Method of producing ergothioneine

Abstract

An object of the present invention is to provide a simple method of producing ergothioneine. The present invention provides a method of producing ergothioneine comprising a step of culturing a microbe belonging to the genus Moniliella in a medium containing a carbon source to allow the microbe to produce ergothioneine.

Claims

1. A method of producing ergothioneine, comprising: culturing a microbe belonging to the genus Moniliella in a medium containing a carbon source to allow the microbe to produce ergothioneine; and collecting ergothioneine from cell fractions obtained in the culturing.

2. The method according to claim 1, wherein the medium contains a carbon source in a concentration of 100 to 500 g/L.

3. The method according to claim 1, wherein the microbe belonging to the genus: Moniliella is at least one selected from the group consisting of Moniliella pollinis, Moniliella megachiliensis, Moniliella acetoabutens, Moniliella suaveolens var. nigra, and Moniliella suaveolens var. suaveolens.

4. The method according to claim 1, wherein the microbe belonging to the genus Moniliella at least one selected from the group consisting of Moniliella pollinis MCI3554 (accession number FERM BP-6170), Moniliella pollinis MCI3555 (accession number FERM BP-6171), Moniliella pollinis MCI3371 (accession number FERM BP-6173), and Moniliella megachiliensis MCI3369 (accession number FERM BP-6172), or a mutant thereof.

5. The method according to claim 1, wherein the step further comprises allowing the microbe to produce at least one selected from the group consisting of sugar alcohols, alcohols, and organic acids.

6. The method according to claim 5, wherein the sugar alcohol is erythritol.

7. The method according to claim 1, further comprising collecting at least one selected from the group consisting of sugar alcohols, alcohols, and organic acids from the culture obtained in the step.

8. The method according to claim 7, wherein the sugar alcohol is erythritol.

Description

EXAMPLES

(1) Hereinafter, the present invention is described in further detail with reference to Examples but is not limited by the following Examples unless it goes beyond the gist thereof.

(2) 1. Production of Ergothioneine by Microbes Belonging to the Genus Moniliella

Examples 1 to 3

(3) Microbes belonging to the genus Moniliella were investigated for the production of ergothioneine.

(4) (1) Strain

(5) For the microbes belonging to the genus Moniliella, Example 1 used Moniliella pollinis (hereinafter, sometimes referred to as “M. Pollinis”) MCI3554 (accession number FERM BP-6170), Example 2 used M. Pollinis MCI3555 (accession number FERM BP-6171), and Example 3 used M. Pollinis MCI3371 (accession number FERM BP-6173). M. pollinis MCI3555 is a mutant strain of a wild-type strain M. pollinis MCI3554, and M. pollinis MCI3371 is a mutant strain of M. pollinis CBS461.67.

(6) (2) Culture and Cell Collection of Each Strain

(7) A 100 g/L solution of a yeast extract (MEAST P1G manufactured by ASAHI FOOD & HEALTHCARE, CO., LTD) and a 333 g/L solution of glucose (anhydrous crystalline dextrose #300 manufactured by NIHON SHOKUHIN KAKO CO., LTD.) were separately sterilized at 120° C. for 20 minutes, subsequently each strain was inoculated to a mixture of 5 ml and 45 ml of each solution in similarly sterilized 500 ml conical flasks with baffles, and cultured at 30° C. at 180 rpm for 3 days. Upon completion of the culture, 5 ml each of the culture solutions was dispensed and centrifuged at 4° C. at 8000×g for 5 minutes. The obtained cells were suspended in 0.9 wt % saline, centrifuged at 4° C. at 8000×g for 5 minutes, suspended again in saline and centrifuged to collect cells. The collected cells were measured for the weight on a wet cell weight basis. Additionally, one tube of the obtained wet cell samples was lyophilized overnight to measure a dry cell weight.

(8) Measurement results of the obtained wet cell weights, dry cell weight, and water contents are shown in Table 1.

(9) TABLE-US-00001 TABLE 1 Wet cell weight Dry cell weight Water Strain (g) (g) content Example 1 MCI 3554 0.799 0.264 33.0% Example 2 MCI 3371 0.453 0.124 27.4% Example 3 MCI 3555 0.736 0.225 30.5%
(3) Investigation of Ergothioneine Production

(10) The wet cells obtained by the collection described above were suspended in water having a weight two times the wet cell weight, and then treated at 95° C. for 20 minutes to extract ergothioneine in the cells. Subsequently, centrifugation was carried out at room temperature at 10000×g for 5 minutes, 1 ml of the supernatant was collected and lyophilized overnight. The dried solid substance obtained by lyophilization was dissolved again in 500 μl of water, subsequently centrifuged at room temperature at 10000×g for 5 minutes, and the supernatant was filtered using a 0.2 μm filter to measure a concentration of ergothioneine in the filtrate.

(11) In the present Example, an amount of ergothioneine was determined by HPLC using HILICpak VG-50 4E (SHOWA DENKO K.K., 4.6×250 mm). Ergothioneine was eluted using acetonitrile/5 mM ammonium acetate aqueous solution=70/30 as an eluent at a flow rate of 0.6 ml/min and measured with an absorbance at a wavelength of 260 nm. Under the present measurement conditions, the elution of ergothioneine was confirmed after 7.99 min.

(12) Table 2 shows the amount of ergothioneine production by the dry cell weight for each strain of Examples 1 to 3. As a result of culture for 3 days in the medium described above, all the strains were confirmed to have produced ergothioneine.

