KR19990065347A - Mutant strain Candida Tropicalis SdID-101 and a method for preparing xylitol - Google Patents

Abstract

The present invention relates to a novel microorganism and a method for producing xylitol using the same, and to a method for producing a high yield of xylitol from xylose using a novel mutant strain, Candida Tropicalis SDB-101.

There are two methods for producing xylitol, which is used as a sweetener, but there are chemical methods and methods using microorganisms.However, chemical methods are difficult to purify, low yield, risk and waste problems. This inexpensive and easy separation and purification process, but still low yield, the present invention was invented to solve this conventional problem.

The present invention is to produce a mutant strain producing high yield of xylitol from xylose and to produce xylitol in high yield using the same. In the present invention, the mutant strain Candida Tropicalis SDB-101 is the first selection of a fast growth strain using a sample of sludge from the xylose company, and the medium consisting of xylose, yeast extract, potassium monophosphate After selecting the strain with high yield of xylitol at 2nd time and spreading it in YM medium added with NTG, the resulting colonies were separated again and repeated twice or more, and the resulting mutant strains were smeared on YM medium containing xylose and colonies. After isolation again, a strain with high yield and high heat resistance is selected. The mutant strain is cultured in a YM medium and then cultured in a fermentation medium having xylose to obtain a high yield of xylitol.

Description

Mutant strain Candida Tropicalis SDB-101 and a method for preparing xylitol

The present invention relates to a novel microorganism and a method for preparing xylitol using such a novel microorganism, and more particularly, a novel Candida tropicalis SDB-101 (mutation number KFCC-) 11005) and a method for obtaining xylitol from xylose using the same.

Xylitol is a sugar alcohol that has been used as a sweetener since 1960, first known by chemist Emil Fisher in 1891, and its sweetness is the same as sugar and has a great latent heat of dissolution. In addition to inhibiting the growth of (Streptococos mutans), it is known to have the effect of suppressing the development of tooth decay.In addition, it is used as a substitute sugar for diabetic patients due to the independence of insulin in the metabolic process of human body, especially gum and toothpaste. As such, active interest in xylitol has increased.

However, although xylitol is present in vegetables or fruits, it is not very economical to separate it naturally because it is a trace amount.

As a method for producing xylitol, chemical methods and methods using microorganisms are known.

As a chemical production method, a method of reducing hemicellulose hydrolysate containing a large amount of xylose is known, but after the reaction, separation and purification of xylitol and remaining hydrolyzate are difficult, yield is low, and high pressure. As a response, there are risks and waste issues.

The production of xylitol by microorganisms is carried out using yeast blankii, parapsilosis, tropicalalis, and pombe in Saccharomyces. In the case of bacteria, there are methods using Enterbacter liqufaciens, Corynebacterium and Mycobacterium smegmatis. Microbial methods are cheaper than chemical methods and can be selectively converted from xylose-containing hydrolyzate of plant raw material to xylitol, which facilitates the separation and purification of xylitol after reaction, but the yield is low. The disadvantages made it difficult to industrialize.

The present invention devised to solve the problems as described above is an object of obtaining a high yield and high productivity xylitol from xylose using the Candida tropicalis SDB-101 which is a mutation strain.

Another object of the present invention to provide a novel mutation strain Candida Tropicalis SDB-101.

In order to achieve the above object, the present invention induces cell growth using glucose and obtains xylitol from xylose using the same. The strain isolates the bacteria producing xylitol by decomposing xylose from the sludge of Xylose Chemical Co., Ltd., Changchang, China, and first selects and mutants with high yield and productivity from the wild strain. The variant strain is improved to 50% and 70% productivity to complete the present invention.

The method for obtaining Candida Tropicalis SDB-101, a novel strain of the present invention, will be described in detail by way of examples, but is not limited thereto.

Example 1

After a suitable dilution of the sludge sample of the xylose company, a high growth rate monarch was grown at 30 ° C. using a plate containing 10 g / l xylose, 5 g / l peptone, 3 g / l yeast extract, and 15 g / l agar. After primary selection, these selected strains were fermented in a medium consisting of 100 g / l xylose, 5 g / l yeast extract, and 5 g / l potassium monophosphate, and a second high strain of xylitol was selected after 30 hours of fermentation. After spreading wild strains selected secondly in YM medium (glucose 20g / l, peptone 5g / l, yeast extract 3g / l, malt extract 3g / l) with 0.05-0.1% NTG (Nitroso Methyl Guanidine) Colonies produced by incubation at < RTI ID = 0.0 > The mutant strain obtained by repeating this operation two or more times was irradiated with ultraviolet rays to smear YM medium containing xylose to separate the resulting colonies. From the isolated colonies, strains having a high yield and productivity and a sugar tolerance of 30% or more were finally selected.

