JPH07274978A - Production of ang-khak colorant - Google Patents

Abstract

PURPOSE:To obtain an ang-khak colorant not containing citrinin and excellent in safety as a food additive. CONSTITUTION:This method for producing an ang-khak colorant substantially not containing citrinin is carried out by using a strain belonging to Monascus pilosus group or Monascus rubber group or by using a strain belonging to Monascus selected from Monascus purpureus, Monascus albidus, Monascus rubiginosus and Monasucus major. A method for producing a mutant maintaining a red color productivity without producing citrinin by mutating a strain belonging to Monascus anka and capable of producing a citrinin-containing ang-khak color is also provided.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to red yeast rice pigments. More specifically, the present invention relates to the production of red yeast rice pigment using red yeast mold which is incapable of producing citrinin.

[0002]

BACKGROUND OF THE INVENTION Red yeast rice pigment is a pigment obtained by extracting a reddish pigment produced by culturing red yeast rice fungus (genus Monascus). Conventionally, Monascus anka has been exclusively used for the production of red yeast rice pigment.
The reason is that this species contains strains with high pigment-producing ability. The red yeast rice obtained by culturing Monascus anka has been widely used for a long time in the Orient, particularly in China and Southeast Asian countries, for the production of Chinese herbs, food preservatives, and alcoholic beverages and pickles. In recent years, the red-colored pigment produced by this fungus has been evaluated for its value as a natural pigment and has come to be used as a colorant for many foods including processed seafood (Akira Endo: Fermentation and Industry, Vol. 43). , Pp.5
44-552, 1985).

Recently, the present inventors have found that most of Aspergillus oryzae, which has been conventionally used for the production of Aspergillus oryzae pigment, is known as a toxin produced by yellow mold of rice (Citrinin: 3R-).
trans-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-
3H-2-benzopyran-7-carboxylic acid: Merck Index 11t
h edition, 2329) is newly discovered. Citrinin is a powerful toxin (for review, see Saito et al. "Yellowed Rice Toxins," Microbial Toxins: A. Ci.
(See egler, S. Kadis, A. Aji, Academic Press, New York, 1971, Volume VI, pp.357-367.)
Originally, it is a substance that should not be contained in the red yeast rice pigment, which should be added to food, even in a trace amount. However, many of the red yeast rice pigments that have been conventionally used for food contain citrinin in a small amount. This fact is extremely serious, and there is an urgent need to provide a red yeast rice pigment containing no citrinin instead of the red yeast rice pigment.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a red yeast rice pigment containing no citrinin. Another object of the present invention is to provide a red mold of Aspergillus niger, which produces a red mold as a pigment containing no citrinin. Another object of the present invention is to provide a method for culturing a red mold yeast that produces a red mold yeast which does not contain citrinin, and for separating and purifying the red mold yeast which does not contain citrinin from the culture.

[Means for Solving the Problems] In order to achieve the above object, the present inventor collected various red mold yeast strains and searched for a strain that does not produce citrinin. As a result, they found that some red yeast strains did not produce citrinin, and completed the present invention.

That is, the present invention provides a method for producing a red yeast rice pigment substantially free of citrinin using a strain belonging to the Monascus pirosus group or the Monascus ruber group, and Monascus perpleus species, Monascus albizus species. , A Monascus rubiginosus species, and a Monascus major species selected from the group consisting of Monascus major species are used to provide a method for producing red yeast rice pigment substantially free of citrinin.

Another aspect of the present invention is characterized in that a red yeast rice pigment containing substantially no citrinin is separated and collected from a culture obtained by culturing a strain belonging to the Monascus pirosus group or the Monascus ruber group. And a method for producing red yeast rice pigment, and substantially from a culture obtained by culturing a strain belonging to the genus Monascus selected from the group consisting of Monascus perpleus species, Monascus albizus species, Monascus rubiginosus species, and Monascus major species Provided is a method for producing red yeast rice pigment, which comprises separating and collecting red yeast rice pigment not containing citrinin.

