KR100643793B1 - 200 A novel flocculating yeast strain Candida tropicalis HY200 its cultivation and production of xylitol using repeated-batch fermentation - Google Patents

Abstract

A novel flocculating yeast strain, Candida tropicalis HY200, its cultivation method and a production of xylitol by repeated-batch fermentation of the same strain are provided to cheaply produce xylitol by precipitating the flocculating yeast strain without a further process. The novel flocculating yeast strain, Candida tropicalis HY200(KCTC 10813BP) is precipitated after finishing the stirring of fermentation medium. The xylitol is produced by (i) culturing Candida tropicalis HY200(KCTC 10813BP), (ii) inoculating the cultured medium into the fermentation medium containing xylose, and culturing it by regulating the air supply speed, stirring speed and temperature, and (iii) purifying xylitol from the cultured fermentation medium, wherein the cultivation method of step(ii) is repeated-batch fermentation; the air supply speed is 0.5-1.5 vvm, stirring speed is 250-500 rpm, and fermentation temperature is 28-31 deg.C; and the initial xylose concentration is 100-300 g/l.

Description

Novel flocculating yeast strain, Candida tropicalis HY200, its cultivation and production of xylitol using repeated-batch fermentation}

1 is a photograph showing a phenomenon that the HY200 strain of the present invention is precipitated with only the cohesiveness of the strain itself after 15 minutes when the mixture is stirred with a medium in a 2.5l fermenter,

(A) 0 minutes after stopping stirring (B) 15 minutes

2Is a photograph showing the results of comparing the flocculation of the HY200 strain of the present invention (right) with another Candida tropicalis (ATCC 20336, left),

(A) 0 minutes after stopping stirring (B) 1 minute (C) 2 minutes (D) 3 minutes later

FIG. 3 is a profile showing the results of batch fermentation for xylitol production in a medium comprising 200 g / l xylose, 10 g / l yeast extract and 7 g / l tryptone.

▼: dry cell weight ●: xylose ○: xylitol

Figure 4 is a profile showing the results of repeated batch culture at 420 rpm, 0.5 vvm conditions at 30 ℃.

DCW: ▼ Xylose: ● Xylitol: ○ pH: △ Cohesive (%):. ■

The present invention relates to a novel Candida genus strain HY200 and a method for producing xylitol using the same, and more specifically, Candida Tropicalis HY200 having resistance to high concentrations of xylose and having cohesive D-xylose using the same It relates to a method for producing xylitol from.

Xylitol is a pentose alcohol found in various fruits and some vegetables (Washuttle. J. et al., J. Food Sci. 38: 1262, 1973) because it has high sugar content and does not require insulin to break down. It has been in the spotlight as a sugar substitute for treating diabetes and glucose-6-phosphate dehydrogenase-deficient diseases (Maekinen. KK Adv. Food Res. 25: 137-158, 1979; Ylikahri, R. Adv. Food Res. 25: 159 -180, 1979). In addition, it is used as a carbon source of the dental caries Streptococcus mutans and Streptococcus sobrinus , and when it is metabolized, it does not generate an acid causing tooth decay and thus prevents secondary caries (anticariogenic) of teeth (Maekinen.KK Adv. Food Res. 25: 137-158, 1979) Prolongs shelf life of food by inhibiting saprophytes such as Clostiridium butyrican and Salmonella typhii Emodi, A. Food Thechol. 32: 20-32, 1978). It is therefore added to various products such as chewing gum, soft drinks, ice cream and toothpaste.

Xylitol is generally produced by chemical reduction of xylose, mainly derived from hemicellulose hydrolysate of xylose-rich substances such as birch. Although xylitol can be obtained from the hemicellulose portion of these natural substances, various sugars and their polymers are included together, so that only xylitol can be obtained through several costly separation steps (Hyvoenen L., Adv. Food Res. 28: 373-403, 1983). On the other hand, the method of producing xylitol as a microorganism is a method that is attracting much attention because the process is simple and a large amount of xylitol can be obtained.

