Fermentation process for preparing erythritol using mother...

Details Fermentation process for preparing erythritol using mother... Fermentation process for preparing erythritol using mother...

1999-12-21

2001-08-07

Hendricks, Keith (Department: 1761)

Food or edible material: processes, compositions, and products

Fermentation processes

Of or with yeast or mold

C435S158000, C435S255100

Reexamination Certificate

active

06270815

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a fermentation process for preparing erythritol in high productivity with a novel mutant of Torula sp. DS101, more specifically, for preparing erythritol using mother liquor produced from purification process of palatinose, which comprises 30~60% of trehalulose, 10~30% of palatinose, 5~15% of fructose, 5~25% of glucose and 0~10% of sucrose.
Erythritol is a four-carbon polyol with property similar to other polyols currently used as food ingredients, such as, xylitol, sorbitol, manintol, maltitol, lactitol and isomalt. It is a naturally occurring substance and is widely distributed in nature. Erythritol is a metabolite or storage compound for seaweed and mushrooms. Fruits like melons, grapes and pears contain erythritol. It occurs frequently in fermented foods including wines and beers, and processed vegetables, such as, soy sauce and oriental miso bean paste.
Erythritol has sweetness with 60~70% of sucrose in a 10% solution. Its high negative heat of solution provides the crystalline material with a strong cooling effect. Erythritol can be safely used in foods to make them tooth-friendly. This property causes the inability of dental caries by developing bacteria to use erythritol as a fermentation substrate. Erythritol as a small molecule has strong colligative properties, such as, strong freezing point depression, boiling point elevation and high osmotic pressure. With its low hygroscopicity and viscosity in solution, it is very useful to reduce and control the water activity of foodstuff.
Erythritol can be produced by microbial methods using the osmophilic yeasts, especially species of the genus Torulopsis such as
T. magnoliae, T. veratilis,
and
T. candida; Endonycopsis chodati; Hansenula supelliculsa; Pichia miso; Monilliella tomentosa
var.
pollinis; Trigonopsis variabilis;
Trichosporonoides;
Candida zeylanoides;
and Aureobasidium sp.
Monilliella tomentosa
var.
pollinis
on the medium containing 33~37% of glucose with 43~48% of erythritol yield. However, erythritol production using such strains could not be applied to industrial scale due to the formation of by-products, such as, glycerol and ribitol.
On the other hand, industrial production of erythritol has been performed using a mutant of Aureobasidium. The mutant was isolated and developed by cooperative study of Nikken Chemical and National Food Research Institute of Japan. The mutant produced erythritol with 1.8~1.9 g/L·h volumetric productivity and 42~44% yield in the medium containing 40% of glucose. This has been regarded as the highest report of erythritol productivity and yield among the erythritol-producing microorganisms.
It has not been previously reported that Torula sp. produces erythritol. However, the inventors found that a mutant of Torula sp. isolated from a 40% of sucrose solution produces erythritol.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel mutants cells of Torula sp. DS101, which were deposited to Korean Culture Center of Microorganism with accession number KCCM-10171 on Sep. 7, 1999 under Budapest treaty, for preparing erythritol with high productivity.
Another object of the present invention is to provide the optimal fermentation conditions for maximum production of erythritol using mutant cells of Torula sp. DS101 (KCCM-10171) by controlling following conditions;
i) fermenting the medium consisting of mother liquor produced from purification process of palatinose as carbon source, 1.8~2.2% of yeast extract as nitrogen source and 0.2~0.4% of KH
2
PO
4
with mutant cells wherein
a) said mother liquor contains 20~50% of sugar as total sugar
b) pH of culture medium is 4.5~6.5;
c) temperature of cultivation is 28~38° C.