The present invention also provides a method for mutating a strain belonging to Monascus anka species that produces a citrinin-containing red yeast rice pigment to produce a mutant strain that does not produce citrinin and retains a reddish color-producing ability. As one aspect thereof, the above method is provided, wherein the mutant strain is Monascus anchor 4478A or Monascus anchor 4478B. Further provided are strains belonging to Monascus Anka species that do not produce citrinin and retain a red-colored productivity, and the above strains that are Monascus anchor 4478A or Monascus anchor 4478B.

Comparison of citrinin productivity of Monascus sucrose 3%, polypeptone 1%, tartaric acid 0.1%,
L- asparagine _{0.3%, KH 2 PO 4 0.1} %, MgSO 4 · 7 H 2 O
Dispense 100 ml of the medium consisting of 0.05% and antifoaming agent (CB442) 0.01% (pH 4.4) into Sakaguchi flask (500 ml volume).
Sterilized at 120 ℃ for 20 minutes. This medium was inoculated with various slant-cultured strains of the genus Monascus and cultivated with shaking at 25 ° C for 10 days. 10 ml of the culture broth was filtered, the liquid was adjusted to pH 2-3 and extracted twice with 10 ml of ethyl acetate. The extract was dehydrated with anhydrous sodium sulfate, dried under reduced pressure, and dissolved in 1 ml of methanol. Centrifuge the methanol solution at 0 ° C for 10 minutes (1
The supernatant (0.00000 g) was used for the assay.

[0009] When the culture solution contains a small amount of contaminants, such as when using a synthetic medium, the culture filtrate is adjusted to 10 ml, then 10 ml of methanol is added to cool the mixture, and the mixture is centrifuged for 10 minutes.
(10,000 × g) and the supernatant was taken and used for the assay. The bacterial cells (corresponding to 10 ml of culture solution) from which the mother liquor was sufficiently removed were extracted twice with 5 ml of methanol, and the extract was dried under reduced pressure and dissolved in 1 ml of methanol. The supernatant obtained by cooling and centrifugation under the above conditions was used for the assay. The samples prepared under the above conditions (10 to 50 μl as a culture solution equivalent) were used and analyzed in the following cases. Column: Silica C ₁₈ (Inertsil ODS 6 × 250 mm) Fluid phase: Acetonitrile: 0.1% Phosphoric acid water = 55:45 Flow rate: 1.0 ml / min. Detector: UV multi-channel detector, 235 nm

Under the above conditions, the retention time of citrinin is 14 to 15
It was a minute. The citrinin in the sample was confirmed by the retention time of the peak and the UV absorption pattern of the peak and quantified by the peak area, and the identification was performed by mass spectrometry, NMR analysis or the like. The detection limit of the standard citrinin sample under the above conditions is 0.0
It was below 5 μg / injection. Table 1 shows the detection results of citrinin. In addition, by the proposal of Hawksworth and others,
The Monascus genus is shown in three groups, the Monascus Pirosus Group, the Monascus Perpleus Group, and the Monascus Ruber Group (DL Hawksworth, JI Pitt, Australian Journal of Botany, Vol. 31, 51. Page, 1983). further,
The productivity of citrinin is different from that of other media with different media compositions (2 types)
As a result of the examination, similar results were obtained. In addition, there was no change in the productivity of citrinin even when the culture temperature was 30 ° C.