Several microorganisms have been screened for xylitol-producing strains and most of them are C. boidinii , C. gulliermondii , C. parapsilosis , C. pelliculosa , C. shehatae C. and contained in tropicalis) or related species Debaryomyces genus Candida Hansenii and Pichia stipitis (Barbosa, MF S et al., J. Ind. Microbiol. 3; 241-251, 1988; Du Preez, JC et al., Enzyme Microb. Technol. 32: 20-32, 1978; Jeffries, TW Biotechnol. Bioeng. 24: 371-384, 1981; Kim, SY et al., J. Ferment. Bioeng. 83: 267-270, 1997; Kitpreechsvanich, V. et al., Biotechnol. 6: 651-656, 1984; Ligthelm, ME, et al., Appl. Microbiol. Biotechnol. 28: 63-68, 1988; Ligthelm, ME, et al., Appl. Microbiol. Biotechnol. 28: 193-296, 1988; Vongsuvalert, V. and Y. Tani.J Ferment.Bioeng. 67: 35-39, 1989). Xylitol is a major xylose reduction product of the yeast, but the factors regulating production and emission are not yet clear. Among them, Candida Tropicalis, Pichia stipitis and recombinant Saccharomyces cerevisiae have been mainly used in batch or fed-batch cultures for xylitol production (Kim, MS et al., J. Microbiol. Biotechol. 11: 564-569, 2001; Lee, WJ et al., J. Microbiol. Biotechnol. 13: 725-730, 2003; Vandeska, ES et al., J. Microbiol 11: 213-218, 1995) A high yield of about 80% of xylitol was obtained using this strain.

An important step in the batch culture process is the time it takes to wash, sterilize the fermentor and then regrow the cells. Fed-batch cultures inevitably result in cell loss during the process and lead to variations in readjustment time during operation. In order to solve this problem and improve the xylitol yield, alternative strategies for using cohesive cells or freezing the cells, which are easy to separate the cells from the culture medium, are considered as alternatives, which also easily separate the cells from the medium and contaminate the cells. And risk of loss and loss (Jang, SH et al., J. Microbiol. Biotechnol. 13: 501-508, 2003; Roca, E. et al., Biotechnol. Bioeng. 51: 317-326, 1996; Yahashi, Y. et al., Biotechnol. Lett. 18: 1395-1400, 1996).

Republic of Korea application 10-1997-0010244, 10-1997-009717, 10-1998-0020034 describes a method for producing xylitol by the new strain Candida tropicalis, but the strain itself is different from the present invention and the batch or Problems encountered during the fed-batch cultivation process could not be solved.

Thus, the present inventors have attempted to isolate novel strains that are cohesive and resistant to high xylose concentrations that can result in increased xylitol production and simplified processes in repeated batch cultures. The HY200 strain of Candida genus of the present invention isolated and identified in soil has been found to have the above characteristics, and it is easy to maintain and reuse the cells with repeated batch manipulation with high cell mass, and to have high xylitol yield and Confirmed that the production is possible to complete the present invention.

It is an object of the present invention to provide a novel Candida genus strain.

The present invention also aims to provide a method for producing xylitol from D-xylose using the strain.

In order to achieve the above object, the present invention provides a novel Candida Tropicalis HY200.

The present invention also provides a method for producing xylitol using the strain.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Candida Tropicalis HY200.

The present inventors obtained various soil samples from the rice paddy, and then, the cells attached thereto were collected and cultured in a enrichment medium containing a high concentration of xylose. These were plated on agar medium containing xylose and cultured to select colonies showing marked cell growth and tested for cohesiveness in xylitol liquid medium. The cohesive culture medium was diluted properly and plated on a solid medium, and five strains that grew rapidly to form large colonies were selected. Selected strains were cultured in 100 g / l xylose liquid medium to compare xylitol-producing ability and aggregation. Among them, HY200, one species with the highest yield, was finally selected, and the maximum cell concentration was 10.8 g when cultured in enrichment medium supplemented with 100 g / l xylose, 100 g / l yeast extract and 10 g / l bactopeptone. / l and xylitol concentration was 73.3 g / l. The selected strains were cultured in low concentration xylose (100g / l) because they had relatively high cell growth and low lag time and were systematically identified according to the genotype and phenotype characteristics for species classification.

The strain was an aerobic microorganism with no mobility, creamy color, and a slight mycelial border, showing catalase and oxidant positive reactions. Experiments using Biolog Microlog ^™ (Biolog) confirmed that HY200 uses glucose, fructose, maltose mannitol, xylose and inositol raffinose as carbon sources and the optimum growth temperature range is 30 to 34 ° C.

In addition to the above physiological classification methods, 18S ribosomal RNA (rRNA) sequence information, which is a powerful means of measuring the lineage relationship, was analyzed, and the 18S rRNA sequence of HY200 was highly homologous to typical Candida strains, especially Candida tropicalis . 99% or more).

Therefore, from the homology level and physiological classification of 18S rRNA, it was confirmed that HY200 was Candida tropicalis species and the strain was named Candida tropicallis HY200. Candida Tropicalis HY200 cell line of the present invention was deposited on June 2, 2005 at the Korea Research Institute of Bioscience and Biotechnology Center and accession number is KCTC 10813BP.

