KR0180986B1 - Preparation process of xylitol by microbial fermentation from hydrolysate - Google Patents

Abstract

The present invention hydrolyzes corncob in 0.01 ~ 0.2M sulfuric acid solution, centrifuged to remove pulp, neutralized with NaOH, removed salt and passed through ion exchange resin to purify high concentration of xylose syrup. It relates to a method for producing xylitol, characterized in that the fermentation is carried out using a Candida paroxypsis KFCC-10875 cells concentrated to 15 to 35g / L as a substrate.

Description

Method for producing xylitol by microbial fermentation from hydrolyzate of corn cob

1 is a diagram showing an acid decomposition process for obtaining a high concentration of xylose from corn cobs.

The present invention relates to a method for producing xylitol using as a substrate a concentrate of xylose obtained from corncob by Candida parapsilosis KFCC 10875.

Xylitol is a sugar alcohol with five carbon atoms discovered by the chemist Emil Fisher in 1891, and is represented by the formula C ₅ H ₁₂ O ₅ , mainly obtained by reduction of xylose. Xylitol has been of interest to hygienists and nutritionists since the 1970s because its sweetness and nutritional value is very similar to sugar and has some important physical, chemical and biochemical properties. In particular, xylitol can be used by diabetics because it causes a great deal of cooling in the mouth because of heat loss when dissolved, and does not require insulin during metabolism after ingestion. It inhibits the growth of bacteria and inhibits the development of tooth decay. It is used as a substitute for diabetics, sugar-free raw materials and toothpaste of products.

In addition, the chemically non-reactive reaction to the Maillard reaction is a remarkable property, and since it is a monosaccharide, it cannot be converted unlike sugar, so it can be used in an acidic environment without fear of alteration, and because the break point is 95 ° C. It can be reached without breaking down, so it can be used to apply sugar-coating, so it does not need to be dissolved in water.

Xylitol exists naturally in various fruits and vegetables, but in this case only a very small amount, and thus extracting from fruits or vegetables is not economically feasible. In consideration of the fact that xylitol cannot be produced by the extraction method, the method of synthesizing xylitol from xylose was considered.In the industrial method of producing xylitol by synthesis, xylan contained in the plant raw material is carried out through the following four steps. (xylan) is made using xylose that is hydrolyzed.

Acid hydrolysis of vegetable raw materials

Separation and Purification of Xylose from Mixtures Obtained by Hydrolysis

Synthesis of Xylitol by Hydrogenation Reduction of Xylose

Separation and Crystallization of Xylitol

In theory, xylitol can be obtained by xylitol reduction from xylose by three methods: chemical, biochemical and biological methods.

The chemical method is currently industrially producing xylitol, and is produced by hydrogenation of xylose contained in a hydrolyzate of a plant raw material under a catalyst of sodium hydrogen borohydride compound or Raney Nickel at high temperature and high pressure. Regardless of the technical details, chemical methods produce a mixture of polyalcohols, which must be crystallized after multistage chromatography to obtain high purity xylitol. Therefore, large-scale facility investment and inevitable cost increase should be paid. Therefore, it is very important industrially to selectively convert only xylitol from hydrolyzate of vegetable raw materials. It is a biochemical method to selectively convert only xylitol from hydrolyzate of vegetable raw materials, but there is a problem that the enzyme used is unstable and NADPH or NADH used as a coenzyme is expensive. On the other hand, the biological method is not an expensive method, and can only selectively convert xylitol from hydrolyzate of a vegetable raw material, thereby facilitating the separation and purification of xylitol after the reaction.

Gong's paper Biotechnol. Bioeng., 25, 87 (1983) and Preez, Enzyme Microb. According to Technol., 16, 944 (1994), yeast that can produce relatively high amounts of xylitol are blankii, guilliermndii, tropicalis, utilis and saccharomyces of Kendida. Baili in the Saccharomyces, rouxii, uvarium and pombe in the Schizosaccharomyces. These strains have a relatively higher yield of xylitol than other strains, but the productivity is difficult to industrialize because of the low 0.3 ~ 1.5 g / L-h.

Several attempts have been made to look for wild or mutant types of yeast as a microorganism that selectively makes xylose only from xylose-containing mixtures of sugars. Gong's paper Biotechnol. Lett. 3, 130 (1981) reported that Candida tropicalis strains were obtained in xylos containing yeast-malt extract-peptone (YME) media, yielding 96% yield of xylitol from xylose. Expensive medium containing 3 g / L yeast extract, 3 g / L malt extract, 5 g / L peptone, etc., making it impossible to apply industrially and the 250 mL flask scale result. have.

