U.S. patent application number 09/931044 was filed with the patent office on 2001-12-27 for fermentation process for preparing erythritol by a high salt tolerant mutant of candida sp..
This patent application is currently assigned to Bolak Co. Ltd.. Invention is credited to Jung, Soo Ryun, Kim, Sang Yong, Ryu, Yeon Woo, Seo, Jin Ho.
Application Number | 20010055796 09/931044 |
Document ID | / |
Family ID | 19544590 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010055796 |
Kind Code |
A1 |
Seo, Jin Ho ; et
al. |
December 27, 2001 |
Fermentation process for preparing erythritol by a high salt
tolerant mutant of Candida sp.
Abstract
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.
Inventors: |
Seo, Jin Ho; (Sungnam-Shi,
KR) ; Ryu, Yeon Woo; (Seoul, KR) ; Jung, Soo
Ryun; (Seoul, KR) ; Kim, Sang Yong;
(Kwangmyung-Shi, KR) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Bolak Co. Ltd.
|
Family ID: |
19544590 |
Appl. No.: |
09/931044 |
Filed: |
August 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09931044 |
Aug 17, 2001 |
|
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09344609 |
Jun 25, 1999 |
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6287830 |
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Current U.S.
Class: |
435/158 ;
435/255.4 |
Current CPC
Class: |
C12N 1/165 20210501;
C12R 2001/72 20210501; C12P 7/18 20130101 |
Class at
Publication: |
435/158 ;
435/255.4 |
International
Class: |
C12P 007/18; C12N
001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 20, 1998 |
KR |
98-29083 |
Claims
We claim:
1. A fermentation process for 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 KH2P0.sub.4, 0.1-5.0 (w/v) % of
(N.sub.4).sub.2S0.sub.4 and 0.01-1.0 (w/v) % of
MgS0.sub.4.7H.sub.20, ii) pH of culture medium is 6-8, iii)
temperature of cultivation is 26-30.degree. 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).
2. The cells of Candida magnoliae SR101 [KCCM-10160].
3. A fermentation process for producing of erythritol using mutant
cells of Candida magnoliae SR101 deposited to Korean Culture Center
of Microorganism with accession number KCCM-10160 comprising: a)
fermenting monosaccharide or disaccharide medium with cells of the
Candida magnoliae SR101 deposited to Korean Culture Center of
Microorganism; and b) feeding a solution containing KCl
continuously or intermittently fed into the culture broth during
erythritol production phase to be 2-10% of its concentration.
4. The process of claim 3 wherein the conditions for fermenting the
monosaccharide or disaccharide medium are: i) medium composition
consisting of 10-50 (w/v) % of glucose, 0.2-2.0 (w/v) % of yeast
extract, 0.1-10 (w/v) % of KH.sub.2P0.sub.4, 0.1-5.0 (w/v) % of
(NH.sub.4).sub.2S0.sub.4 and 0.01-1.0 (w/v) % of
MgS0.sub.4.7H.sub.20, ii) pH of culture medium is 6-8, iii)
temperature of cultivation is 26-30.degree. 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.
5. A fermentation process for producing of erythritol using mutant
cells of Candida magnoliae SR101 deposited to Korean Culture Center
of Microorganism with accession number KCCM-10160 comprising: a)
fermenting monosaccharide or disaccharide medium with cells of the
Candida magnoliae SR101 deposited to Korean Culture Center of
Microorganism; and b) feeding a solution containing KCl
continuously or intermittently fed into the culture broth.
6. The process of claim 5 wherein the conditions for fermenting the
monosaccharide or disaccharide medium are: i) medium composition
consisting of 10-50 (w/v) % of glucose, 0.2-2.0 (w/v) % of yeast
extract, 0.1-10 (w/v) % of KH.sub.2P0.sub.4, 0.1-5.0 (w/v) % of
(NH.sub.4).sub.2SO.sub.4 and 0.01-1.0 (w/v) % of
MgSO.sub.4.7H.sub.20, ii) pH of culture medium is 6-8, iii)
temperature of cultivation is 26-30.degree. 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.
