JP2003516155A - Fermentative production method of erythritol using mother liquor generated in the process of palatinose purification - Google Patents

Abstract

  (57) [Summary] SOLUTION: Trehalulose 30 to 60%, palatinose 10 to 30%, fructose 5 to 15%, glucose 5 to 25%, glucose A fermentation production method for obtaining erythritol with high productivity using a mother liquor consisting of sugars 0 to 10%.

Description

Detailed Description of the Invention TECHNICAL FIELD OF THE INVENTION

The present invention relates to a fermentation method for obtaining erythritol with high productivity by a novel mutant strain Torula genus DS101, and more specifically, 30-60% trehalulose, 10-30% palatinose, and 5-fructose produced in the process of purifying palatinose. It relates to a method for producing erythritol using a mother liquor composed of 15%, glucose 5 to 25%, and sucrose 0 to 10%.

[0002]

[Technical background]

Erythritol is a four-carbon polyol and has properties similar to those of xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt and the like currently used as food additives. A naturally produced substance that is widely distributed in nature. Erythritol is a metabolite or accumulator of seaweed and mushrooms. Fruits such as melons, grapes and (pear) pears contain erythritol.
It is often contained in fermented foods such as wine and beer, and processed plant foods such as soy sauce and miso.

Erythritol has a sweetness of 60-70% of a 10% sucrose solution. Since its heat of solution takes a large negative value, it is a crystalline substance having a strong cooling effect. Erythritol can be safely used as a tooth-friendly food. Due to this property, erythritol does not become a fermentation substrate for bacterial growth and does not cause tooth decay. As a small molecule, erythritol has strong freezing point depression, boiling point elevation, and strong cohesion such as high osmotic pressure. The hygroscopicity and viscosity of the solution are low, which is extremely useful for reducing or adjusting the water activity of foods.

Erythritol is a yeast of high osmotic affinity, especially Torrhopsis magnoliae (To
rulopsis magnoliae), torlopsis veratilis (T. veratilis), torlopsis candida (T.candida), and other species of the genus Tolulopsis, Endomycopsis chodati, Hansenula spericlesa (Hans)
enula supelliculsa), Pichia miso, Monilliella tomentosa var. pollinis, Trigonopsis variabilis, Trichosporonoides spp.
ichosporonoides), Candida zeylanoides
, And Aureobasidium sp. Monilliella tomentosa var. Monilliella tomentosa
var. pollinis) yields of erythritol 43-48% in medium containing 33-37% glucose. However, the above-mentioned erythritol production method using microorganisms cannot be used on an industrial scale because by-products such as glycerin and ribitol are produced.

In contrast, the industrial production of erythritol is based on the genus Aureobasidium (Aure
obasidium) mutants. This mutant was isolated and developed by joint research between Nikken Chemicals and Japan National Food Research Institute (National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries). This mutant is 1.8-1.9 g / L in a medium containing 40% glucose.
-Erythritol was produced with a volumetric productivity of h and a yield of 42-44%. this is,
It is considered to report the highest erythritol productivity and yield among the erythritol-producing microorganisms.

[0006]

[Means for solving problems]

Heretofore, it has not been reported that Torula produces erythritol. However, the inventors have discovered that a Torula mutant isolated from a 40% sucrose solution produces erythritol.

The object of the present invention is to provide a novel Torula DS that produces erythritol with high productivity.
To provide 101 mutant strains. This mutant strain is 19
It was deposited at the Microbial Culture Center of the Republic of Korea on September 7, 1999 under the accession number KCCM-10171.

[0008] Another object of the present invention is to provide appropriate fermentation conditions by adjusting the following conditions for maximally producing erythritol using a Torula genus mutant strain DS101 (KCCM-10171). That is, i) In a medium comprising a mother liquor produced in the process of purifying palatinose as a carbon source, 1.8 to 2.2% of yeast extract as a nitrogen source, and 0.2 to 0.4% of KH ₂ PO ₄ a) The mother liquor has a total sugar content of 10 to 50%. %) Sugar b) Medium pH is 4.5-6.5 c) Culture temperature is 28-38 ° C d) Aeration rate of medium is 0.1-1.0 volume per minute of medium volume e) Medium agitation rate is 300- The mutant strain is cultivated under the condition of 1200 rpm, ii) the mutant strain and other residues are removed from the fermentation medium, and iii) erythritol is separated and recovered from the fermentation medium of ii).

Still another object of the present invention is a fermentation method in which the composition of the mother liquor is 30-60% trehalulose, 10-30% palatinose, 5-15% fructose, 5-25% glucose and 0-10% sucrose. Is to provide.