Various efforts have been made to increase the production of xylitol, which aims to prevent the use of xylitol as an energy source for xylitol metabolism and cell growth, and to increase the initial xylose concentration to enable much xylitol production. Since xylitol, as well as xylose, is known to be used as a carbon source for xylitol production by Candida species, it is necessary to reduce the reduction of xylitol by preventing metabolism of xylitol by the dehydrogenase. In addition, it has been believed that the amount of xylitol obtainable will increase if the alternative carbon sources used for cell growth are identified to reduce the amount of xylitol used for cell growth.

The inventors first tested the activity of assimilation on 18 carbon sources to find a carbon source that HY200 can metabolize in addition to xylose. As a result, HY200 metabolized fructose, glucose, maltose and mannitol well even at relatively high cell concentrations, and inositol and raffinose rhamnose could be used as carbon sources, but xylitol was not used as a carbon source. As a result, HY200 could use other carbon sources in addition to xylose but could not metabolize xylitol, thus confirming that HY200 is an excellent candidate to produce xylitol without loss from xylose.

As such, HY200 consumes xylose faster, is resistant to high concentrations of xylose, and is cohesive without using xylitol as a carbon source, so it easily sinks in culture, so that cells and aqueous products can be separated without additional processes such as centrifugation. Since it has a large difference from other Candida strains (see Figs. 1 and 2).

In addition, the present invention

1) species culturing the HY200 strain;

2) inoculating the culture solution of step 1 into the fermentation medium and culturing by adjusting the air supply amount, stirring speed and temperature; And

3) provides a xylitol production method comprising the step of purifying xylitol from the culture medium of step 2.

The present inventors have studied a method for optimizing the medium conditions to effectively produce xylitol from the HY200 of the present invention.

First, in order to select a nitrogen source suitable for xylitol production at the highest yield, tests with liquid starch waste, yeast extract and soytone confirmed that HY200 exhibited high productivity and cohesiveness in yeast extract, tryptone or mixtures thereof. It was.

Increasing the concentration of xylose increases the yield of xylitol, but decreases the ethanol yield. Nolleau et al. Reported that initial growth of xylose inhibited cell growth by substrate inhibition and / or osmotic pressure, while Sirisansanneeyakul et al. Increasing xylose concentrations at certain growth rates reported increased xylose uptake and xylitol production. Therefore, the initial xylose concentration suitable for HY200 culture for cell growth and maximum xylitol yield was determined. To this end, after observing the results after adding the cells in fermenters with different initial xylose concentrations, when the xylose concentration was less than 200 g / l, the cell concentration and productivity increased as the xylose concentration increased, while the xylitol yield was increased. There was only a slight difference. When the xylose concentration was higher than 200 g / l (200 to 300 g / l), the incubation time increased drastically and the cell growth and productivity decreased (Table 2). This result is consistent with Gong et al. And Ikeuchi's report that initial xylose concentrations yield the highest yields at 200 g / l.

While there is an opinion that excessive aeration in cell proliferation, xylose consumption, and xylitol production reduces cell volume or xylitol production, research suggests that lower oxygen levels limit cell growth and reduce xylitol production. Furlan, SA et al., Process Biochem. 29: 657-662, 1994; Furlan, SA et al., Biotechnol. Lett. 13: 203-206, 1991; Roseiro, J. c. ET AL., 156: 484 -490, 1991; Walther.T. Et al. Biores. Technol. 76: 213-220, 2001). Therefore, the present inventors observed a change in xylitol production when various air amounts were added to find an optimum air amount. As a result, the cell concentration reached maximum at aeration rate of 1.5 vvm, but the yield of xylose to xylitol was relatively lower than other conditions. At 0.5 vvm aeration, xylitol productivity and yield were high, but the incubation time was very long. Based on the above results, the optimum aeration rate was determined to be about 1.0 vvm (Table 3).

On the other hand, the final cell volume and xylitol productivity increased in proportion to the increase in the stirring speed, but the incubation time and the xylitol yield decreased as the stirring speed increased (Table 4), and the stirring speed of the maximum xylitol production was 330 rpm. The maximum xylitol yield and productivity were maintained when the incubation temperature was maintained at 30 ° C. The cell growth was abruptly decreased below 27 ° C. The cell growth rate was lowered and the xylitol productivity was decreased below 32 ° C. Initial pH had little effect on xylitol production, but cell growth was highest at pH 4.0.

