ES2363002T3 - NEW CROPICALIS CJ-FID (KCTC 10457BP) AND METHOD TO PRODUCE XYLITOL USING THE SAME. - Google Patents

Abstract

An alginate globule comprising Candida tropicalis CJ-FID (KCTC 10457BP), prepared by adding Candida tropicalis CJ-FID (KCTC 10457BP) to an aqueous solution of alginic acid with stirring to obtain a uniform solution, and to drip the uniform solution on a calcium salt.

Description

Technical field

The present invention relates to a new Candida tropicalis CJ-FID and a method of producing xylitol using it. More particularly, the present invention relates to a new Candida tropicalis CJ-FID from honey and a method for producing high purity xylitol with high yield and productivity, which includes cultivating Candida tropicalis CJ-FID itself, or a Candida CJ-FID tropicalis immobilized in alginate, in a fermentation medium containing sucrose and xylose in high concentration to obtain a culture containing xylitol, followed by purification with an activated charcoal column and an anionic column, to finally obtain xylitol in powder.

Fundamental technique

Xylitol is a sugar alcohol produced by the conversion of a terminal reducing group of xylose, which is a sugar with five carbon atoms, into an alcohol group by hydrogenation, and is found in small quantities in natural plants such as fruits, vegetables and mushrooms. Xylitol is also known as an intermediate product in mammalian carbohydrate metabolism. Xylitol is more stable than other sugars because of its chemical structure, has a high affinity for water and does not experience a darkening reaction.

Xylitol has a sweetness equal to that of sucrose but contains few calories and does not cause elevated blood sugar or tooth decay. In this regard, xylitol has become very popular as a sugar substitute [K. McNutt and A. Sentki, 1996, "Sugar replacers: a growing group of sweeteners in the United States", Nutr. Today 31 (6): 255-261]. Xylitol is absorbed in the human intestinal tract by active diffusion. However, since the rate of absorption is quite small, xylitol is incompletely absorbed by the intestinal tract and is excreted from the body. The absorbed xylitol is partially broken down by intestinal microorganisms and only a small fraction is used in the human body. In this regard, xylitol falls into the category of low-calorie sweeteners.

In particular, since it does not require insulin when it is being absorbed by cellular tissues, xylitol is used as a sugar substitute for diabetic patients. In addition, because of its sweetness equivalent to that of sucrose and its heat of negative dissolution, xylitol imparts a refreshing and fresh sensation in the oral cavity. Therefore, xylitol is used in various food industries, including confectionery and chewing gum. In addition, xylitol can prevent tooth decay by inhibiting the growth of Streptococcus mutans, which is responsible for tooth decay, and, therefore, is used as toothpaste material.

Xylitol is found in vegetables and fruits, but the extraction of xylitol from these sources is not economical because of its low concentration. In this regard, the chemical production of xylitol has been used primarily. According to the method of chemical production, xylitol is produced by chemical reduction of xylose, which is a product of hydrolysis of hemicellulose present in wood, rice straw, millet, etc., under conditions of high temperature and high pressure. However, the separation and purification of xylose or xylitol from hydrolysis products of other polymeric sugars derived from hemicellulose fractions are difficult, and the yield in xylose or xylitol is as small as 50-60%. In addition, problems arise such as the risk of a reaction at high temperature and high pressure and the removal of waste due to the use of acid or alkali.

The production of xylitol using a microorganism as a biocatalyst for the conversion of xylose into xylitol in a medium containing xylose is also known. That is, in the microorganism, the xylose of a medium containing xylose is transported through the cell membrane and is then converted into xylitol by a xylose reductase (XR) dependent on the NADPH coenzyme. The abundantly accumulated xylitol is released from the cells of the microorganism. The production of xylitol using a microorganism has advantages since the separation of xylitol from the xylose is not required due to the complete consumption of the xylose after fermentation and mild conditions such as ambient temperature and atmospheric pressure are used. However, since it is difficult to efficiently and selectively separate by-products such as organic acids and glycerol, the ingredients of the medium and substances derived from the microorganism, which are generally contained in a fermentation medium, it is difficult to purify xylitol with high purity, That results in poor performance.