[0011]

[Table 1] Productivity of citrinin of the genus Monascus ─────────────────────────────────── Monascus Species citrinin production (μg / ml) ──────────────────────────────────── M. pirosus (pilosus) Group M. pilosus IFO 4480, 4520 0 M. pubigerus IFO 4521 0 M. serorubescens IFO 4487, 4525 0 ─────────────── ───────────────────── M. purpureus group M. purpureus IFO 4513, AHU 9096, 9451 ATCC 6405, 16436, 16385, 16386, 16365, 16427 All 0 M. Anka IFO 4478 235 IFO 6540 ~ 1 IFO 30873 35 IFO 32228 18 IFO 32316 13 M. Anka var. Rubellus IFO 5965 16 IFO 6085 0 M. Kaoliang iang) MO-F1 47 M. albidus IFO 4489 0 M. rubiginosus IFO 4484 0 M. major IFO 4485 0 ────────────────── ───────────────────────── M. ruber (ruber) group M. ruber (FOB) IFO 4492 and other 10 shares All 0 M. paxii IFO 8201 0 M. Phryginosus (fuliginosus) IFO 4483 0 M. vitreus IFO 4532, 7537 Both 0 M. barkeri ATCC 16966 0 ─────────────────────── ───────────── Other than those belonging to M. anka, which is currently used in the production of red yeast rice pigments in Japan and neighboring countries. M. sp. T1 (Thailand) 112 343 (Japan) 156 030 (Japan) 277 12N (Japan) 61 202-13 (Japan) 13 H4 (Taiwan) 3 T4 (Taiwan) 32 C2 (Cambodia) 102 VN-3 (Vietnam) 2 ───── ───── ────────────────────────

As is clear from the results shown in Table 1,
As for the strains belonging to Monascus anchor, all the tested strains were found to have the ability to produce citrinin. All of the strains currently used for producing red yeast rice pigments are classified as Monascus anchor. On the contrary, the strains belonging to the Monascus pirosus group and the Monascus ruber group did not have citrinin productivity at all.
Therefore, any strain belonging to the Monascus pirosus group or the Monascus rubus group can be used in the present invention. Also, Monascus Perpleus
Within the group, all nine Monascus perpleus strains tested did not produce citrinin. Therefore, strains belonging to the Monascus perpleus species can also be used in the present invention. Furthermore, strains belonging to Monascus albizus sp., Monascus rubiginosus sp., Or Monascus major sp. Do not produce citrinin in the above test, and can be used in the present invention.

Among the strains shown in Table 1 above, the strains that do not produce citrinin (the ones in which the citrinin production is described as 0) are all specific examples of the strains that can be preferably used in the present invention. However, the strain used in the present invention is not limited to these. In the present invention, a strain in which citrinin is not substantially produced, specifically, the amount of citrinin produced is 1 μg / ml or less, preferably 0.1 μg / ml
In the following, particularly preferably, there is no production of citrinin,
Alternatively, a strain that produces less than 0.05 μg / ml can be used. From the viewpoint that citrinnin is not produced and the color of the red yeast rice pigment is good, strains belonging to the Monascus perpleus species can be preferably used in the present invention. For example, the Monascus purpleus IFO 4513, AHU 9096, 9451, ATCC 6405, 16436, listed in Table 1
16385, 16386, 16365, 16427 and the like are strains that can be particularly preferably used in the present invention.

According to the present invention, for producing a red yeast rice pigment containing no citrinin using the above strains, the above strains are cultured by a method well known to those skilled in the art, and red yeast rice is produced from the culture by a method known per se. The dye should be separated and collected.
The examples are specifically shown in the following examples, but the method of the present invention is not limited to these methods, and those skilled in the art can appropriately select the culture conditions depending on the strain based on the examples. ,
It is possible to produce red yeast rice pigment that does not contain citrinin.

According to another aspect of the present invention, Monascus anchor having the ability to produce citrinin is treated with various mutagens,
For example, a method for producing a mutant strain which retains the reddish-color-producing ability without producing citrinin by treating with ultraviolet irradiation, nitrosoguanidine, nitrous acid, an alkylating agent, hydroxyamine or the like alone or in combination, and the method. A mutant strain produced by

The production of citrinin by Monascus anchor was first revealed by the research of the present invention, and has never been known. Further, there has been no knowledge of the mutual relationship between the biosynthesis pathway of citrinin and the biosynthesis pathway of red pigment by Monascus anchor. Therefore, whether or not the citrinin-producing ability of the bacterium can be lost to maintain the red-colored pigment-producing ability cannot be predicted by a person skilled in the art from the conventional knowledge.
As a method for treating Monascus anchor having citrinin-producing ability by the method of the present invention, a method for producing a mutant strain known per se can be used. For example, various mutagens such as UV irradiation, nitrosoguanidine, nitrous acid, alkylating agents, and hydroxyamine may be used alone or in combination, but the treatment is not limited thereto. Whether or not the ability to produce citrinin has been lost may be assayed by the above method.