d) aeration rate of the medium is 0.1~1.0 volume of air per volume of medium per minute; and
e) agitation speed of the medium is 300~1200 rpm;
ii) removing the mutant cells and other residue from the fermentation medium; and
iii) separating and recovering erythritol from the fermentation medium of step (ii).
The further object of the present invention is to provide a fermentation process wherein the composition of said mother liquor comprises 30~60% of trehalulose, 10~30% of palatinose, 5~15% of fructose, 5~25% of glucose and 0~10% of sucrose.
The further object of the present invention is to provide a method of isolating Torula sp. DS101 (KCCM-10171) comprising the steps of:
i) spreading and culturing a wild type Torula sp. on growth medium (18~22% of glucose and 0.9~1.1% of yeast extract) containing 0.01% of N-methyl-N′-nitro-N-nitroguanidine FNTG);
ii) isolating the produced colonies at least three times on growth medium;
iii) spreading and culturing the colonies of step (ii) on growth medium under UV illumination of 250~270 nm; and
iv) isolating the growing colonies.
DETAILED DESCRIPTION OF THE INVENTION
The mutant cells used for the present invention are isolated by following method.
Isolation of a High Erythritol Producing Mutant
A wild strain from the air in 40% sucrose solution at Bolak Co., Osan, Kyunggi-Do, Korea was selected to produce erythritol. A single colony was incubated in a 250-mL flask containing 50 mL of growth medium (18~22% of glucose and 0.9~1.1% of yeast extract). It was incubated at 28~32° C. and 230~270 rpm until the optical density of culture broth at 600 nm reached at 1.0. The grown cells were collected by centrifugation at 3,000 g for 20 min and washed with 0.1 M citrate buffer pH 5.5.
The collected cells were resuspended in the buffer solution containing 0.01% of N-methyl-N′-nitro-N-nitroguanidine (NTG) and incubated at 28~32° C. for 25~35 mmn. After NTG treatment, the cells were incubated at 28~32° C. for 8~12 hours in YM broth and plated on the agar plate containing 38~42% of glucose and 1.8~2.2% of yeast extract for the selection of a high erythritol producing mutant. Single colony was selected as fast growing mutants. The selected colony was transferred on the fermentation medium containing 18~22% of glucose and 0.9~1.1% of yeast extract to test erythritol producing activity in shake flask. After incubating at 28~32° C. and 230~270 rpm in 100~160 hours, a high erythritol producing mutant was selected and colony produced was separated by repeating separation method more than 3 times.
The obtained colony was again spread and cultured to the medium containing 18~22% of glucose and 0.9~1.1% of yeast extract under UV illumination of 250~270 nm. Finally, growing colony was isolated and obtained as mutant cells and used as strain in this invention.
This mutant has superior properties compared to the those of wild strain in erythritol yield from glucose, volumetric productivity, and sugar tolerance.
Characterization of a High Erythritol Producing Mutant
Microorganism which can produce erythritol from glucose was selected and was identified as Torula sp. Microbial characteristics of a high erythritol producing mutant are as follows;
1) Description
Cream colonies; vegetative reproduction by budding; no filamentous no sexual reproduction.
2) Fermentation
The sugar which can be used for the fermentation of such mutant is illustrated in following Table 1.
TABLE 1
Material
Ferment.
Material
Ferment.
D-Glucose
+
Lactose
−
D-Galactose
−
Cellobiose
−
D-Mannose
+
Melezitose
−
D-Fructose
+
Raffinose
−
Maltose
−
Starch
−
Sucrose
+
Inulin
−
Trehalose
−
D-xylose
−
Melibiose
−
* + means can be used for fermentation,
  − means cannot be used for fermentation.
3) Growth
Following materials are tested if they can be used as growth medium. Table 2 shows the result.
TABLE 2
Material
Growth
Material
Growth
D-Glucose
+
D-Mannitol
+
D-Galactose
+
Galactitol
−
D-Mannose
+
myo-Inositol
−
D-Fructose
+
D-Gluconate
+
L-Sorbose
+
D-Glucuronate
−
D-Glucosamine
−
D-Galacturonate
−
D-Ribose
+
DL-Lactate
−
D-Xylose
+
Succinate
+
D-Arabinose
+
Citrate
+
L-Rhamnose
+
Methan

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