In Korean Patent Publication No. 10-2001-49918, exposed to public consultation, a method for producing xylitol from arabitol using Gluconobacter oxydans ATCC 621 is described. According to the description of the patent document, xylitol is purified from 200 ml of a finally produced solution of 45 g / l of xylitol. However, no exact results of purification performance are described in this document. In addition, since xylitol is produced from a low concentration xylitol solution (50 g / l), a production of xylitol with high concentration and efficacy is not ensured, which makes mass production difficult. To solve these problems, many studies have recently been conducted on the separation of a new strain of yeast and on a method for producing xylitol using the new strain of yeast.

DESCRIPTION OF THE INVENTION

In view of these problems, the present description provides a new Candida tropicalis CJ-FID. The present invention also provides a method for producing high purity xylitol with high yield and productivity.

The above and other objects of the present invention may be carried out by embodiments of the present invention, as will be described later.

Therefore, according to one aspect of the present description, a Candida tropicalis CJ-FID (KCTC 10457BP) producing xylitol is provided.

According to another aspect of the present invention, an alginate globule prepared by adding Candida tropicalis CJ-FID (KCTC 10457BP) is provided to an aqueous solution of alginic acid, with stirring to obtain a uniform solution, and dripping the uniform solution over a calcium salt

In accordance with yet another aspect of the present invention, a method of producing xylitol is provided, which includes preparing a fermentation medium containing xylose and sucrose to produce xylitol, inoculating an alginate globule containing Candida tropicalis CJ-FID to the fermentation medium , and make the fermentation medium ferment in a fermenter.

The method may also include the purification of a fermentation culture containing xylitol, produced by fermentation, with a column of activated charcoal, purification, with an anionic column, of a solution of xylitol produced by purification with the column of activated charcoal, and the preparation of a xylitol powder from a solution of xylitol produced by purification with the anionic column.

The present invention will be described in more detail below.

Separation of the yeast strain that produces xylitol

A sample of natural honey from a domestic honey producing area was taken and diluted appropriately in a sterilized tube. The diluted honey sample was grown in a plate medium containing 200-400 g / l of xylose, 5 g / l of a yeast extract, 5 g / l of peptone and 15-20 g / l of agar, at 30 ° C, to obtain individual colonies formed by yeast strains that grew rapidly under the above growing conditions.

Identification of yeast strains that produce xylitol

Among these colonies, yeast strains with excellent sugar tolerance and xylitol productivity were selected under the above growth conditions and their morphological and physiological characteristics were analyzed. As a result of the sequencing of the 26S rRNA, yeast strains were found to have a nucleotide sequence as set forth in the ID. SEC. No. 1. The yeast strains were named Candida tropicalis CJFID and deposited at the Gene Bank of Korea Research Institute of Bioscience and Biotechnology (Accession No.: KCTC 10457BP).

Xylitol Productivity Assessment

To evaluate the xylitol productivity of the selected yeast strains, the selected yeast strains were inoculated into a 50 ml planting medium containing 20 g / l of xylose, 5 g / l of yeast extract, 5 g / l of peptone and 2 g / l of sucrose, in a 250 ml capacity flask, and then a shake culture was carried out at 200 rpm and 30 ° C for 12 hours. Then 5% of the sowing culture was inoculated into a 100 ml main medium containing 100 g / l of xylose, 5 g / l of yeast extract, 5 g / l of peptone and 10 g / l of sucrose, in a 500 ml capacity flask, and then a shake culture was carried out at 150 rpm and 30 ° C for 42 hours. Culture samples were taken from the resulting culture at predetermined intervals and then the production of xylitol in each culture sample was determined.

Xylose and xylitol analysis

For the analysis of xylose and xylitol, the culture samples taken in the previous Section were centrifuged at

12,000 rpm to separate cells. The supernatants were analyzed in a device (Shimadzu, Japan) for high performance liquid chromatography (HPLC), equipped with a Kromasil 100-10 NH2 column (Denmark), using an index detector of refraction (Shimadzu C-R6A, Japan). At this time, the solvent was 90% acetonitrile and the flow rate was 2.0 ml / min. The density of yeast cells was estimated by measuring the optical density at 600 nm using a turbidimeter and comparing the value with a previously prepared standard curve in which the optical density is related to the weight of the cells in the dry state.