7. The process of claim 5 wherein the solution containing KCl is
continuously or intermittenly fed into the culture broth during the
erythritol production phase.
8. The process of step 5 wherein the solution containing KCl is fed
into the culture to be 2-10% of its concentration.
Description
BACKGROUND OF THE INVENTION
[0001] 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.
[0002] 1. Field of the Invention
[0003] 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.
[0004] 2. Background Art
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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
[0011] The present invention provides novel mutants cells of
Candida magnoliae SR101, which were deposited to Korean Culture
Center of Microorganism with accession number KCCM-10160 on May 21,
1999 under Budapest treaty, which cells are used for preparing
erythritol with high productivity.
[0012] 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:
[0013] a) fermenting monosaccharide or disaccharide medium with
cells by controlling following fermentation conditions:
[0014] 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.sub.2PO.sub.4, 0.1-5.0 (w/v) %
of (NH.sub.4).sub.2SO.sub.4 and 0.01-1.0 (w/v) % of
MgSO.sub.4.7H.sub.2O,
[0015] ii) pH of culture medium is 6-8,
[0016] iii) temperature of cultivation is 26-30.degree. C.,
[0017] iv) aeration rate is 0.75-2.0 volume of air per volume of
medium per minute, and
[0018] v) agitation speed is 300-1200 rpm;
[0019] b) feeding solution containing KCl continuously or
intermittently fed into the culture broth during erythritol
production phase to be 2-10% of its concentration;
[0020] c) removing cells from the fermentation medium; and
[0021] d) separating and recovering erythritol from the
fermentation medium of step c).
[0022] Various other objectives and advantages of the present
invention will become apparent from the following description.
DETAILED DESCRIPTION OF THE INVENTION
[0023] As used in the claims, "a" can include multiples.
[0024] 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.
[0025] The mutant cells of the present invention are isolated by
following method.
[0026] 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.degree. C. The broth was diluted
and incubated at 30.degree. 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 magnoliae by the Microcheck Co.
[0027] 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.
[0028] The selected spore was treated by EMS (ethyhnethanol
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.
[0029] After incubating at 30.degree. 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 with accession
number KCCM-10160under Budapest treaty.
[0030] The following is fermentation method for producing
erythritol using mutants cells.
[0031] Seed Culture
[0032] The cells of Candida magnoliae [KCCM-10160] are cultivated
in a 250-mL flask containing 40-60 mL of growth medium (2.0% of
glucose, 1.0% of peptone, 1.0% of yeast extract) at 30.degree. C.
and 240 rpm for 48 hours and the seed culture was transferred to a
250-ml flask or a 5-L fermentor for producing erythritol in a main
culture.
[0033] Main Culture
[0034] Flask experiments with fermentation medium were performed at
26-30.degree. C. and 300-1200 rpm in 60-100 hours. The fermentation
medium consisted of 10-50% of glucose as carbon source and 0.2-2.0%
of yeast extract, 0.1-15.0% of(NH.sub.4).sub.2SO.sub.4, 0.1-10% of
KH.sub.2PO.sub.4 and 0.01-1.0% of Mg.sub.2S0.sub.4.7H.sub.20 were
used to be inorganic sources. For the experimental purpose, glucose
concentration was adjusted. Batch and fed-batch culture in the
fermentor were performed at 26-30.degree. C. and initial pH 7.
Aeration rate was in the range of 0.75-2.0 vvm. Agitation speed was
300-1200 rpm. Fed-batch culture was performed with 5-10% of glucose
by adding continuously or intermittently 10-40% of glucose.
[0035] The fermentation process is preferably by fed-batch process.
After glucose was completely consumed in the medium, the amount of
erythritol is measured by high performance liquid chromatography
equipped with Carbohydrate Analysis column. Dry cell weight is
estimated by using a calibration curve made from relationship
between optical density at 600 nm and dry cell weight. Glucose is
measured by dinitrosalicylic acid method.
[0036] The measured yield of erythritol is 35-55% of glucose
consumption and volumetric productivity is 0.7 g/L-h.
[0037] Finally the fermentation medium is centrifuged for removing
cells and other residue, and the supernatant is filtered and
dialyzed for obtaining erythritol.