Gong also investigated the possibility of obtaining xylitol from sugarcane hydrolyzate (J. Food Sci. 50, 226 (1985)). Candida sp., Adapted by hydrolysis products. A high yield of 91.9% was obtained using B-22, but the culture medium used contained 30 g / L of glucose and 47 g / L of arabinose, and the medium used was expensive (3 g / L). Yeast extract, 3 g / L malt extract, 5 g / L peptone), and the amount of contact used is 1 to 3 × 10 ⁸ cells / mL, so the presence of cells of this sliding amount is expensive. The experiment was also carried out in a 250 mL flask, which is far from industrialization.

As such, to date laboratory reports on the production of xylitol from xylose by biological methods have shown the possibility of selectively converting sugar mixtures to xylitol, but industrially available processes ( it does not create a process.

The inventors have found a strain from a natural species of Candida parapsilosis (disclosed in patent application No. 95-37516), and the patent application No. 96- about the optimal medium and culture conditions for the production of the mutant xylitol. It was disclosed in Korean Patent Application No. 13638 and disclosed in Patent Application No. 96-30577 by increasing the productivity of xylitol by concentrating the cells after fermentation or concentrating the cells during fermentation, and optimizing xylitol by controlling oxygen partial pressure during fermentation Patent Application No. 95-37516 discloses a patent for a method for preparing xylitol by controlling the redox potential, which has a constant relationship with acid high partial pressure and is easy to control because the control range of oxygen partial pressure is narrow. It was disclosed in the application 96-30578, and the related mutant strain was deposited with KFCC-10875 in the Korean spawn consultation.

Most fermentation methods to date have been carried out with commercial products of xylose, which has the disadvantage of not being able to meet the cost of production due to the high price of the finished product of xylose. Therefore, the present inventors have completed a method of drawing xylitol from the hydrolyzate of corn corp, which contains relatively high amounts of xylose, using Candida paroxylosis KFCC-10875.

An object of the present invention is to hydrolyze the corncob in 0.01 ~ 0.2M sulfuric acid solution and centrifuged to remove the pulp, neutralize with NaOH, remove the salt component, and then pass through an ion exchange resin to purify the highly concentrated xylose syrup. It is to provide a method for obtaining xylitol with high productivity using a Candida paroxypsis KFCC-10875 cells concentrated at 15 to 35 g / L as a substrate. At this time, the amount of xylose used as the substrate is added to the xylose by dividing the oil price in order to prevent the initial amount of xylose from becoming high. At this time, the water for purifying xylose from the sugar mixture is characterized in that the strong acid cation exchange resin in the form of sulfated polystyrene cross-linked divinylbenzene.

The present invention is described in more detail as follows.

Acid hydrolysis of the corncob was carried out using a 25 L volume of stainless steel reactor with stirring at 140-150 ° C. for 18-22 minutes. Add 65-75 g of concentrated sulfuric acid to 1000 g of corncob, add 9-10 L of sufficient water to make the ratio of liquid and solid to 10: 1, and centrifuge at 4500-5500 rpm for 12-18 minutes to remove pulp. It was. The hydrolyzate of corncob of 0.8-1.2 was neutralized with NaOH to adjust the pH to around 7.0, and then concentrated to dryness at 65-75 ° C. The pH of the concentrated hydrolyzate was raised to 9.5-10.5 and then lowered to 5.0-5.5 to remove the precipitated solid component (mainly salt) by centrifugation at 4500-5500 rpm for 12-18 minutes. The desalted hydrolyzate was concentrated to dryness at 65-75 ° C. to 65-70% dryness. This hydrolyzate is obtained to produce xylitol. The corncob hydrolyzate obtained by the above method was adjusted to a concentration of 20-30%, and then passed through a strong acid cation exchange resin in the form of sulfated polystyrene cross-linked with 3-4% divinylbenzene to increase the concentration of xylose 80-. Concentrated to 95%.

The method for fermenting xylitol using the concentrated xylose is as follows.

Strained Candida Parasilosis KFCC-10875, frozen strain (-70 ° C), contains 250 mL of 50 ml of YM medium (20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract). Inoculated in a mL flask was performed at 240 rpm, 30 ℃ in a shaking incubator until the cell concentration grew to 3-4 g / L (about 14-16 hours). In order to examine the effect of cell concentration on the production of xylitol, the seed culture medium was fermented medium (xylose 50 g / L, yeast extract 5g / L, ammonium sulfate 5g / L, potassium diphosphate 5g / L, ammonium sulfate 0.2g / 10L fermenter containing 6L) was inoculated and cultured at 30 ° C., and cultured until the cell concentration reached about 10 g / L, followed by centrifugation.