Immobilization of Candida tropicalis CJ-FID

To prepare immobilized cells, a solution of alginic acid (3% by weight / volume) used in food was prepared. Then a cell concentrate with the same concentration was added thereto and the mixture was stirred. The resulting solution was allowed to drip onto a 100 mM solution of calcium chloride in a blood cell generator to form alginate blood cells. The alginate globules thus formed were stored at 4 ° C for 12 hours to increase the resistance of the alginate globules for subsequent experiments.

Xylitol fermentation by Candida tropicalis CJ-FID

A fermentation medium containing xylose, 0.1-10 g / l of yeast extract, 0.1-10 g / l of peptone and 0.1-50 g / l of sucrose was introduced into a fermenter. Then the Candida tropicalis CJ-FID itself (KCTC 10457BP) or the Candida tropicalis CJ-FID immobilized in alginate was inoculated into the fermentation medium and a culture under aeration was carried out with a flow rate of 0.5-2 vvm (volume of gas per volume of liquid per minute) at 200-300 rpm and 25-35 ° C for a period of 60 to 100 hours. After fermentation, the culture was centrifuged by fermentation to separate the cells and the xylitol was recovered from the supernatants.

Preferably, the xylose is contained in the fermentation medium in an amount of 50 to 200 g / l and, more preferably, in an amount of 80 to 150 g / l. If the xylose content is less than 50 g / l, the yield in xylitol may be reduced. On the other hand, if it exceeds 200 g / l, productivity enhancement can be insignificant. For productivity enhancement, the fermentation medium can be supplemented with 0.1-5 g / l potassium phosphate (KH2PO4) and 0.5-5 g / l magnesium sulfate (MgSO4).

Purification of the xylitol culture by fermentation

To produce high purity xylitol powders from the fermentation culture obtained in the previous Section, 300 g of an activated charcoal was packed in a column (60 cm high x 10 cm in diameter) and then the culture was allowed to by fermentation it will cross the column to obtain discolored and tasteless fractions containing xylitol. The obtained fractions containing xylitol were allowed to pass through an anionic column (60 cm high x 10 cm in diameter) packed with anionic resin, to remove impurities. The resulting xylitol containing fractions were analyzed by HPLC. As a result, pure xylitol was present in the fractions containing xylitol.

The fractions containing xylitol were concentrated to more than 800 g / l in a concentrator and were crystallized at 4 ° C, which was followed by the addition of an alcoholic ethanol with stirring to obtain fine xylitol crystals. The xylitol crystals were filtered off under vacuum to partially remove ethanol and water. Finally, the residual ethanol and water were removed by means of a vacuum dryer to obtain xylitol powders.

Brief description of the drawings

Figure 1 is a graph illustrating the productivity of xylitol with respect to an initial concentration of xylose.

Figure 2 is a graph illustrating the productivity of xylitol with respect to an initial cell density.

Figure 3 is a graph illustrating the yield and productivity of xylitol with respect to the density of alginate immobilized cells.

Figure 4 is a graph illustrating the yield and productivity of xylitol with respect to the mass of alginate globules.

Figure 5 is a graph illustrating the yield and productivity of xylitol with respect to the time of yeast cells in the sowing culture to be immobilized with alginate.

Figure 6 is a graph illustrating the yield and productivity of xylitol with the fermentation time of the yeast cells immobilized in alginate, in a fermenter of 2 l capacity adjusted to the optimal fermentation conditions.

Figure 7 is a graph illustrating the yield and productivity of xylitol with the fermentation time of the yeast cells immobilized in alginate, in a 17 l fermenter of capacity adjusted to the optimal fermentation conditions.

Figure 8 is a graph illustrating the change in the concentration of xylitol in the xylitol-containing fractions obtained by applying a culture containing xylitol to a column of activated charcoal.