(13) TABLE-US-00002 TABLE 2 Amount of ergothioneine production Strain (μg/gDCW/h) Example 1 MCI 3554 12.1 Example 2 MCI 3371 4.1 Example 3 MCI 3555 4.2

Examples 4 to 8

(14) Production test of ergothioneine under conditions with different glucose concentrations

(15) M. Pollinis MCI3554 was compared in the ergothioneine productivity under the conditions based on Example 1 in which initial glucose concentrations during the flask culture were changed in the range of 100 to 500 g/L.

(16) M. Pollinis MCI3554 was cultured under the same conditions as in Example 1 except the initial glucose concentration, and the results of ergothioneine production are shown in Table 3. In the present Example, the ergothioneine production was maximized when an initial glucose concentration was 300 g/L.

(17) TABLE-US-00003 TABLE 3 Amount of Wet cell Dry cell ergothioneine Glucose weight weight Water production (g/L) (g) (g) content (μg/gDCW/h) Example 4 100 0.460 0.129 28.2% 9.2 Example 5 200 0.683 0.211 30.9% 9.5 Example 6 300 0.799 0.264 33.0% 12.1 Example 7 400 0.774 0.266 34.3% 8.0 Example 8 500 0.693 0.239 34.4% 1.4

Example 9

(18) (1) Strain

(19) In Example 9, Moniliella megachiliensis (hereinafter, sometimes referred to as “M. Megachiliensis”) MCI3369 (accession number FERM BP-6172) was used as the microbe belonging to the genus Moniliella. M. megachiliensis MCI3369 is a mutant strain of M. megachiliensis CBS567.85.

(20) (2) Production Test of Ergothioneine Under Conditions with Different Glucose Concentrations

(21) M. megachiliensis MCI3369 was compared in the ergothioneine productivity under the conditions in which initial glucose concentrations during the flask culture were changed in the range of 100 to 500 g/L as in the conditions in Examples 4 to 8. M. megachiliensis MCI3369 was cultured and ergothioneine production was investigated. As a result, it was confirmed that ergothioneine was produced at all concentrations.

(22) The above Examples 1 to 9 verified that when a microbe belonging to the genus Moniliella is cultured in a medium containing a carbon source and allowed to produce ergothioneine, ergothioneine can be produced simply and in a short period of time.

(23) 2. Investigation of Erythritol Production

(24) The supernatant collected after centrifuging the culture solution of each strain in the above item “1” was collected to measure a concentration of erythritol.

(25) An amount of erythritol was determined by HPLC using MCI GEL™ CK08EH (Mitsubishi Chemical Corporation, 8×300 mm). Erythritol was eluted using a 1.175 g/L phosphoric acid aqueous solution as an eluent at a flow rate of 0.6 ml/min at a column temperature of 50° C. and measured by detection using a refractive index detector (RI). Under the present measurement conditions, the elution of erythritol was confirmed after 13.02 min.

(26) Table 4 shows the amount of erythritol production in the culture solution of each strain. As shown in Table 4, all the strains measured were confirmed to have produced erythritol.

(27) TABLE-US-00004 TABLE 4 3-Day (72-h) culture Amount of erythritol accumulated Strain (g/L) Example 1 MCI 3554 58.3 Example 2 MCI 3371 4.7 Example 3 MCI 3555 44.2 Example 4 MCI 3554 23.2 Example 5 MCI 3554 68.8 Example 6 MCI 3554 58.3 Example 7 MCI 3554 50.0 Example 8 MCI 3554 28.1

(28) The above results revealed that, in the method of the present invention, when a microbe belonging to the genus Moniliella is cultured in a medium containing a carbon source, the microbe can be allowed to not only produce ergothioneine but also to produce a sugar alcohol. Additionally, it was verified that when a sugar alcohol is collected from the culture supernatant of the ergothioneine producing strain, a sugar alcohol in addition to ergothioneine can be produced.

(29) These results further revealed that, in the method of the present invention, ergothioneine can be obtained from cells and organic compounds such as sugar alcohols, alcohols, and organic acids can be obtained from the culture supernatant in a single culture solution.

(30) As evident in the above results, according to the method of the present invention, ergothioneine and a sugar alcohol can be obtained from a single (same) culture solution. Therefore, the method of the present invention is extremely economic- and time-efficient and hence an industrially useful method. Further, according to the method of the present invention, ergothioneine can be obtained from cells which have been otherwise discarded. Therefore, the method of the present invention is an extremely useful method capable of reducing the burden on environment by effectively utilizing wastes.

(31) Moniliella pollinis MCI 3554 and Moniliella pollinis MCI 3555 described in the present specification were deposited internationally under accession numbers FERM BP-6170 and FERM BP-6171, respectively, as of Nov. 19, 1997 in the International Patent Organism Depositary, National Institute of Technology and Evaluation (NITE-IPOD) (former Life Engineering Research Institute of the Agency of Industrial Science and Technology, Ministry of International Trade and Industry) located at #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba (zip code: 292-0818). Moniliella pollinis MCI 3371 was transferred to the international depositary under accession number FERM BP-6173 as of Nov. 19, 1997 in the same Center. Moniliella megachiliensis MCI 3369 was transferred to the international depositary under accession number FERM BP-6172 as of Nov. 19, 1997 in the same depositary.