Candida tropicalis HY200, the isolated strain, was seeded in 100 ml xylose medium (300 g / l xylose, 10 g / l yeast extract, 10 g / l bactopeptone) and incubated for 24 hours at 30 ° C. at a stirring speed of 200 rpm. The seed culture was incubated by mixing the fermentation medium with 1-l working volume. As described above, the stirring speed was 330 rpm, the aeration amount was 1.0 vvm, the initial pH was 4.0, the incubation temperature was 30 ° C., the initial xylose concentration was 200 g / l, and the HY200 strain was cultured after setting the optimum medium and culture conditions. After incubation, the cells were checked by spectroscopy after centrifugation and the culture medium was analyzed by high performance liquid chromatograpy (Waters). As a result, as shown in FIG. 3, the xylitol production of the HY200 increased linearly, the xylose consumption rate decreased linearly, and when the initial xylose concentration was supplied at 200 g / l, the xylitol yield was 77% and the productivity was 2.57 g. It was confirmed that /l.h was the highest.

Xylitol produced by the above method can be obtained by separating from the medium in a conventional manner. For example, the remaining solution after removing the medium by a method such as centrifugation may be crystallized after desalting by activated carbon, ion-exchange resin or the like.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only for illustrating the present invention, but the present invention is not limited thereto.

< Example  1> Enzyme and Chemical Preparation

D-xylose and xylitol were purchased from Sigma. Restriction enzymes, Taq DNA polymerase, pGEM-T vectors and genomic DNA isolation kits were purchased from Promega and all other reagents and chemicals were used for analytical grade.

< Example  2> Enrichment Culture  And screening

After obtaining various soil samples from rice paddies located in Hwaseong, Suwon, Korea, they were suspended overnight in saline buffer or enrichment medium to remove most adherent cells. Suspension cells were recovered by centrifugation or seeded in enrichment medium (pH 6.0) containing 300 g / l xylose, 10 g / l yeast extract and 10 g / l bacto peptone (Difco). Ten samples showed marked cell growth in enriched medium supplemented with 300 g / l xylose and these species were inoculated with the same medium with 100 g / l xylose and analyzed for flocculation.

As the flocculation phenomenon was observed in some flasks, the culture medium was properly diluted and then plated on the same solid medium. As a result, about 38 colonies were preferentially screened and they were plated on solid medium of high xylose concentration (300 g / l), and then five strains were selected that grew rapidly to form large colonies. Selected strains were cultured in 100 g / l xylose liquid medium to compare the potential xylitol-producing ability and aggregation.

The cultured cells were collected by centrifugation (4000 rpm, 5 minutes), and then confirmed by spectroscopy (600 nm, UV-1201 Shimazu), and the culture medium was analyzed by high performance liquid chromatography (Waters). A carbohydrate analysis column (Waters) was used for the analysis of xylose and xylitol, which was equilibrated with a solution of acetonitrile and distilled water at 80:20, and then operated at a constant flow rate of 2 ml / min and the mobile phase solvent (eluent) Recording was done using RI (Refractive Index) detector (Waters).

As a result, the maximum cell volume and xylitol concentration were 10.8 g / l when cultured in a enrichment medium containing 100 g / l xylose, 10 g / l yeast extract and 10 g / l bacto peptone for 24 hours at 30 ° C. And HY200, which was the highest at 73.3 g / l, was finally selected.

< Example  3> species identification

As in < Example 2> , HY200 isolated from various soils through a continuous screening step was systematically identified according to genotype and phenotype characteristics for species classification. This strain is an aerobic microorganism with no mobility, creamy color, and a slight mycelial border, which has catalase and oxidant positive reactions. Experiments using Biolog Microlog ^TM (Biolog) showed that HY200 used glucose, fructose, maltose, mannitol, xylose and inositol raffinose as carbon sources, and the optimum growth temperature ranged from 30 to 34 ℃ and did not grow at 40 ℃.

In addition to the above physiological classification, ribosomal RNA gene sequence provides a powerful means of measuring the lineage relationship, so 18S ribosomal RNA (rRNA) sequence information was analyzed to identify the species of HY200. Each 18S rRNA gene (1,785 bp) was amplified using a common primer set described in SEQ ID NOs: 1 and 2, respectively. PCR was performed by a thermal cycler (Techne) and the reaction conditions are as follows. After an initial denaturation step at 96 degrees for 3 minutes, denaturing at 96 degrees for 30 seconds, annealing at 56 degrees for 40 seconds and extending at 72 degrees for 1 minute, followed by 35 cycles for 3 minutes at 72 degrees The last extension step was performed. Amplified DNA fragments were loaded into 0.8% agarose gel, eluted, purified, subcloned into pGEM-T vector and sequenced. As a result, it was confirmed that the 18S rRNA sequence of HY200 shows very high homology (more than 99%) with a typical Candida strain, particularly Candida tropicalis . The homology level of the 18S rRNA, along with the physiological classification results, indicates that HY200 is a Candida tropical species, so the strain was named Candida tropicalis HY200.