Figure 9 is a graph illustrating the change in the concentration of xylitol in the xylitol-containing fractions obtained by applying the fractions of Figure 8 to an anionic column.

Figure 10 is a chromatogram of the high-performance liquid phase chromatography (HPLC) of the fractions of Figure 9.

Figure 11 is a graph illustrating the yield in xylitol crystallization of the fractions of Figure 9 concentrated to different concentrations.

Best way to carry out the invention

The present invention will be described more specifically below by way of examples. However, the following examples are provided for illustrations only and, therefore, the present invention is not limited to them or limited by them.

[Example 1] Productivity of xylitol with respect to the initial concentration of xylose

After a sowing culture was carried out in the manner described above, the sowing culture was inoculated into a main medium of 10 l to produce xylitol containing 0.5 g / l of yeast extract, 5 g / l of peptone , 10-30 g / l of sucrose and 100-300 g / l of xylose and the cultivation was carried out in a 15 l capacity fermenter (Korea Fermentation Co., Ltd.). The fermentation conditions were as follows: stirring speed of 250 rpm, aeration rate of 1.0 vvm, and culture temperature of 30 ° C. The productivity of xylitol was measured with respect to the initial concentration of xylose, and the results are shown in Figure 1.

As shown in Figure 1, the maximum production of xylitol was 187 g / l. At this time, the yield in xylitol production was excellent, 93.5%. It can be seen that the addition of sucrose increased the yield in xylitol.

[Example 2] Productivity of xylitol with respect to initial cell density

Once a sowing culture was carried out in the manner described above, the sowing culture was inoculated into a main medium of 10 l to produce xylitol containing 5 g / l of yeast extract, 5 g / l of peptone, 10 g / l of sucrose and 100 g / l of xylose and the cultivation was carried out in a 15 l capacity fermenter (Korea Fermentation Co., Ltd.). The fermentation conditions were as follows: stirring speed of 250 rpm, aeration rate of 1.0 vvm, and culture temperature of 30 ° C. The productivity of xylitol was measured with respect to the initial cell density, and the results are shown in Figure 2.

As shown in Figure 2, the maximum production of xylitol was 132 g / l. At this time, the ratio of xylitol produced to xylose used was excellent, 98%. However, it can be seen that the use of xylose in high concentration delayed the rate of production of xylitol.

[Example 3] Sugar tolerance test for conventional xylitol producing strains and for Candida tropicalis CJ-FID (KCTC 10457BP)

Candida tropicalis CJ-FID was evaluated in accordance with the present invention and conventional xylitol producing strains for sugar tolerance, and the results of the evaluation are presented in Table 1 below.

Table 1: Results of the sugar tolerance test for yeast strains producing xylitol

Yeast strain

Xylose concentration (g / l)

100

 200 300  400

Candida tropicalis KCTC 7101

+++ +++ + -

Debaryomyces hanseni KCTC 7128

+++ +++ - -

Candida tropicalis KCTC 7212

+++ +++ - -

Candida parapsilosis KCTC 7653

+++ +++ - -

Candida tropicalis KCTC 7725

+++ +++ + -

Candida tropicalis KCTC 7901

+++ +++ - -

Candida sp. KCTC 17041

+++ +++ - -

Candida shehata KCCM 11895

+++ +++ + -

Candida guilliermondii KCCM 50050

+++ +++ - -

Candida tropicalis KCCM 50091

+++  +++ + -

Candida mogii KCCM 50199

+++  +++ - -

Candida guilliermondii KCCM 50631

+++  +++ - -

Candida utilis KCCM 50667

+++  +++ - -

Candida tropicalis CJ-FID KCTC 10457BP

+++  +++ ++ +

5 (+++: excellent growth; ++: good growth; +: slight growth; -: no growth)

As can be seen in Table 1, the Candida tropicalis CJ-FID (KCTC 10457BP) of the present invention exhibited excellent tolerance to xylose compared to the currently known yeast strains. This means that the Candida tropicalis CJ-FID (KCTC 10457BP) of the present invention can produce large-scale xylitol in a

10 medium containing xylose in high concentration.