[0038] The present invention is more particularly described in the
following examples which are intended as illustrative only since
numerous modifications and can be explained more specifically by
following examples.
EXAMPLES
Example I
[0039] Isolation of Mutant Cells
[0040] Candia 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.degree. C. The broth was diluted
and incubated at 30.degree. 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 supernant was analyzed for
erythritol determination. A high erythritol producing strain was
selected for erythritol production. The strain was identified to
Candida magnoliae by Microcheck Co.
[0041] This strain was incubated at 30.degree. C. for 48 hours 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 agar plate
containing 10% of glucose, 0.8% of yeast extract, 0.3% of peptone
and 2.0% of agar at 30.degree. C. for 36 hours, and then obtained
colony was transferrred on sporulation medium containing 0.1% of
glucose, 1.0% of yeast extract and 2.0% of agar, and spores were
formed at 4.degree. C. after 4 days. 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 (100,000 units) for 4 hours. The selected spore was treated
for 30 minutes by EMS. The reaction was terminated by adding 5% of
thiosulfate. The spore was incubated on the medium containing 30%
of glucose, 18% of KCl, 0.1% of yeast extract and 2.0% of agar for
the selection of a high salt tolerant mutant. Single colony was
selected as fast growing mutants. The selected colonies were
transferred on the fermentation medium of 50 mL to test erythritol
producing activity in 250 mL-shake flask. After incubating at
30.degree. C. and 240 rpm in 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 Microorganism with accession number KCCM-10160.
Example II
[0042] Erythritol Production by the Fermentation of Mutant
Cells
[0043] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
30.degree. C. and 240 rpm for 48 hours and this seed culture was
transferred to a 250-ml flask for producing erythritol. Flask
experiments with fermentation medium were performed at 280.degree.
C., initial pH 7, and 240 rpm for 84 hours. The fermentation medium
consisted of 25% of glucose as carbon source and 0.5% of yeast
extract, 0.2% of (NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4
and 0.04% of MgS0.7H.sub.2O.
[0044] After 84 hours fermentation, the amount of erythritol from
10% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 25 g/L and volumetric
productivity is 0.30 g/L-h.
Comparative Example I
[0045] Erythritol Production by the Fermentation of Wild Type of
Cells
[0046] The wild type of cells of Candida magnoliae are cultivated
in a 250-mL flask containing 50 mL of growth medium (2.0% of
glucose, 1.0% of peptone, 1.0% of yeast extract) at 30.degree. C.
and 240 rpm for 48 hours, and this seed culture was transferred to
a 250-ml flask for producing erythritol. Flask experiments with
fermentation medium were performed at 28.degree. C., initial pH 7,
and 240 rpm for 108 hours. The fermentation medium consisted of 25%
of glucose as carbon source and 0.5% of yeast extract, 0.2% of
(NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4 and 0.04% of
MgSO.sub.4.7H.sub.2O.
[0047] After 108 hours fermentation, the amount of erythritol from
10% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 17 g/L and volumetric
productivity is 0.16 g/L-h.
Example III
[0048] The Effect of Initial pH for Erthritol Production
[0049] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
300.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 250-ml flask for producing erythritol. Flask
experiments with fermentation medium were performed at 280.degree.
C. and 240 rpm for 84 hours. The fermentation medium consisted of
25% of glucose as carbon source and 0.5% of yeast extract, 0.2% of
(NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4 and 0.04% of
MgSO.sub.4.7H.sub.2O. Effect of pH on erythritol production was
investigated.
[0050] After 84 hours fermentation, the amount of erythritol at
initial pH of 5.0 is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 21.4 g/L and volumetric
productivity is 0.25 g/L-h.
[0051] After 84 hours fermentation, the amount of erythritol at
initial pH of 6.0 is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 23.6 g/L and volumetric
productivity is 0.28 g/L-h.
[0052] After 84 hours fermentation, the amount of erythritol at
initial pH of 7.0 is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 25.0 g/L and volumetric
productivity is 0.30 g/L-h.
[0053] After 84 hours fermentation, the amount of erythritol at
initial pH of 5.0 is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 18.6 g/L and volumetric
productivity is 0.22 g/L-h.