< Example  4> Cohesive  Test

The HY200 was stirred in a 2.5 L fermenter containing 1-1 working volume, and the cohesion of HY200 was confirmed immediately after stopping the stirring and 15 minutes after the stirring was stopped. As a result, immediately after the stirring was stopped, HY200 cells began to aggregate as shown in FIG. 1A, and after 15 minutes, most of the HY200 aggregated and settled as shown in FIG.

In addition, the HY200 strain of the present invention exhibits a distinct cohesiveness, C. which is a Candida genus strain generally used for xylitol production. tropicalis It settled faster upon stirring compared to ATCC20336 (left of FIG. 2) (right of FIG. 2).

The strain screened as above was identified as Candida tropical species but HY200 consumes xylose faster than other Candida tropical species ATCC 20336 and is resistant to high xylose concentrations of 300 g / l or more, so that HY200 cells grow at this concentration. Is not severely inhibited and has a merit in that aggregation occurs easily during cultivation, so that it is easily distinguished from tropicalis (ATCC 20336) which is generally used for industrial use (FIG. 2).

< Example  5> Medium and culture condition optimization

The isolated strain Candida Tropicalis HY200 was transferred to a medium containing 100 g / l xylose 10 g / l yeast extract, 10 g / l bacto peptone and 20 g / l agar periodically at 30 ° C. when grown to a certain degree. It was seeded at 200 rpm at 30 ° C. in a 500 ml Erlenmeyer flask containing 100 ml of xylose medium containing 300 g / l xylose, 10 g / l bacto peptone and 10 g / l yeast extract.

The cultured medium was mixed with fermentation broths containing different carbon and nitrogen sources as in the following <5-1> to <5-6> , or cultured under various aeration, stirring speed, or temperature conditions. The cultured cells were collected by centrifugation (4000 rpm, 5 minutes), and then confirmed by spectroscopy (600 nm, UV-1201 Shimazu), and the culture medium was analyzed by high performance liquid chromatography (Waters). A carbohydrate analysis column (Waters) was used for the analysis of xylose and xylitol, which was equilibrated with a solution of acetonitrile and distilled water at 80:20, and then operated at a constant flow rate of 2 ml / min and the mobile phase solvent (eluent) Recording was done using RI (Refractive Index) detector (Waters).

The results were analyzed to establish optimal media and culture conditions with high xylitol yield and productivity.

<5-1> Carbon source

The inventors tested the activity of assimilation on 18 carbon sources to find a carbon source that HY200 can metabolize in addition to xylose. As a result, HY200 metabolized well fructose, glucose, maltose and mannitol even at relatively high cell concentrations. Cells did not grow when gluconic acid and lactose were carbon sources, and in contrast to previous studies on Candida Tropicalis (Gong, CS et al., Biotech. Lett. 3: 130-135, 1981; Horitsu, et al. , Biotechnol.Bioeng. 40: 1085-1091, 1992; Lu, Jean et al., Biotechnol.Lett. 17: 167-170, 1995; Oh, DK and SY Kim Appl.Microbiol.Biotechnol. 50: 419-425, 1998; Walther, T. Biores. Technol. 76: 213-220, 2001) HY 200 was able to utilize inositol, raffinose and rhamnose as carbon sources. Notably, HY200 did not use xylitol as its sole carbon source, which strongly supports HY200 as a good candidate to produce xylitol from xylose.

<5-2> nitrogen source

The present inventors studied a method for optimizing the medium conditions in consideration of high xylose concentration and cell volume in order to effectively produce xylitol from the HY200 of the present invention. First, in order to select a nitrogen source suitable for xylitol production at the highest yield, , Several representative nitrogen sources were tested (Table 1). Testing with commercially available liquid starch waste, yeast extract and soytone showed that liquid starch waste had the highest cell concentration (11.5 g / l) and yeast extract had the highest xylitol yield. Soyton had low cell concentration but no flocculation. Tryptone had a cell concentration twice as high as peptone and showed a pronounced aggregation reaction. These results indicate that HY200 shows high productivity and aggregation in yeast extract or tryptone. The use of yeast extract (10 g / l) and tryptone (7 g / l) also showed high productivity and agglutination, and the inorganic nitrogen source was inadequate for cell growth and xylitol production, which is consistent with the results of the study by Sirisansanneeyakul. Sirisansanneeyakul, S. et al., J. Ferment.Bioeng. 80: 565-570, 1995).