[Example 4] Evaluation of the productivity of xylitol with respect to the density of cells immobilized in alginate

To evaluate the productivity of xylitol with respect to the density of the yeast cells Candida tropicalis CJ

15 FID trapped in alginate globules, once a sowing culture was carried out in the manner described above, alginate globules were prepared by mixing an alginate solution with the seeding culture with variation in cell density {from 10 to 100 [ optical density value (OD)]}. Then, the alginate globules were inoculated into a main medium of 10 l to produce xylitol containing 5 g / l of yeast extract, 5 g / l of peptone and 100 g / l of xylose and carried out in culture in a 15 l capacity fermenter (Korea Fermenta

20 tion Co., Ltd.). The fermentation conditions were as follows: stirring speed of 250 rpm, aeration rate of 1.0 vvm, and culture temperature of 30 ° C. The productivity of xylitol was measured with respect to the initial cell density, and the results are shown in Figure 3.

As shown in Figure 3, when the cell density was 10 to 20 (OD value), the productivity of xylitol was approximately 0.5 g / l · h. With a cell density greater than 50 (OD value), the productivity of xylitol was greater than 1.25 g / l · h. With a cell density of 100 (OD value), the productivity of xylitol was maximum, 1.65 g / l · h.

[Example 5] Evaluation of the productivity of xylitol with respect to the alginate blood cell mass

Alginate globules prepared in the same manner as in Example 4, with variable mass (2.5 to 50 g), were inoculated into a fermentation medium to produce xylitol in a fermenter of 2 l capacity and the culture under the same fermentation conditions as in Example 4. The productivity of xylitol was measured with respect to the mass of the alginate globules, and the results are shown in Figure 4.

As shown in Figure 4, the productivity of xylitol remained constant regardless of the change in the mass of the alginate globules.

[Example 6] Evaluation of xylitol productivity with respect to planting time

To evaluate the productivity of xylitol with respect to the sowing culture time of the sowing culture cells used for the preparation of the alginate globules, yeast strains were grown in seeding media with variation of the cultivation time (from 12 to 60 hours) and then the fermentation was carried out under the same fermentation conditions as in Example 4. The productivity of xylitol was measured with respect to the planting culture time, and the results are shown in Figure 5.

As shown in Figure 5, the productivity of xylitol remained constant regardless of the planting culture time. However, yeast strains that had been grown for planting for 24 hours had the maximum productivity of xylitol.

[Example 7] Evaluation of the yield and productivity of xylitol with respect to the repetitive use of immobilized cells

Candida tropicalis CJ-FID cells immobilized in alginate globules were prepared and fermentation was carried out in the same manner as in Example 4 except that the alginate globules were used repetitively (one to five times). The yields and productivity in xylitol of the Candida tropicalis CJ-FID cells immobilized in alginate globules were evaluated by comparison with the values of the currently known Candida guilliermondii FTI 20037 immobilized in alginate globules, and the results are presented below in Table 2

5

10

fifteen

twenty

25

30

35

Table 2: Yields and productivity of xylitol with respect to the repetitive use of immobilized yeast cells

Strain

Section Number of repetitions

one

2 3 4 5

Candida tropicalis

Productivity (g / l · h) 3.31 2.95  3.04  3.11 2.99

CJ-FID (KCTC 10457BP)

Performance (%) 89.5  92.3  88.6  90.4 90.6

Candida guilliermondii

Productivity (g / l · h) 0.5 0.59 0.6 0.6 0.58

FTI 20037

Performance (%) 53.2  59.5  62.6  62.4 61.4

As shown in Table 2, no reduction in yields or productivity of xylitol was observed even when immobilized cells were used five times. In particular, the Candida tropicalis CJ-FID cells had a five-fold increase in the productivity of xylitol and a greater than 45% increase in the yield of xylitol, compared to the Candida guilliermondii FTI 20037 cells.