The bacteriological properties of Candida Tropicalis SDB-101 selected in this way are as follows.

1. Growth of bacteria by temperature

20 ℃: + 30 ℃: + 37 ℃: + 42 ℃:-

2. Growth of bacteria

40% D-glucose: +, 50% D-glucose:-, devitaminized medium:-

3. Decomposition of elements:-

4. Starch Formation:-

5.Diazonium blue B formation reaction:-

6. Use of Nitrate:-

7. Filament:-

8. Use of Carbon Sources

Glucose: + Galactose: + L-Solvoze: + Sucrose: +

Maltose: + Lactose:-Trehalose: + Cellobiose: +

Melibiose:-Raffinose: + Merezitoz: + Soluble Starch: +

D-Xylose: + L-Aribinose: + D-Arabinose:-D-Ribose:-

D-Ramnose:-Glycerol: + Erythritol:-Ribitol: +

Galactitol: + D-mannitol: + D-sorbitol: + D-glutitol:-

Xylitol:-Inositol:-Salicyline:-Inulin:-

DL-lactic acid:-Succinic acid: + Citric acid: + Formic acid:-

Ethanol: + Methanol:-

Xylitol is produced using the novel Candida Tropicalis SDB-101 as follows.

Species cultures were inoculated in a 250 ml flask containing 50 ml of YM medium incubated for 10 hours at 220 rpm. 10% of these species cultures were fermentation broth (optimal medium, i.e., 100g / l of xylose was used as a carbon source, and 10g / l of yeast extract, 5g / l of potassium monophosphate, 0.2g / l of magnesium sulfate as a nitrogen source). After inoculation, incubate at 220 rpm using a shaker until the xylose is completely consumed. Fermenter culture is used a 5L fermenter (Korea Fermenter Co., Ltd.) having a medium volume of 3L containing 200g of xylose at the beginning of fermentation.

The concentration of xylose, xylitol, and cells were measured using a Refractive Index Detector of HPLC (WATERS, USA) equipped with Carbohydrate Aminex-87C column (Boi-Rad, USA). At this time, the solvent was water, the temperature is 85 ℃ and the flow rate is 0.6ml / min.

Cell concentration was converted to dry weight using a standard curve measured in advance by measuring the suspension at 600mm using a turbidimeter. Dissolved oxygen was measured using an Ingold (Swiss, polarographic type) dissolved oxygen electrode.

The present invention will be described in more detail with reference to the following examples.

Example 2

Candida Tropicalis SDB-101 (KFCC-11005), a novel mutant strain of the present invention, was inoculated into a 250 ml flask containing 50 m of YM medium and incubated at 220 rpm for 30 hours for 10 hours. This seed culture solution was then inoculated into a 250 ml flask containing 50 ml of fermentation medium (optimal medium) and then incubated with a shake incubator at 220 rpm at 30 ° C. until the xylose was completely consumed. The results of examining the production of xylitol over time are shown in Table 1.

(Generation of xylitol over time of the isolated strain when the initial xylose concentration is 100 g / l in a 250 ml flask) Incubation time (hours) Cell concentration (g / l) Xylose (g / l) Xylitol (g / l) 0 0.03 100 10 3.5 92.0 0 20 5.21 52.7 37 30 5.50 12.6 73 32 5.37 0.0 82

The yield of xylitol was high when the initial concentration of xylose was 50-150 g / l.

Example 3

The cultivation method is the same as in Example 2, and the result of examining the effect of the nitrogen source on the production of xylitol after 30 hours of fermentation in a fermentation medium consisting of 100 g / l of xylose, 5 g / l of nitrogen and 5 g / l of potassium phosphate Same as

Effect of Nitrogen Source on the Production of Xylitol in a 250ml Flask Nitrogen source Cell concentration (g / l) Xylitol (g / l) Yeast extract 5.10 72.8 Yeast nitrogen 6.90 62.4 Trypton 4.53 15.9 Soybean meal 6.51 9.4 Corn Soak 7.24 5.4 Malt Extract 4.85 5.4 peptone 3.31 6.8

Yeast extract is the best nitrogen source among the nitrogen source.