<5-3> first Xylose  density

In order to examine the effect of the initial concentration of xylose on cell growth, the present inventors found fermentation broth with 10 g / l yeast extract, 7 g / l tryptone and different initial xylose concentrations of 100 to 300 g / l in 2.5 l fermenter. And HY200 strain culture medium was mixed with 1-1 working volume (working volume) and observed the growth of cells. As a result, when the initial xylose concentration was 200 g / l or less, productivity increased as the xylose concentration increased. When the xylose concentration was higher than 200 g / l (200 to 300 g / l), the incubation time was greatly increased and the cell growth. And productivity fell. Incubation time represents the time for complete consumption of xylose in batch culture (Table 2).

<5-4> aeration

Some researchers report that excessive aeration in cell proliferation, xylose consumption, and xylitol production increases cell volume but reduces xylitol production, and some researchers report that lower oxygen levels limit cell growth and decrease xylitol production (Furlan). , SA et al. Process Biochem, 29: 657-662, 1994; Furlan, SA et al., Biotechnol. Lett. 13: 203-206, 1991; Roseiro, JC et al., Arch.Microbiol., 156: 484 -490, 1991; Walther, T. et al., Biores. Technol. 76: 213-220, 2001).

In order to optimize the aeration conditions, the present inventors 1-1 work volume of a fermentation medium containing 100 g / l xylose, 10 g / l yeast extract, 7 g / l tryptone and a seed culture medium of HY200 strain in a 2.5 l fermenter. volume), and then cultured the HY200 strain under various aeration conditions of 0.5-1.5 vvm at a stirring speed of 300rpm, and analyzed the xylitol production.

As a result, the cell concentration reached a maximum of about 14.8 g / l at an aeration rate of 1.5 vvm, but the yield of xylose to xylitol was relatively lower than other conditions. Xylitol productivity and yield at 0.5vvm aeration rate were 1.15 g / l · h and 0.72 g xylitol per 1 g xylose with high productivity and yield but very long incubation time. Based on the above results, the optimum aeration rate was determined to be about 1.0 vvm (Table 3).

<5-5> Stirring  speed

In order to optimize the stirring speed, the present inventors 1-1 work volume of a fermentation medium containing 100 g / l xylose, 10 g / l yeast extract, 7 g / l tryptone and a culture medium of HY200 strain in a 2.5 l fermenter. volume), and then cultured the HY200 strain under various agitation conditions with an aeration of 1.0 vvm, and analyzed the xylitol production.

As a result, the final cell mass and xylitol productivity increased in proportion to the increase in the stirring speed, but the incubation time and the xylitol yield were observed to decrease as the stirring speed increased (Table 4). This is due to oxygen-mediated NADH consumption, which is thought to be because NADH consumption lowers the level of NADH / NAD + and promotes the further metabolism of xylitol to xylulose.

The agitation rate of maximal xylitol production was 330 rpm and the aggregate phenotype was well observed under these conditions and the grown cells easily settled.

<5-6> incubation temperature and pH

In order to optimize the culture temperature, the present inventors 1-1 work volume of a seed culture medium of HY200 strain and a fermentation medium containing 100 g / l xylose, 10 g / l yeast extract, 7 g / l tryptone in a 2.5 l fermenter. volume), and then cultured the HY200 strain at various temperature conditions with aeration rate of 1.0 vvm and agitation speed of 330rpm, xylitol production was analyzed.

As a result, the maximum xylitol yield and productivity were 1.72g / lxh and 0.72g xylitol / g xylose, respectively, when the incubation temperature was maintained at 30 ° C. Has brought a change in appearance. On the other hand, when the incubation temperature was higher than 32 ° C., the cell growth rate was lowered and the xylitol productivity was also decreased.

On the other hand, the same experiment was conducted on the initial pH, but the initial pH had little effect on xylitol production, but the highest cell growth was when the pH was 4.0.

< Example  6> under optimized conditions HY200 On by Xylitol  Production analysis

Yeast culture for the production of xylitol is a fermentation medium containing 10g / l yeast extract, 7g / l tryptone in 2.5l fermenter and HY200 strain seed culture medium mixed in 1-1 working volume, the stirring speed Is 330rpm, aeration amount is I.0 vvm, the initial pH is 4.0 culture temperature 30 ℃, the initial xylose concentration of 200g / l was optimized according to the results of < Example 5> .

