (1982)].

[0003] Natural dyes have higher safety to human body than artificial dyes, and may have pharmacological activities such as vitamins, and have the advantage of exhibiting more natural colors. However, the production cost is high and the color tone is stable. It also has the disadvantage that the properties are not good. Currently used natural coloring agents include paprika extract (paprika extrac).
t), deoresin calcium
lcium), carotene, chlorophyll, monascus pigment and the like. In particular, Monascus pigment is the only pigment of microbial origin and is mainly used for processing kamaboko, meat, soft marine products and the like. Research for the industrial production of this dye is continuing without delay, and it has already been commercialized to a level at which it can be produced.

[0004] Monascus spp.
It is a microorganism that has been used in the fermentation of traditional foods such as red wine and red tofu in East Asian countries such as China, Japan and Indonesia, and includes fungi, ascomycetes (Ascomycet)
es) Net, Plectascales
s) eyes, Aspergillacea
e) Classified into family. In an embodiment of the present invention, nitrosoguanidine:
NTA), Monascus s (Monascus s) having improved pigment-producing ability through mutation processes such as UV irradiation.
p. ) J101 strain (KCCM-10093) [Ref: Journal of the Society of Industrial Microbiology, 22:85 (199)
4)].

Monascus pigments are known to be excellent in heat resistance, usable in a wide pH range, and excellent in dyeing properties, and have been found to contain more than 10 kinds of components. I have. Among them, the chemical structural formulas were clarified in six kinds in total, and monascin (monasci) was used.
n), ankaflavin, monascorbrin, rubropunktatin, monascorbramin
And rubropunctamine
ine) and the like [Ref: J. Chem. Soc. ,
3577 (1971)]. It is known that these dye components have different solubility characteristics in various solvents, and their production ratios and production amounts are different depending on the used strain, nutrient substrate, culture conditions and the like.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have found that in Monascus cultivation, a phenomenon in which pigment production is relatively remarkably increased by mixed cultivation with other microorganisms such as yeast and mold. Saccharomyces cerevisiae (Sacc.)
haromyces cerevisiae). By continuing further research, it was clarified that the effect of enhancing the pigment production by the mixed culture described above was due to the action of extracellular enzymes such as amylase, cellulase, protease, and chitinase produced by the mixed culture strain. I made it. According to this result, the culture filtrate of Saccharomyces cerevisiae was used to adjust and optimize the effect of the mixed culture, while the effect of enhancing Monascus pigment production was improved using a commercially available enzyme derived from a strain similar to Saccharomyces cerevisiae. After confirmation, the present invention was completed.

Accordingly, the present invention provides a method for increasing the production of Monascus red pigment and / or yellow pigment by adding a yeast or mold-derived hydrolase at the time of pigment production by culturing Monascus. Is what you do.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically. As the pigment-producing strain of the present invention, various fungi of the genus Monascus can be used, and Monascus sp. J101 having excellent pigment productivity as described above.
(KCCM-10093) is preferably used. In an embodiment of the present invention, 0.7% physiological saline was added to the slant culture medium for preservation of the strain, the surface was scraped off with a glass rod, and a spore suspension adjusted to 10 ⁵ CFU / mL was produced. Used. Seed culture for pigment production was as follows: glucose 5.0%, peptone 2.0%, KH ₂ PO _{4
0.8%, MgSO 4 · 7H 2} O 0.05%, CH 3
5 mL of the spore suspension was inoculated into a 500 mL Erlenmeyer flask containing 75 mL of pH 6.6 medium composed of 0.2% COOK and 0.1% NaCl, and cultured in a reciprocating shaking incubator at 30 ° C. and 120 rpm for 48 hours. . The main culture in liquid fermentation was sucrose 10.0%, yeast extract 0.1%.
3%, casamino acid 0.
5%, NaNO ₃ 0.2%, KH ₂ PO ₄ 0.1
_{%, MgSO 4 · 7H 2 O0.05} %, KCl 0.0
75 mL composed of 5% and 0.001% of FeSO ₄
Inoculate the seed culture solution to 5% in the main culture medium of
Rotational shaking culture was performed at 30 ° C. and 180 rpm for 96 hours. The main culture in solid fermentation is performed by adding 2% agar to a liquid fermentation medium, dispensing the mixture into a plate, producing a gel state, applying 1 mL of the seed culture solution to the plate, and incubating at 30 ° C. incubator for 7 days. Cultured. In the culture of the Monascus strain of the present invention,
Glucose, sucrose, starch, glycerol or dextrin is used as a carbon source.

