Fermentation process for preparing erythritol by a high salt...

Details Fermentation process for preparing erythritol by a high salt... Fermentation process for preparing erythritol by a high salt...

1999-06-25

2001-09-11

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S254100, C435S255100, C435S255210

Reexamination Certificate

active

06287830

ABSTRACT:

BACKGROUND OF THE INVENTION
This application claims priority to Korean Patent Application Number 98-29083, filed Jul. 20, 1998, and the 98-29083 application is herein incorporated by this reference in its entirety.
1. Field of the Invention
The present invention relates to a fermentation process for preparing a high yield erythritol using a salt tolerant mutant of Candida sp. [
Candida magnoliae
, SR101:KCCM-10160]. More specifically, the present invention relates to a process for preparing erythritol under optimal fermentation conditions for maximal erythritol production by optimizing the environmental conditions of culture, including medium component, pH, temperature, aeration rate and agitation speed.
2. Background Art
Erythritol, a four carbon sugar alcohol, is a naturally occurring substance and is widely distributed in nature. Like most of the other polyols, it is a metabolite or storage compound for seaweeds and mushrooms. Fruits like melons, grapes and pears also contain erythritol. As it is often produced by bacteria, fungi, and yeasts, erythritol also occurs frequently in fermented food systems like wines or beers, and in processed vegetables, such as soy sauce or the oriental miso bean paste.
Erythritol is a moderately sweet bulking agent with 60-70 percent of the sweetness of sucrose in a ten percent solution. Its high positive enthalpy of solution provides the crystalline material with a strong cooling effect. As it has a taste which is very close to sucrose without bitter aftertaste, it is ideal to improve the taste of a combination with intense sweeteners like aspartame.
As a small molecule, erythritol also has strong colligative properties, i.e. a strong freezing point depression and boiling point elevation effect as well as a high osmotic pressure. In combination with its low hygroscopicity and viscosity in solution, it is also very useful to reduce and control the water activity of foodstuffs.
Erythritol produced from its natural sources, such as fruits and vegetables, occurs in relatively small amounts. Consequently, these natural sources are impractical for the high yield production of erythritol. Other methods of producing erythritol include chemical manipulation and the use of micro-organisms. Chemically, erythritol is produced by reduction of meso-tartarate, oxidation and reduction of 4,6-o-ethylidene-D-glucose, hydrolysis of dealdehyde starch, or addition of hydrogen. The production of erythritol by this and other related chemical processes, however, is expensive.
Erythritol produced by microbial methods is typically grown by use of osmophilic yeasts such as with species of the genus Torulopsis, such as
T. magnoliae, T. veratilis
, and
T candida; Endomycopsis chodati; Hansenula supelliculsa; Pichia miso; Monilliella tomentosa var. pollinis; Trigonopsis variabilis
; Trichosporonoides;
Candida zeylanoides
; and Aureobasidium. Some bacteria such as
Leuconostoc oenos
can also produce erythritol.
Monilliella toiiientosa var. pollinis
produced erythritol on a medium containing 35.7% glucose with 45.6% yield. Erythritol production using this strain did not apply to industrial scale due to by-products such as glycerol and ribitol. Industrial production of erythritol has been performed by a mutant of Aureobasidium. The mutant was isolated and developed by cooperative study of Nikken Chemical and National Research Institute of Japan. The mutant produced erythritol with 47.6% yield on a medium containing 22.5% glucose and 2 g/L-h volumetric productivity. However, the culture with this fungus had more difficultly than that with yeast.
The present invention presents a novel process for producing a high yield erythritol by isolating a wild yeast strain of Candida sp. from nature and mutating the yeast with EMS (Ethyl-methanol sulfonate) treatment. One of the mutants has superior properties to the wild strain in erythritol yield from glucose, volumetric productivity, and salt tolerance. By using the mutant of Candida sp., the optimization of the environmental conditions of culture was performed for maximal erythritol production.