As a result, under optimized conditions, the xylitol production and xylose consumption rates of HY200 increased and decreased linearly as shown in FIG. 3, which means that the higher the xylitol concentration, the higher the obtained xylitol concentration. Indeed, when the initial xylose concentration was 200 g / l, it was confirmed that the xylitol yield (0.77 g xylitol / g xylose) and productivity (2.57 g / l · h) were considerably high.

< Example  7> HY200 Repetitive Batch  By fermentation process Xylitol  production

Repetitive batch fermentation is a very efficient method because it can reduce costs in terms of the amount of medium added and the cost of operating the device, rather than several batch fermentation processes individually if only the strain can be sustained without loss. However, general strains are not suitable for repetitive batch fermentation process because the loss of strain occurs due to the delayed time through the process of centrifugation or medium exchange, etc. . On the other hand, the HY200 strain of the present invention is cohesive and does not require a separate process such as centrifugation, so it is considered to be very suitable because it can be quickly converted from one batch to the next batch during the repeated batch fermentation process.

Thus, the present inventors found the optimum conditions that can actually improve the productivity when the batch batch fermentation process using the cohesiveness of the HY200 strain within the range suitably set for the batch fermentation process in the above embodiment. . Initial xylose yields the highest yield and productivity at 200 g / l, but maximizes the effect on the cohesiveness of this strain, which is characteristic of the repeated batch fermentation process, and proceeds to the optimal level of 100 g / l considering the xylitol production xylose unit price. .

The present inventors cultured the HY200 strain by a repeated batch fermentation process, which is actually used when producing xylitol, under conditions of agitation speed 360 rpm, aeration rate 1 vvm, temperature 30 ° C., and initial xylitol concentration of 100 g / l.

The suspension of the frozen HY200 strain was first placed in a 250 ml Erlenmeyer flask containing 50 ml of growth medium consisting of 5 g / l yeast extract, 3 g / l malt extract, 3 g / l peptone and 20 g / l glucose. Preculture was carried out for 9 hours at 30 ℃, 200 rpm. This was transferred to a 2.5l fermenter containing 1-1 working medium consisting of 100g / l xylose, 10g / l yeast extract, and 10g / l bacto-peptone, stirring speed 360 rpm, aeration rate 1 vvm, temperature 30 ° C. And cultured under conditions of an initial xylitol concentration of 100 g / l. Antifoaming agent (antifoam 289, Sigma) was added to prevent foaming, and after confirming that the amount of xylose was 5g / l or less at the end of each batch fermentation process, the agitation was stopped, and the strain was allowed to settle.

When sedimented naturally by cohesion, 0.8 l of the supernatant was pumped out of the cells so as not to mix the cells, leaving only 0.2 l and adding 0.8 l of fresh medium thereto to resuspend the aggregated cells. Sedimentation occurred by cohesion for 20 minutes, draining the supernatant for 10 minutes and filling fresh medium. The transition from one batch to the next was completed in 30 minutes. The same procedure was repeated five times.

As a result, xylitol yield was 0.59 g / g and productivity was 2.18 g / l · h in the first batch fermentation, while xylitol yield was 0.64 g / g and productivity was 4.01 g / l · h and cell mass in the final batch fermentation. The cell mass also increased to 21.28 g / l (Table 5).

 In addition, the present inventors have to reduce the operating (fermentation) time to increase the productivity while maintaining the existing yield, thereby increasing the stirring speed and reducing the aeration amount to optimize the optimum conditions of xylitol production for the strain of the present invention 420 rpm, 0.5 Determined by vvm, the batch batch fermentation process, that is, the batch fermentation was carried out six times under conditions of 100 g / l.

As a result, the xylitol yield was 0.64 g / g and 3.02 g / l · h in the first batch fermentation, while it increased to 0.67 g / g and 6.28 g / l · h in the last batch fermentation. As a result of the six cultures, the average yield and productivity were 0.66 g / g, 4.6 g / l · h, and the cell mass was 26.32 g / l, which was very high (Table 5 and FIG. 4).

In conclusion, in the batch batch fermentation process, it was confirmed that the culture condition of 420 rpm, 0.5 vvm is an optimized condition to maintain the existing yield and secure the highest productivity.

< Example  8> HY200 Produced by Xylitol  refine

Xylitol produced by the above method was purified by the following method.

<8-1> removing impurities and Color  remove

   After the completion of the culture, the cells were removed by centrifugation at 4,500 rpm for 10 minutes, and only the supernatant was taken out to remove impurities by membrane filtration. After passing through activated carbon (100 g, 50 mesh) to remove organic impurities (proteins, lipids, dyes) and filtered again using a glass filter paper. In order to investigate the extent of deodorization and decolorization by activated carbon, it was measured using spectrophotometer and conductivity (P. V. Gurgel. Et al., Bioresource Technology, 52: 219-223, 1995).