[0017]

【Example】

Example 1 Sucrose 10%, peptone 1%, KH ₂ PO ₄ 0.1%, M
_{gSO 4 · 7H 2 O 0.05%} , 0.1% tartaric acid, asparagine 0.3
% (pH 4.4) liquid medium 100 ml in Sakaguchi flask
(500 ml volume), after sterilization, Monascus pirosus I
The FO 4480 strain was inoculated and cultured at 30 ° C. for 10 days with shaking. The culture solution was filtered to collect the cells. 70% in this cell
50 ml of ethanol was added, and the mixture was extracted with stirring for 3 hours, and the extract was filtered to collect the filtrate. The filtrate was concentrated to dryness under reduced pressure to obtain 110 mg of crude red yeast rice pigment containing no citrinin. Example 2 100 ml of liquid medium consisting of 5% bread crumbs and 3% glucose
In a 500 ml Erlenmeyer flask and after sterilization by the usual method,
Monascus perpleus IFO 4513 inoculated at 30 ℃
Cultivated for 10 days. According to the method of Example 1, 180 mg of crude red yeast rice pigment containing no citrinin was obtained from the culture.

Example 3 Monascus anchor IF0 4478 strain (Table 1, citrinin producing ability of 235 μg / ml) was treated with ultraviolet rays by a conventional method. One of 1,200 grown strains, which has the ability to produce red yeast rice pigment and has a citrinin production ability of 1 μg / ml or less.
(4478A strain) was obtained. Example 4 The 4478A strain obtained in Example 3 was treated with nitrosoguanidine by a conventional method. From about 2,000 strains thus obtained, a mutant strain (4478B) having a citrinin production ability of 0.1 μg / ml or less was obtained.

[0019]

INDUSTRIAL APPLICABILITY The red yeast rice pigment produced by the method of the present invention is useful because it does not contain citrinin and is excellent in safety as a food additive.

Claims (8)

[Claims] 1. A method for producing a red mold yeast substantially free of citrinin using a strain belonging to the Monascus pirosus group or the Monascus ruber group. 2. A red yeast rice pigment containing substantially no citrinin is obtained by using a strain belonging to the genus Monascus selected from the group consisting of Monascus perpleus, Monascus albizus, Monascus rubiginosus and Monascus major. Method of manufacturing. 3. Production of red yeast rice pigment, characterized in that the red yeast rice pigment substantially free of citrinin is separated and collected from a culture obtained by culturing a strain belonging to the Monascus pirosus group or the Monascus luber group. Method. 4. A red that is substantially free of citrinin from a culture obtained by culturing a strain belonging to the genus Monascus selected from the group consisting of Monascus perpleus, Monascus albizus, Monascus rubiginosus, and Monascus major. A method for producing red yeast rice pigment, which comprises separating and collecting the yeast rice pigment. 5. A method for producing a mutant strain which belongs to Monascus anka species and which produces a citrinin-containing red yeast rice pigment is mutated to produce a citrinin-producing mutant which retains a reddish color-producing ability. 6. The method according to claim 5, wherein the mutant strain is Monascus anchor 4478A or Monascus anchor 4478B. 7. A strain belonging to Monascus anka species, which does not produce citrinin and retains a reddish-colored productivity. 8. The strain according to claim 7, which is Monascus anchor 4478A or Monascus anchor 4478B.