Example 4

The culture method and fermentation medium are the same as in Example 2, and the result of incubating for 30 hours by changing the concentration of the double yeast extract is shown in Table 3.

(Production amount of xylitol by yeast extract concentration in 250ml flask) Yeast Extract (g / l) Cell concentration (g / l) Xylitol (g / l) One 1.2 0.0 5 5.5 73.0 10 5.2 84.8 20 3.4 72.8

The yield of xylitol was high when the concentration of yeast extract was 5-25 g / l.

Example 5

The culture method and the culture medium are the same as in Example 2, and the results of incubation for 30 hours while changing the concentration of potassium monophosphate in the fermentation medium in this culture are shown in Table 4.

(Production amount of xylitol by potassium monophosphate concentration in 250ml flask) Potassium monophosphate (g / l) Cell concentration (g / l) Xylitol (g / l) 0 0.03 0.0 2 5.3 56.8 5 5.5 84.9 7 5.2 80.15

When the concentration of potassium monophosphate was 2-25 g / l, the yield of xylitol was high.

Example 6

The culture method and the culture medium were the same as in Example 2, and the results of incubation for 30 hours while changing the concentration of magnesium sulfate in the fermentation medium in this culture were shown in Table 5.

(Production amount of xylitol by magnesium sulfate concentration in 250ml flask Magnesium sulfate (g / l) Cell concentration (g / l) Xylitol (g / l) 0.0 5.4 84 0.1 5.38 85.5 0.2 5.7 87.0 1.04 6.0 83.7

When the concentration of magnesium sulfate was 0.1-5 g / l, the yield of xylitol was high.

Example 7

Species culture was the same as in Example 2, this culture was 3 l with an optimal fermentation medium (xylose 100 g / l, yeast extract 10 g / l, potassium monophosphate 5 g / l, magnesium sulfate 0.2 g / l, ammonium sulfate 5 g / l) Culture was carried out using a phosphorus 5 l fermentation tank. Several pH-specific experiments were performed at a culture temperature of 30 ° C. The result of incubation until the xylose was completely consumed by changing the pH is shown in Table 6.

(Production amount of xylitol by pH in 5l fermentation tank) pH Cell concentration (g / l) Xylitol (g / l) Incubation time (hours) 3.5 5.2 62.2 43 4.5 5.35 86.5 29 5.5 6.5 81.6 26 6.5 7.3 50.7 28

The yield of xylitol was high at pH 4.0-6.0.

Example 8

The culturing method and the fermentation medium were the same as in Example 7, and the experiments were carried out according to the culture temperature at the culture pH of 4.5 in this culture, and the culture results are shown in Table 7.

(Production amount of xylitol by temperature in 5l fermenter) Temperature Cell concentration (g / l) Xylitol concentration (g / l) Incubation time (hours) 27 7.4 85.1 33 30 5.8 86.2 30 33 5.4 76.7 36 36 4.5 57.9 43 39 1.5 3.50 82

The highest yield of xylitol was produced at 23-35 ℃.

Example 9

The fed-batch culture of xylose was carried out in a fermenter by increasing the concentration of xylose to 300 g / l in the selected optimum medium. Cultivation was carried out using a 5 l fermentation tank (sold by Korea Fermentor Co., Ltd.) having a medium volume of 21 containing 200 xylose at the beginning of fermentation. During the fermentation, a 250 ml solution containing 175 g of xylose was added intermittently four times (19.5 hours, 26 hours, 32 hours, 37.5 hours) and the final culture volume was 3 L. Dissolved oxygen was controlled to 300-600rpm to initially maintain 10% or more, and the dissolved oxygen was limited by changing the stirring rate to 150-400rpm when the cell concentration was about 10g / l. The production of xylitol over time is shown in Table 8. In this way, 252 g / l xylitol was obtained in 4 hours from 300 g / l xylose.