In the examples of the present invention, a yeast culture filtrate was produced by the following method. Yeast Saccharomyces cerevisiae
Inoculate 500 mL Erlenmeyer flask containing M medium
Rotational shaking culture was performed at 180 ° C. and 180 rpm for 24 hours. After taking out the culture solution and centrifuging at 3000 rpm for 30 minutes to remove the cells, the MFS syringe filter unit (syr
inge filter unit) (pore size 0.45μ)
m) to obtain a filtrate, which was stored at 4 ° C. and used for the experiment. To measure the amount of pigment production in liquid fermentation, 5 mL of the fermented culture was taken, placed in a 100 mL Erlenmeyer flask together with 15 mL of 95% ethanol, and stirred and extracted at 180 rpm for 1 hour using a 30 ° C reciprocal shaker. Using. The amount of the dye was determined by subjecting the extract to 10,000 rpm at 10 rpm.
After removing the cells by centrifugation for a minute, the cells were diluted at an appropriate magnification, and the absorbance was measured at 510 nm and 400 nm using a spectroscope, and the results were indicated by the amounts of red and yellow pigments, respectively. The measurement of the pigment production amount in the solid fermentation was performed by taking five cylindrical sections of 4 mm in diameter and 3 mm in height from the plate after culturing, placing the sections in a 250 mL Erlenmeyer flask with 95% ethyl alcohol, and using a 30 ° C reciprocal shaker. The cells were removed by extraction at 180 rpm for 1 hour, and the pigment content was measured by the method described above.

In the cultivation of Monascus, the hydrolase is prepared by mixing and culturing yeast or fungi producing amylase, cellulase, protease, chitinase, etc. with the Monascus strain,
Alternatively, it is supplied by a method such as directly adding the above-mentioned yeast or mold-derived hydrolase to the culture solution of Monascus.

[0011]

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to these examples. Example 1 A 500 ml Erlenmeyer flask containing 75 mL of a main culture medium for liquid fermentation (containing 10% sucrose as a carbon source) was placed in a 500 ml Erlenmeyer flask, and the number of Monascus sp. J101 (accession number based on the Budapest Treaty: KCCM-1)
0093; Trustee: Korean Culture
Center of Microorganisms,
Seoul, Korea; accession date: December 23, 1996), and cultured for 24 hours.
% (V / v) of Saccharomyces cerevisiae, and mixed culture was performed for 96 hours. As a result, when the yeast culture solution is not added, the red pigment 8.
5 OD units and 9.1 OD units of the yellow pigment were produced, but when yeast culture was added, the red pigment 11
9.0 OD units and 109.3 OD units of yellow pigment were obtained, and when Monascus was mixed and cultured with Saccharomyces cerevisiae, the production of red pigment and yellow pigment was respectively smaller than that when only Monascus was monocultured. 14 times,
It was confirmed that the increase was 12 times.

Example 2 A solid fermentation medium containing sucrose as a carbon source was inoculated with Monascus sp. J101 (KCCM-10093) and cultured for 24 hours. Then, 1 mL of yeast culture was spread on a plate and mixed culture was performed for 7 days. Done. In the case of pure culture of only Monascus without adding a yeast culture solution, the red pigment 0.073
OD unit and yellow pigment 0.058 OD unit were obtained. However, when yeast culture solution was added and mixed culture was performed, red pigment 3.00 OD unit and yellow pigment 3.62 OD unit were obtained. When the culture and the pure culture are compared, the production amounts of the red pigment and the yellow pigment are 41% respectively.
It was confirmed that the number increased by a factor of 62 and 62.