Cells concentrated to about 20 g / L was inoculated into a 5 L fermenter containing 3 L of fermentation broth. In the early stage of culture, 2L of culture medium containing 300g of xylose was incubated, and 500mL solution containing 300g of xylose was added twice during the fermentation process, and the final culture solution had a volume of 3L (final concentration of 300g of xylose added). Fed-batch cultivation to become / L). The pH was constantly adjusted to 4.5-5.5 during the whole fermentation process, the temperature was 28-32 ° C., the aeration rate was 0.8 ° C. 1.2 vvm, and the dissolved oxygen was adjusted to 350-380 rpm to adjust the dissolved oxygen concentration. The culture was maintained at about 0.7-1.5% (redox potential corresponds to 80-110 mV) until the xylose was consumed completely.

The concentrations of xylose and xylitol were measured by HPLC (Shimadzu C-R7A, Japan) equipped with Sugar-Pak I column. The solvent was water, the temperature was 90 ° C, and the flow rate was 0.5 mL / min. The detector used RI. Cell concentration was converted to dry weight using a standard curve measured in advance by measuring the suspension at a wavelength of 600nm using a turbidimeter. The concentration of dissolved oxygen during fermentation was measured by using dissolved oxygen electrodes (Ingold, Swiss).

Hereinafter, the present invention will be described in detail according to the embodiment.

Example 1

The mass balance of the process for obtaining high concentration of xylose from corn cobs is shown in FIG. Acid hydrolysis of corncob was performed by stirring at 145 ° C for 20 minutes using a 25 L volume of stainless steel reactor. 70 g of concentrated sulfuric acid was added to 1000 g of corncob and sufficient water was added to make the ratio of liquid and solid to 10: 1 and centrifuged at 5000 rpm for 15 minutes to remove pulp. The pH of the corncob of 0.90 was neutralized with NaOH to adjust the pH to around 7.0, and the dry drying was concentrated at 70 ° C. The pH of the concentrated hydrolyzate was raised to 10.0 and then lowered to 5.3 to remove the precipitated solid component (mainly salt) by centrifugation at 5000 rpm for 15 minutes. The dehydrated hydrolyzate was concentrated to dryness at 70 ° C. to about 68% dryness. To produce xylitol, this hydrolyzate was used in a fermenter.

Example 2

The corncob hydrolyzate obtained in Example 1 was adjusted to a concentration of 25% and then treated with a strong acid cation exchange resin in the form of sulfated polystyrene crosslinked with 3.5% divinylbenzene. At this time, looking at the composition of the sugar component before and after the resin treatment is shown in Table 1.

Example 3

Using the material obtained in Example 2 as a substrate, using 20 g / L of the concentrated strain of KFCC-10875 Candida paraxilosis mutant KFCC-10875, on the other hand, 50 g / L for reagents, 5 g / L yeast extract, 5 g / L ammonium sulfate, In a 5 L fermentation tank containing 3 g medium containing 5 g / L potassium diphosphate and 0.2 g / L ammonium sulfate, on the other hand, 50 g / L xylose obtained from the hydrolysate of corn cob, 5 g / L yeast extract, 5 g / L ammonium sulfate Xylitol production yield and xylitol productivity from xylose when incubated at 30 ° C, 350-380 rpm, pH 4.5-5.0 in a 5 L fermentation tank containing 3 g medium containing 5 g / L ammonium sulfate 2 g / L potassium diphosphate Table 2 shows.

Example 4

Under the experimental conditions described in Example 3, 300 g / L of xylose was added by a fed-batch method, incubated with 20 g / L of concentrated cells, and then centrifuged at a time point when all of the xylose was consumed. The nutrient solution was removed and cells were added into a 5L fermentor containing fresh medium for reuse. Xylitol production, yield and productivity are shown in Table 3 when the strain is used as it is, and only the medium is replaced to produce xylitol five times. The concentration of total xylose added during one repetition period was 300 g / L and the volume of medium at the end of one repetition period was 3 L.

Claims (3)

The corncob is hydrolyzed with 0.01 ~ 0.2M sulfuric acid solution, centrifuged to remove pulp, neutralized with NaOH, salt removed and passed through ion exchange resin as a substrate. A method for producing xylitol, characterized in that the fermentation is carried out using the Candida parapsilocysis KFCC-10875 cells concentrated at 15 to 35 g / L. The method for preparing xylitol according to claim 1, wherein the ion exchange resin is a strong acid cation exchange resin in the form of sulfated polystyrene crosslinked with divinylbenzene. The method for producing xylitol according to claim 1, wherein the added amount of xylose obtained by hydrolysis of corn cob is added and fermented by dividing xylose into a fed-batch formula so that the amount of initial xylose does not become a high concentration.