[Example 8] Productivity of xylitol with respect to fermentation time

Candida tropicalis CJ-FID cells immobilized in alginate globules were prepared and fermentation was carried out in fermenters of 2 l and 17 l capacity in the same manner as in Example 4. The productivity of xylitol was measured by varying the time of fermentation, and the results are shown in Figures 6 and 7.

As shown in Figures 6 and 7, the concentration of xylitol was increased after 6 hours of fermentation. With respect to fermentation in the 2 l capacity fermenter (Figure 6), the productivity of xylitol was maximum at 24 hours of fermentation. With respect to fermentation in the fermenter of 17 l capacity, the productivity of xylitol was maximum at 36 hours of fermentation.

[Example 9] Purification of the xylitol culture by fermentation

The concentrations of xylitol in the fractions of a culture of xylitol were measured by fermentation that had passed through a column of activated charcoal and an anionic column. The results are shown in Figures 8 and 9. The fractions of Figure 9 were analyzed by high performance liquid chromatography (HPLC), and the results are shown in Figure 10. The fractions of Figure 9 were concentrated until different concentrations and the yields in the crystallization of xylitol were measured with respect to the concentrations. The results are shown in Figure 11.

According to the purification results when using the activated charcoal column, as shown in Figure 8, fraction 4 had the highest concentration of xylitol. According to the purification results when using the activated charcoal column and the anionic column, as shown in Figure 9, fractions 3 to 5 had the highest concentration of xylitol. In the chromatogram of Figure 10 it can be seen that the fractions that had crossed the activated charcoal column and the anionic column contained highly purified xylitol. The purified xylitol solutions were crystallized to different concentrations. As the concentration of xylitol solutions increased, the yield in crystallization of xylitol increased proportionally.

[Example 10] Yield and concentration of xylitol according to the fermentation and purification of xylitol

Table 3 below summarizes the concentrations of xylitol and the yields measured after fermentation and purification.

Table 3: Purification yields according to the processes of purification of the xylitol culture by fermentation

Section

Xylitol concentration (g / l) Performance (%)

Xylitol culture by fermentation (300 g / l)

270 90

Activated charcoal column

270 90

Anionic column

262 87.3

Concentration

 257 84.7

Crystallization

 244 80.4

Dust formation

230 76.4

Final Purification Performance

77.4

As shown in Table 3, the final yield in xylitol after fermentation and purification was as high as 77.4% and very little loss of xylitol was observed during each purification process.

Industrial applicability

As is evident from the above description, the new Candida tropicalis CJ-FID (KCTC 10457BP) of the present invention and a method for producing xylitol using it can produce very pure xylitol with high yield and productivity under culture conditions. and a composition of the optimized medium.

SEQUENCE LIST

<110> CJ Corp. KIM, Jung-hoon SOHN, Young-rok LEE, Woon-hwa PARK, Seung-won PARK, Kang-june LEE, Ki-chang LIM, Jae-kag

<120> New Candida tropicalis CJ-FID (KCTC 10457BP) and method of manufacturing xylitol through it

<130> PP04-0114

<150> KR10-2003-0051593 <151> 2003-07-28

<150> KR10-2004-0033733 <151> 2004-05-13

<160> 1

<170> KopatentIn 1.71

<210> 1 <211> 576

<212> DNA

<213> Candida tropicalis

<400> 1

image 1

Claims (3)

1. An alginate globule comprising Candida tropicalis CJ-FID (KCTC 10457BP), prepared by adding Candi give CJ-FID tropicalis (KCTC 10457BP) to an aqueous solution of alginic acid with stirring to obtain a uniform solution, and drip the uniform solution over a calcium salt. 2. A method of producing xylitol from xylose, comprising: prepare a fermentation medium to produce xylitol, containing xylose and sucrose; inoculate the alginate globule of Claim 1 into the fermentation medium; Y 10 ferment the fermentation medium in a fermenter. 3. The method of Claim 2, further comprising: purify a fermentation culture containing xylitol, produced by fermentation, with a carbon column activated vegetable; Purify, with an anionic column, a solution of xylitol produced by purification with the activated charcoal column; and preparing a xylitol powder from a solution of xylitol produced by purification with the anionic column.