(Production amount of xylitol per hour from 300 g / l xylose in fermentation bath) Incubation time (hours) Cell concentration (g / l) Xylose (g / l) Xylitol (g / l) 0 0.1 100 6 0.56 99.5 12 14.0 70 20 21.9 2 58.2 20 17.8 79 52 25 21.8 2.1 119.5 25 18.5 73 106.3 32 22.6 2.8 175.0 32 19.8 67 159.6 36 21.8 5.7 211.6 36 18.6 63.9 195.5 41 20.5 0.0 252.0

By adjusting the dissolved oxygen as in the present embodiment, a high yield of 84% can be obtained, which is an improved yield of 34% compared to the yield of 50% or less when the dissolved oxygen is not controlled. Yields can be increased further.

Example 10

The culture method and the culture medium were the same as in Example 2, after inoculating the flasks with cells and incubated at 30 ° C. for 30 hours, and the pH of the medium was not adjusted after adjusting to 5.0 at the beginning of fermentation. At this time, the media components were glucose 0-30g / l, xylose 100 g / l, yeast extract 5g / l, ammonium sulfate 5g / l, potassium monophosphate 5g / l. Table 8 shows the results of xylitol production according to the concentration of glucose added to the fermentation medium.

(Production of Xylitol According to Glucose Concentration in 250ml Flask) Glucose (g / l) Cell concentration (g / l) Xylitol (g / l) 0 5.25 75.1 5 5.71 91.3 10 6.09 94.4 15 6.23 89.7 20 6.50 80.3 30 7.42 58.9

As a result of the culture, when 5-20 g / l of glucose was added, the yield was higher than that of the control group.

Example 11

Species culture was the same as in Example 2, 5L fermentation tank containing 3L of glucose medium (glucose 30g / l, yeast extract 10g / l, potassium monophosphate 5g / l, magnesium sulfate 0.2g / l) Incubated for 12 hours. At this time, the culture pH and temperature were 5.0 and 30 ℃, respectively. Cell concentration, xylose concentration and xylitol concentration over time are shown in Table 10.

(Production amount of xylitol using concentrated bacteria of Candida Tropicalis SDB-101 grown with glucose) Hour Cell concentration (g / l) Xylose concentration (g / l) Xylitol concentration (g / l) 0 20.6 100.0 0.0 3.5 21.3 75.6 13.5 5 21.5 44.3 49.5 6 21.7 22.0 68.0 7 21.1 0.0 88.2

Xylitol production yield by Candida Tropicalis SDB-101 was 88% and the average volumetric productivity was 14.5g-xylitol / Lh. These results were proportional to the cell concentration when the cells grown in glucose instead of xylose were concentrated. It indicates that the productivity of xylitol increases.

As described above, xylitol, which is used as a sweetener, is a compound that is difficult to produce naturally, but many conventional techniques for producing the same are known, but both chemical methods and methods using microorganisms have disadvantages. Therefore, according to the present invention, not only can xylose be used as a carbon source, but also to produce xylitol in high yield by producing Candida Tropicalis SDB-101, a mutant strain having a high yield of xylitol production. In addition, it has a low cost and a very easy purification process compared to the chemical method.

Claims (8)

Mutant strain Candida Tropicalis SDB-101 (KFCC-11005). A method of producing xylitol from xylose using the Candida Tropicalis SDB-101 of claim 1. The culture medium of Candida Tropicalis SDB-101 is a YM medium consisting of glucose, peptone, yeast extract and malt extract, and the fermentation medium is xylose, yeast extract as a nitrogen source, potassium monophosphate and sulfuric acid as inorganic salts. A method for producing xylitol from xylose, characterized in that magnesium is used. 4. The xylitol is produced from xylose according to claim 3, wherein the composition of the fermentation medium is xylose 5-15%, yeast extract 0.1-2.5%, potassium monophosphate 0.1-2.5% and magnesium sulfate 0.001-0.5%. How to. The method for producing xylitol from xylose according to claim 3, wherein xylitol productivity is increased by adding 5-20 g / l of glucose to the xylose medium. 4. The method of claim 3, wherein the xylose medium has a culture pH of 4.0-6.0 and a culture temperature of 25-35 ° C. 4. The method for producing xylitol from xylose according to claim 3, wherein the productivity of xylitol is increased by intermittently adding xylose to the xylose medium. 4. The method for producing xylitol from xylose according to claim 3, wherein the cell growth stage at the beginning of fermentation maintains a high dissolved oxygen concentration and the xylitol producer increases the productivity of xylitol by limiting the dissolved oxygen concentration.