SUMMARY OF THE INVENTION
The present invention provides novel mutants cells of
Candida magnoliae
SR101, which were deposited to Korean Culture Center of Microorganism 361-221, Yurim Building, Hongje-1-dong, Seodacmun-gu, Seoul 120-091, Korea with accession number KCCM-10160 on May 17, 1999 under Budapest treaty, which cells are used for preparing erythritol with high productivity.
The present invention also provides an optimal fermentation process for maximum production of erythritol using mutant cells of
Candida magnoliae
SR101 deposited to Korean Culture Center of Microorganism with accession number KCCM-10160 comprising the steps of:
a) fermenting monosaccharide or disaccharide medium with cells by controlling following fermentation conditions:
i) composition of medium for maximum production of erythritol consists of 10-50 (w/v)% of glucose, 0.2-2.0 (w/v)% of yeast extract, 0.1-10 (w/v)% of KH
2
PO
4
, 0.1-5.0 (w/v)% of (NH
4
)
2
SO
4
and 0.01-1.0 (w/v)% of MgSO
4
.7H
2
O,
ii) pH of culture medium is 6-8,
iii) temperature of cultivation is 26-30° C.,
iv) aeration rate is 0.75-2.0 volume of air per volume of medium per minute, and
v) agitation speed is 300-1200 rpm;
b) feeding solution containing KCl continuously or intermittently fed into the culture broth during erythritol production phase to be 2-10% of its concentration;
c) removing cells from the fermentation medium; and
d) separating and recovering erythritol from the fermentation medium of step c).
Various other objectives and advantages of the present invention will become apparent from the following description.
DETAILED DESCRIPTION OF THE INVENTION
As used in the claims, “a” can include multiples.
The present invention concerns a method for obtaining erythritol with a high yield and a high volumetric productivity using mutant cells of
Candida magnoliae
by optimizing culture conditions.
The mutant cells of the present invention are isolated by following method.
Candida sp. was screened from a comb. A piece of comb was transferred into the medium containing 40% of glucose and 1.0% of yeast extract, and incubated at 30° C. The broth was diluted and incubated at 30° C. on agar plate containing 20% of glucose, 1.0% of yeast extract and 2.0% of agar. After obtained colonies were incubated on fermentation medium, which consisted of 10% of glucose and 1.0% of yeast extract, the culture broth was centrifuged to remove cells, and the supernatant was analyzed for erythritol determination. A high erythritol producing strain was selected for erythritol production. The strain was identified to
Candida magnotiae
by the Microcheck Co.
The
Candida magnoliae
strain was incubated on growth medium containing 2.0% of glucose, 1.0% of yeast extract and 1.0% of peptone. After growth, the broth was spread on an agar plate containing 10% of glucose, 0.8% of yeast extract, 0.3% of peptone and 2.0% of agar, and the obtained colony was transferred on sporulation medium containing 0.1% of glucose, 1.0% of yeast extract and 2.0% of agar. The formed spore was harvested by autoclaved distilled water and was selected by adding 10 mM of 2-mercaptoethanol for 30 minutes and treating with lyticase 0.5 mg/ml for 4 hours.
The selected spore was treated by EMS (ethylmethanol sulfonate), and was incubated on the medium containing 30% of glucose, 18% of KCl, 0.1% of yeast extract and 2.0% of agar. Single colony was selected as fast growing mutants for the selection of a high salt tolerant mutant. The selected colony was transferred on the fermentation medium to test erythritol producing activity in shake flask.
After incubating at 30° C. and 240 rpm for 72 hours, a high erythritol producing mutant was selected. Finally, growing colony was isolated and obtained as mutant cells, and used as a producing strain in this invention. These mutant cells were deposited to Korean Culture Center of Microorganisms 361-221, Yurim Building, Hongie-1-dong, Seodaemun-gu, Seoul 120-091, Korea with accession num

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