Yet another object of the present invention is torula genus DS101 (K
CCM-10171) is provided. That is, i) wild strain of the genus Torula was treated with N-methyl-N′-nitro-N-nitroguanidine (
Growth medium containing 0.01% of NTG (glucose 18-22% and yeast extract 0.9
˜1.1%) and cultivated, ii) the colonies formed in the growth medium are isolated at least three times, and iii) the colonies of ii) above are spread in the growth medium and cultured under UV irradiation of 250 to 270 nm. Iv) Isolate grown colonies.

DETAILED DESCRIPTION OF THE INVENTION

The mutant strain used in the present invention was isolated by the following method. Isolation of mutant strains that highly produce erythritol At Bolak Co., Osan, Gyeonggi-do, Republic of Korea, a wild strain from the atmosphere contained in a 40% sucrose solution was selected for erythritol production. A single colony
Incubation was carried out in a 250 mL flask containing 50 mL of growth medium (glucose 18-22% and yeast extract 0.9-1.1%). Incubation is 28-32 ℃,
It was carried out at 230-270 rpm until the optical density at 600 nm of the culture broth reached 1.0. The grown cells were collected by centrifugation at 3000 G for 20 minutes, and the pH was adjusted to 5.
5 was washed with 0.1 M citrate buffer.

The collected cells were treated with 0.01% N-methyl-N′-nitro-N-nitroguanidine (N
TG) and incubated at 28-32 ° C for 25-35 minutes. After NTG treatment, cells were incubated in YM broth at 28-32 ° C. for 8-12 hours and spread on agar plates containing glucose 38-42%, yeast extract 1.8-2.2% to produce erythritol highly producing mutations. Selected strains. One colony was chosen as a fast growing mutant. The selected colonies were transferred to a fermentation medium containing glucose 18 to 22% and yeast extract 0.9 to 1.1%, and the erythritol production activity was examined in a shake flask. After incubating at 28 to 32 ° C. and 230 to 270 rpm for 100 to 160 hours, a high erythritol-producing mutant strain was selected, and the generated colonies were separated by a repeated separation method of three times or more.

The obtained colonies were again subjected to glucose 18 to 22% and yeast extract 0.9 to 1.1%.
The cells were spread on a culture medium containing the cells and cultured under UV irradiation of 250 to 270 nm. Finally, the grown colonies were isolated to obtain the mutant strain used in the present invention.

This mutant strain was superior to the wild strain in terms of erythritol yield from glucose, volumetric productivity, and glucose tolerance. Characteristics of erythritol high-producing mutant strain A microorganism capable of producing erythritol from selected glucose was identified as a species of Torula. The microbiological characteristics of the high erythritol-producing mutant strain are as follows.

[0015]   1) Descriptive features   Creamy colonies, growth and reproduction by germination, not filamentous fungi, not sexual reproduction.

[0016]   2) Fermentation     Table 1 shows sugars that can be used for fermentation of this mutant strain.

[Table 1]

[0017]   3) Growth   The following materials were tested for use in growth media. The results are shown in Table 2.

[Table 2]

4) Other characteristics The tests below show whether the mutant strains have the properties for a particular test. The results are shown in Table 3.

[0019]

[Table 3] According to the Budapest Treaty, this Torula genus DS101 mutant is located in Seoul, Republic of Korea 12
0-091, Sodamung-gu, Hongye-1-dong, Yurim B / D 361-221 (361-221 Yurim B / D
Hongje-1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea) September 1999 at Korean Culture Center of Microorganisms
It was deposited on March 7th under the deposit number KCCM-10171.

Using this strain, a culture test for erythritol production was performed. Glucose, which is the main compound for erythritol production, affects the production cost of erythritol by fermentation. To reduce production costs, glucose costs must be reduced. However, glucose cost reduction is extremely difficult because it leads to a reduction in erythritol production. If the by-product of the sugar production process is used as the main raw material for the production of erythritol, the production cost could be significantly reduced.

Therefore, in the present invention, a wild strain of the genus Torula isolated from the atmosphere in a 40% sucrose solution at Borak Co., Ltd., Osan, Gyeonggi-do, Republic of Korea was selected for erythritol production. This wild strain was mutated by N-methyl-N'-nitro-N-nitroguanidine (NTG) treatment. One of the mutants had characteristics superior to the wild type in terms of erythritol yield from glucose, volumetric productivity, and glucose tolerance.

Using this Torula mutant strain, erythritol was produced in a mother liquor composed of palatinose, trehalulose, sucrose, fructose, and glucose produced in the process of purifying palatinose.

The present invention relates to a method for obtaining erythritol from a Torula genus mutant strain in high yield and high volume productivity, using a mother liquor produced in a palatinose purification process. The following is the fermentation production method of erythritol using the mutant strain. Seed culture A frozen (-70 ° C) Torula genus mutant strain was added to 50 mL of a growth medium, 250
The seed culture was cultivated in a mL flask at 28 to 32 ° C. and 230 to 270 rpm for 43 to 53 hours, and this seed culture was transferred to a 5 L fermentor to produce erythritol in the main culture.