<8-2> ion exchange resin

   The amount of ion exchange resin was determined for the experiment using the ion exchange resin. The amount of medium used in the experiment was 1 l and the amount of ion exchange resin was set to 20 ml or more in order to maintain the reliability of the experiment. In addition, the cationic resin and the anionic resin were filled in each column and passed sequentially (Jose M. et al., Bioresouce Technology 61: 85-90, 1997).

   Experiment apparatus using ion exchange resin was made to order. Since no feeding pump was used, the cleaning liquid was positioned about 20 cm above the column. The flow rate was converted by measuring the liquid passing time and the liquid flow rate after opening the lower cork.

   In order to remove the ions contained in the filtrate, which was colored and deodorized through the activated carbon, not only the anion exchange resin but also the cation exchange resin were used to remove the ions. In the experiment, C100H (Samyang Diaion.Co.), A strong cation exchange resin, WA30 (Samyang Diaion.Co.), A strong anion exchange resin, and PA412 (Samyang Diaion.Co.), A weak anion exchange resin, were used. After pretreatment of the ion resin, the column was filled with ion resin, and the cation resin was washed with 1N HCl and distilled water, and the anion exchange resin was washed with 1N NaOH and distilled water, respectively (Jose M. Dominguez et al., Bioresouce Technology 61: 85-). 90, 1997).

<8-3> Crystallization Step

   In order to concentrate the xylitol solution, 90% of the total volume was concentrated using EYERA Rotavapor A-3S (Tokyo Rikikai. Co., Japan) at 300 rpm and 45 ° C. After concentrating the concentration of xylitol up to 100 g / l was shaken intermittently, at this time it was stored at 4 ℃ to obtain a crystal. The crystallized xylitol contains about 20% of water in the powder, so to effectively remove it, the xylitol powder was suspended in 500 ml of 100% ethanol, and water and ethanol were simultaneously removed by a vacuum pump using a filter. Xylitol was then crystallized by drying in a vacuum drying oven for 24 hours (Richard Peter Affleck. Recovery of xylitol from Fermentation of Model Hemicellulose Hydrolysates Using Membrane Technology.Masters thesis, Virginia Polytechnic Institute and State University, Verginia, 2000)

The HY200 strain of the present invention has a high concentration of xylose resistance and cohesion, so the production method using the strain can settle the cells without additional processes, thereby reducing the cost of xylitol production. Therefore, by mass-producing xylitol, which is used as a variety of food additives and most of them depend on imports, can not only replace imports of xylitol, but also have high price competitiveness in overseas countries with a market scale of 30,000 tons per year. It is expected to be.

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Claims (9)

Candida tropicalis HY200 strain (Accession No. KCTC 10813BP) having the following bacteriological properties: Iii) aerobic microorganisms without creamy mobility; Ii) carbon compounds selected from the group consisting of inositol, raffinose, and rhamnose in addition to the carbon source commonly used for cultivating Candida tropical strains, can be used as the carbon source; Iv) As a result of the cohesiveness test, in the fermentation tank in which the fermentation broth containing 100 g / l of xylose and the seed culture solution were mixed in a 1: 1 working volume, the agitation was started immediately after the agitation was stopped and the agitation was stopped. being. 1) species culturing the strain of claim 1; 2) inoculating the culture solution of step 1 into a fermentation broth containing xylose and then incubating by adjusting the air supply amount, the stirring speed and the temperature; And 3) xylitol production method comprising the step of purifying xylitol from the culture medium of step 2. 3. The xylitol production method according to claim 2, wherein the culture method of step 2 is a repeated batch fermentation. The fermentation broth of step 2, wherein the fermentation medium of step 2 is D-xylose or D-xylose selected from the group consisting of D-xylose or inositol, raffinose, rhamnose, fructose, glucose, maltose or mannitol. Xylitol production method characterized in that the liquid medium containing a mixture of. The method of claim 2, wherein the fermentation broth of step 2 is a liquid medium comprising yeast extract, tryptone or a mixture thereof as a nitrogen source. 3. The xylitol production method according to claim 2, wherein the initial xylose concentration of the fermentation broth of step 2 is 100 g / l to 300 g / l. 3. The xylitol production method according to claim 2, wherein the aeration amount in step 2 is 0.5 to 1.5 vvm. The method of claim 2, wherein the stirring speed of step 2 is 250 to 500 rpm. The method of claim 2, wherein the incubation temperature of step 2 is 28 ° C to 31 ° C.

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