Example IV
[0054] The Effect of Temperature for Erythritol Production
[0055] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-ml, flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
30.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 250-ml flask for producing erythritol. Flask
experiments with fermentation medium were performed at initial pH
of 7.0 and 240 rpm for 84 hours. The fermentation medium consisted
of 25% of glucose as carbon source and 0.5% of yeast extract, 0.2%
of (NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4 and 0.04% of
MgSO.sub.4.7H.sub.20. Effect of temperature on erythritol
production was investigated.
[0056] After 84 hours fermentation, the amount of erythritol at
26.degree. C. is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 15.0 g/L and volumetric
productivity is 0.18 g/L-h.
[0057] After 84 hours fermentation, the amount of erythritol at
28.degree. C. is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 25.0 g/L and volumetric
productivity is 0.30 g/L-h.
[0058] After 84 hours fermentation, the amount of erythritol at
30.degree. C. is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 20.7 g/L and volumetric
productivity is 0.25 g/L-h.
Example V
[0059] The Effect of the KCI Concentration for Erythritol
Production
[0060] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
30.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 250-ml flask for producing erythritol. Flask
experiments with fermentation medium were performed at 280.degree.
C. and 240 rpm for 84 hours. The fermentation medium consisted of
25% of glucose as carbon source and 0.5% of yeast extract, 0.2% of
(NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4 and 0.04% of
MgSO.sub.4.7H.sub.2O. Effect of KCl concentration on erythritol
production was investigated.
[0061] After 84 hours fermentation, the amount of erythritol at
0.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 25.0 g/L and volumetric
productivity is 0.30 g/L-h.
[0062] After 84 hours fermentation, the amount of erythritol at
1.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 25.2 g/L and volumetric
productivity is 0.30 g/L-h.
[0063] After 84 hours fermentation, the amount of erythritol at
3.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 23.3 g/L and volumetric
productivity is 0.28 g/L-h.
[0064] After 84 hours fermentation, the amount of erythritol at
5.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 27.6 g/L and volumetric
productivity is 0.33 g/L-h.
[0065] After 84 hours fermentation, the amount of erythritol at
6.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 26.5 g/L and volumetric
productivity is 0.32 g/L-h.
Comparative Example II
[0066] The Effect of the KCl Concentration for Erythritol
Production Using Wild Type of Cells
[0067] The wild type of cells of Candida magnoliae are cultivated
in a 250-mL flask containing 50 mL of growth medium (2.0% of
glucose, 1.0% of peptone, 1.0% of yeast extract) at 30.degree. C.
and 240 rpm for 48 hours, and this seed culture was transferred to
a 250-n-d flask for producing erythritol. Flask experiments with
fermentation medium were performed at 280.degree. C. and 240 rpm
for 108 hours. The fermentation medium consisted of 25% of glucose
as carbon source and 0.5% of yeast extract, 0.2% of
(NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4 and 0.04% of
MgSO4.7H.sub.2O. Effect of KCl concentration on erythritol
production was investigated.
[0068] After 84 hours fermentation, the amount of erythritol at
0.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 17.0 g/L and volumetric
productivity is 0.16 g/L-h.
[0069] After 108 hours fermentation, the amount of erythritol at
1.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 24.0 g/L and volumetric
productivity is 0.22 g/L-h.
[0070] After 108 hours fermentation, the amount of erythritol at
2.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 23.7 g/L and volumetric
productivity is 0.26 g/L-h.
[0071] After 108 hours fermentation, the amount of erythritol at
3.0% KCl is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 14.2 g/L and volumetric
productivity is 0.13 g/L-h.
Example VI
[0072] The Erythritol Production According to the Change of
Aeration n the Fermentor
[0073] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
30.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 5-L, fermentor for producing erythritol. Fermentor
experiments with fermentation medium were performed at 28.degree.
C., initial pH of 7.0, and 500 rpm. The fermentation medium
consisted of 25% of glucose as carbon source and 0.5% of yeast
extract, 0.2% of (NH.sub.4).sub.2SO.sub.4, 0.5% of KH.sub.2PO.sub.4
and 0.04% of MgSO.sub.4.7H.sub.20. Effect of aeration rate on
erythritol production was investigated.