The main culture medium is composed of a mother liquor produced in the process of purifying palatinose as a carbon source, yeast extract of 1.8 to 2.2% as a nitrogen source, and KH ₂ PO ₄ . Mother liquor is trehalulose 30-60
%, Palatinose 10-30%, fructose 5-15%, glucose 5-25
% And sucrose 0-10%. 28-3 during the entire culture period in the fermenter
The cells were cultured at 8 ° C., pH 4.5 to 6.5, and aeration rate 0.1 to 1.0 vvm. Dissolved oxygen level 2
The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain above 20% for 4 hours. After 24 hours, the stirring speed was fixed at 600 rpm.

After 100 to 160 hours, the amounts of erythritol, fructose, glucose, palatinose and trehalulose were measured by HPLC (Shimadzu RID-6A, Japan) equipped with a column for carbohydrate analysis (manufactured by Waters, USA). The dry weight of the bacterium was determined using a calibration curve created from the relationship between the dry weight of the bacterium and the optical density of 600 nm. Finally, the fermentation medium was centrifuged to remove the strain and other residues, and the supernatant was filtered and dialyzed to obtain erythritol.

The invention is explained in more detail by the following examples. However, the following examples are for purposes of illustration and are not intended to limit the invention.

【Example】

Example 1 Frozen (−27 ° C.) Torula mutant strain was added to 250 mL of a growth medium of 250.
The seed culture was cultivated in a mL flask at 30 ° C. and 250 rpm for 48 hours, and this seed culture was transferred to a 5 L fermenter containing 3 L of medium for producing erythritol. This medium contains sucrose as a carbon source, yeast extract as a nitrogen source, and 0.3% K.
It consists of H ₂ PO ₄ . Culturing with a fermenter was carried out under the conditions of 34 ° C., pH 5.5, and aeration rate of 0.5 vvm during the culture. 20 dissolved oxygen levels for 24 hours
The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain above%. After 24 hours, the stirring speed was fixed at 600 rpm.

After culturing for 120 hours, the amount of erythritol from 30% sucrose and 1.5% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 132 g / L and the volumetric productivity was 1.07 g / L · h.

After culturing for 120 hours, the amount of erythritol from 40% sucrose and 2.0% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 190 g / L, and the volumetric productivity was 1.58 g / L · h.

After culturing for 140 hours, the amount of erythritol from 50% sucrose and 2.5% yeast extract was measured by tHPLC with a column for carbohydrate analysis attached. The obtained erythritol was 223 g / L, and the volumetric productivity was 1.55 g / L · h.

Example 2 A frozen (-70 ° C.) Torula mutant strain was used in 250 mL of a growth medium of 250 mL.
The seed culture was cultivated in a mL flask at 30 ° C. and 250 rpm for 48 hours, and this seed culture was transferred to a 5 L fermenter containing 3 L of medium for producing erythritol. This medium contained palatinose as a carbon source, yeast extract as a nitrogen source, and 0.
It consists of 3% KH ₂ PO ₄ . Culture in a fermenter was carried out at 34 ° C., pH 5.5, and aeration rate 0.5 vvm. The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain the dissolved oxygen level above 20% for 24 hours. After 24 hours, the stirring speed was fixed at 600 rpm.

After culturing for 120 hours, the amount of erythritol from 30% palatinose and 1.5% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 110 g / L, and the volumetric productivity was 0.92 g / L · h.

After culturing for 120 hours, the amount of erythritol from 40% palatinose and 2.0% yeast extract was measured by HPLC equipped with a carbohydrate analysis column. The obtained erythritol was 160 g / L, and the volumetric productivity was 1.33 g / L · h.

After culturing for 140 hours, the amount of erythritol from 50% palatinose and 2.5% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 180 g / L and the volumetric productivity was 1.29 g / L · h.

Example 3 A frozen (-70 ° C.) Torula mutant strain was mixed with 50 mL of growth medium 250.
The seed culture was cultivated in a mL flask at 30 ° C. and 250 rpm for 48 hours, and this seed culture was transferred to a 5 L fermenter containing 3 L of medium for producing erythritol. This medium contains trehalulose as a carbon source, yeast extract as a nitrogen source, and 0.
It consists of 3% KH ₂ PO ₄ . Fermenter culture is 34 ℃, pH 5.5, aeration rate
It was performed at 0.5 vvm. The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain the dissolved oxygen level above 20% for 24 hours. After 24 hours, the stirring speed was fixed at 600 rpm.

After culturing for 120 hours, the amount of erythritol from 30% trehalulose and 1.5% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 90 g / L, and the volumetric productivity was 0.75 g / L · h.

After 120 hours of culturing, the amount of erythritol from 40% trehalulose and 2.0% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 140 g / L and the volumetric productivity was 1.17 g / L · h.