Example 3 After culturing Monascus sp. J101 (KCCM-10093) for 24 hours in a liquid fermentation medium using sucrose as a carbon source, 7% (v / v) of a yeast culture was added, followed by culturing for 72 hours. As a result, when the yeast culture filtrate is not added,
While 8.5 OD units of red pigment and 9.1 OD units of yellow pigment were produced, 178.5 OD units of red pigment and 172.9 OD units of yellow pigment were added when yeast culture filtrate was added. A comparison was made between the case where the yeast culture filtrate was produced and the case where the yeast culture filtrate was not added, and it was confirmed that the production amounts of the red pigment and the yellow pigment were increased by 21 times and 19 times, respectively. Further, in the case of solid culture, the production amounts of the red pigment and the yellow pigment were 33 times and 7 times respectively when the yeast culture filtrate was added and when the yeast culture filtrate was not added.
It was confirmed that the increase was 6 times. On the other hand, analysis of the enzyme culture filtrate revealed production of amylase, cellulase, protease and chitinase.

Example 4 Monascus J1 in a solid medium containing sucrose as a carbon source
After culturing 01 (KCCM-10093) for 24 hours,
Aspergillus oriza
amylase, cellulase and protease (Sigma, USA) from S. yzae) and chitinase (Sigma, US) from Saccharomyces cerevisiae.
A. ) Was added at 0.1% (v / v), and fermentation was performed for 7 days, and the amount of pigment production was measured. As a result, when the enzyme was added, the production amounts of the red pigment and the yellow pigment were increased 11-fold and 14-fold, respectively, when the amylase was added, and 27-fold and 58-fold, respectively, when the cellulase was added. The increase was 30-fold and 54-fold when the protease was added, and 25-fold and 21-fold when the chitinase was added. On the other hand, in the case of liquid culture, the production amounts of the red pigment and the yellow pigment were 31 and 37 times when the amylase was added, 27 and 38 times when the cellulase was added, and 38 and 39 times when the protease was added.
And when the chitinase was added, the increase was 35-fold and 39-fold.

Example 5 In a liquid culture, a fermentation medium containing starch or glycerol as a carbon source in addition to sucrose was used.
After culturing 01 (KCCM-10093) for 24 hours,
A 7% (v / v) yeast Saccharomyces cerevisiae culture filtrate was added, and the effect of enhancing pigment production was observed. The production of red pigment using starch as a carbon source is 16.5 OD units when no culture filtrate is added, 150 OD units when culture filtrate is added, and increases 9-fold when culture filtrate is added. did. When using glycerol as a carbon source,
The production of red dye was 67.0 when no culture filtrate was added.
The OD unit was 122.6 OD units when added, which was a 1.8-fold increase.

[0016]

As described above in detail, according to the method of the present invention, in the cultivation of a fungus of the genus Monascus belonging to the genus Monascus which produces edible red pigments and yellow pigments, mixed culture with yeast or fungi, The addition of the fungal culture filtrate and the direct addition of yeast or fungal-derived enzymes, amylase, protease, cellulase or chitinase, to the Monascus culture solution can reduce the production of Monascus red and yellow pigments on liquid and solid media. It can be dramatically improved.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 1/02 C12R 1: 645) (56) References JP-A-51-88519 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 1/02 C09B 61/00 C12N 1/38

Claims (2)

(57) [Claims] 1. A Monascus (Monascus) liquid culture and solid culture chromogenic using strains, Ami
Lase, cellulase, protease or chitinase?
Produce one or more hydrolases selected from the group consisting of
Mixed culture of yeast or mold and Monascus strain
Culture filtrate of these yeasts or molds, or these yeasts
In addition, the above-mentioned hydrolase derived from mold is directly converted into Monascus culture solution.
A method for increasing productivity of Monascus red pigment and / or yellow pigment by contacting and culturing . 2. The Monascus strain is Monascus J101 (KCCM-
10093). The method of claim 1, wherein