[0074] After 205 hours fermentation, the amount of erythritol at
0.75 vvm is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 131.2 g/L and volumetric
productivity is 0.64 g/L-h.
[0075] After 205 hours fermentation, the amount of erythritol at
1.00 vvm is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 143.3 g/L and volumetric
productivity is 0.70 g/L-h.
[0076] After 205 hours fermentation, the amount of erythritol at
1.50 vvm is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 125.0 g/L and volumetric
productivity is 0.61 g/L-h.
Example VII
[0077] The Erythritol Production According to the Change of Glucose
Concentration in the Fermentor
[0078] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
300.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 5-L fermentor for producing erythritol. Fermentor
experiments with fermentation medium were performed at 28.degree.
C., initial pH of 7.0, 1.0 vvm, and 500 rpm for 84 hours. The
fermentation medium consisted of glucose as carbon source and 0.5%
of yeast extract, 0.2% of (NH.sub.4).sub.2SO.sub.4, 0.5% of
KH.sub.2PO.sub.4, 5.0% of KCl and 0.04% Of MgS0.sub.4.7H.sub.20.
Effect of glucose concentration on erythritol production was
investigated.
[0079] After 63 hours fermentation, the amount of erythritol at 10%
of glucose is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 29.1 g/L and volumetric
productivity is 0.46 g/L-h.
[0080] After 120 hours fermentation, the amount of erythritol at
15% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 64.9 g/L and volumetric
productivity is 0.54 g/L-h.
[0081] After 160 hours fermentation, the amount of erythritol at
20% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 87.2 g/L and volumetric
productivity is 0.55 g/L-h.
[0082] After 205 hours fermentation, the amount of erythritol at
25% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 143 g/L and volumetric
productivity is 0.70 g/L-h.
[0083] After 205 hours fermentation, the amount of erythritol at
30% of glucose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 117 g/L and volumetric
productivity is 0.57 g/L-h.
Example VIII
[0084] The Erthritol Production According to the Change of Sucrose
Concentration in the Fermentor
[0085] The mutant cells of Candida magnoliae [KCCM-10160] are
cultivated in a 250-mL flask containing 50 mL of growth medium
(2.0% of glucose, 1.0% of peptone, 1.0% of yeast extract) at
30.degree. C. and 240 rpm for 48 hours, and this seed culture was
transferred to a 5-L fermentor for producing erythritol. Fermentor
experiments with fermentation medium were performed at 28.degree.
C., initial pH of 7.0, 1.0 vvm, and 500 rpm for 84 hours. The
fermentation medium consisted of sucrose as carbon source and 0.5%
of yeast extract, 0.2% of (NH.sub.4).sub.2SO.sub.4, 0.5% of
KH.sub.2PO.sub.4, 5.0% of KCl, and 0.04% of MgSO.sub.4.7H.sub.2O.
Effect of sucrose concentration on erythritol production was
investigated.
[0086] After 65 hours fermentation, the amount of erythritol at 10%
of sucrose is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 26.1 g/L and volumetric
productivity is 0.40 g/L-h.
[0087] After 154 hours fermentation, the amount of erythritol at
20% of sucrose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 78.3 g/L and volumetric
productivity is 0.51 g/L-h.
[0088] After 175 hours fermentation, the amount of erythritol at
20% of sucrose is measured by HPLC equipped with Carbohydrate
Analysis column. The obtained erythritol is 105 g/L and volumetric
productivity is 0.60 g/L-h.
[0089] After 250 hours fermentation, the amount of erythritol at
40% sucrose is measured by HPLC equipped with Carbohydrate Analysis
column. The obtained erythritol is 126 g/L and volumetric
productivity is 0.50 g/L-h.
[0090] Candida magnoliae [KCCM-10160] in the present can produce
erythritol from glucose or sucrose with a high productivity and a
high yield, make little foam, and does not produce by-products such
as glycerol, resulting in easy recovery. These advantages have
considerable importance for the possible commercial manufacture of
erythritol.
* * * * *