After culturing for 140 hours, the amount of erythritol from 50% trehalulose and 2.5% yeast extract was measured by HPLC equipped with a column for carbohydrate analysis. The obtained erythritol was 170 g / L, and the volumetric productivity was 1.21 g / L · h.

Example 4 Frozen (-70 ° C.) Torula mutant strain 250 was added to 50 mL of growth medium.
After culturing in a mL flask at 30 ° C. and 250 rpm for 48 hours, this seed culture was transferred to a 5 L fermenter containing 3 L of medium to produce erythritol. This medium consists of a mother liquor produced in the palatinose purification process as a carbon source, 2.0% yeast extract as a nitrogen source, and 0.3% KH ₂ PO ₄ . This mother liquor contained 47.5% trehalulose, 20% palatinose, 17.5% fructose, and 15% glucose, and the concentration was adjusted to 40% total sugar. Fermenter culture is 34 ℃
, PH 5.5, aeration rate 0.5 vvm. 20 dissolved oxygen levels for 24 hours
The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain above%. After 24 hours, the stirring speed was fixed at 600 rpm. Table 4 shows fermentation profile of strain mass, erythritol production in medium containing mother liquor.

[0040]

[Table 4]

Comparative Example 1 A frozen (-70 ° C.) Torula genus mutant strain was added to 50 mL of a growth medium, 250.
The seed culture was cultivated in a mL flask at 30 ° C. and 250 rpm for 48 hours, and this seed culture was transferred to a 5 L fermenter containing 3 L of medium for producing erythritol. This medium consists of 40% glucose as a carbon source, 2.0% yeast extract as a nitrogen source, and 0.3% KH ₂ PO ₄ . Culture at fermenter at 34 ℃, pH 5
.5, aeration rate 0.5 vvm. The stirring speed was gradually increased from 300 to 1200 rpm in order to maintain the dissolved oxygen level above 20% for 24 hours. After 24 hours, the stirring speed was fixed at 600 rpm. Table 5 shows the fermentation profile of strain mass,
It showed erythritol production in a medium containing glucose.

[0042]

[Table 5]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (C12P 7/18 C12R 1: 645) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Oh Deokkun Republic of Korea, Cheongbuk 561-300, Cheongju, Deokjin-kuk, Songcheon-dong, Sanyoung Apartments 103-1403 (72) Inventor No Bonsu, South Korea, Seoul 139-220, Nowon-ku, Chung-gye-dong, Sioun Apartment 101-1106 (72) Inventor Jun Soo-Lung, South Korea, Seoul 138-220, Song Park, Jamsil-dong, 86, Asia Sansoh 1-901 (72) Inventor Kim Kyun Ah Korea, Seoul 158-075, Yangcheon Park, Shinjung-5-Dong, 934-16 F Term (Reference) 4B064 AC05 CA06 CC03 CD21 CD23 DA10 4B065 AA72X AC14 BA18 BB14 CA05 CA41

Claims (5)

[Claims] 1. A fermentation production method for obtaining a maximum amount of erythritol using a Torula genus DS101 mutant (deposited at Korea Microbial Culture Center under the deposit number KCCM-10171). 2. The fermentation production method according to claim 1, wherein the Torula genus DS101 species (KCCM-10171) mutant is cultured under the following conditions. i) In a medium consisting of a mother liquor produced in the process of purifying palatinose as a carbon source, yeast extract 1.8 to 2.2% as a nitrogen source, and KH ₂ PO ₄ 0.2 to 0.4% a) A mother liquor of 10 to 50% as a total sugar It contains sugar b) The pH of the medium is 4.5 to 6.5 c) The culture temperature is 28 to 38 ° C d) The aeration rate to the medium is 0.1 to 1.0 volume per minute for the medium volume e) The stirring rate of the medium is 300 to 1200 rpm The mutant strain is cultured under the conditions, ii) the mutant strain and other residues are removed from the fermentation medium, and iii) erythritol is separated and recovered from the fermentation medium of ii) above. 3. The composition of mother liquor is trehalulose 30 to 60%, palatinose 1
0-30%, fructose 5-15%, glucose 5-25% and sucrose 0-1
It is 0%, The fermentation manufacturing method of Claim 1. 4. A novel Torla DS101 species (KCCM10171) mutant strain. 5. Torla DS101 species (KCCM-10017) having the following steps:
Isolation method of 1). i) The wild strain of the genus Torula was treated with N-methyl-N'-nitro-N-nitroguanidine (
Growth medium containing 0.01% of NTG (glucose 18-22% and yeast extract 0.9
˜1.1%) and cultivated, ii) colonies formed in the growth medium are isolated at least three times, iii) the colonies of ii) are spread in the growth medium and cultured under UV irradiation of 250 to 270 nm, iv) Isolate grown colonies.