KR19980033943A - Method for preparing xylitol by microbial fermentation from hydrolyzate of corn cob - 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 the Candida paroxypsis KFCC-10875 cells concentrated to 15 to 35 g / 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 suppresses the development of tooth decay. It is used for substitute sugar of diabetics, sugar-free raw materials of confectionery products, toothpaste, etc.

It is also noteworthy that it is chemically non-reactive to Maillard recation, and because it is a monosaccharide, it cannot be converted unlike sugar, so it can be used in an acidic environment without fear of alteration. It can be reached without breaking down, which means that sugar-coating 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, it is not industrially economic to extract from fruits or vegetables. In consideration of the fact that xylitol cannot be produced by the extraction method, we came up with a method of synthesizing xylitol from xylose.In industrially, the method of producing xylitol by synthesis is as follows. (xylan) is made using xylose that is hydrolyzed.

Acid hydrolysis of vegetable raw materials

Separation and Purification of Xylose from the Mixture 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 the hydrolyzate of vegetable raw materials 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. This method has an unstable enzyme and an expensive NADPH or NADH used as a coenzyme. 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, guilliermondii, tropicalis, utilis and saccharomyces of the Kendida genus. Baili in the Saccharomyces, rouxii, uvarium and pombe in the Schizosaccharomyces. These strains have a higher yield of xylitol than other strains, but are difficult to industrialize due to the disadvantage of low productivity of 0.3 to 1.5 g / L-h.

Among the mixtures of sugars, including xylose, microorganisms that selectively make xylose into xylitol have been attempted rather than searching for wild type or mutant type of yeast. Gong's paper, Biotechnol . Lett. 3, 130 (1981), reported that 96% yield of xylitol was obtained from xylose when the Candida tropicalis strain was cultured in xylose-containing yeast-malt extract-peptone medium. Expensive medium containing g / L yeast extract, 3 g / L malt extract, 5 g / L peptone, etc., which is impossible to apply industrially and because of the 250 mL flask scale result, it is not suitable for industrial production. .

Gong (J. Food Sci. 50, 226 (1985)) also studied the possibility of obtaining xylitol from sugar cane hydrolysates. Candida sp., Adapted by hydrolysis products. A high yield of 91.9% was obtained with 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). L yeast extract, 3 g / L malt extract, 5 g / L peptone), and the amount of inoculation used is 1 to 3 × 10 ⁸ cells / mL, thus preparing a large amount of cells for this inoculation amount. The experiment was also carried out in a 250 mL flask, 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. 96-13638 for the optimum medium and culture conditions for the production of the mutant xylitol. It is disclosed in Korean Patent Application No. 96-30577 by increasing the productivity of xylitol by the method of concentrating the cells after fermentation or concentrating the cells during fermentation, and optimizing the xylitol by controlling the oxygen partial pressure during fermentation. Patent Application No. 95-37516 discloses a patent application for a method of preparing xylitol by controlling the redox potential, which has a certain relationship with the oxygen partial pressure and is easy to control because the control range of oxygen partial pressure is narrow. As disclosed in No. 96-30578, the relevant mutant strain was deposited with KFCC-10875 to the Korean spawn association.

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 for preparing 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, centrifuged to remove the pulp, neutralize with NaOH, remove the salinity, 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 the Candida paroxypsis KFCC-10875 cells concentrated to 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 formula in order not to have a high concentration of the initial xylose. At this time, the resin 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 25L volume stainless steel reactor with stirring at 140-150 ° C. for 18-22 minutes. After adding 65-75 g of concentrated sulfuric acid to 1000 g of corncob, 9-10 L of sufficient water was added to make the ratio of liquid and solid to 10: 1, and the pulp was removed by centrifugation at 4500-5500 rpm for 12-18 minutes. . The hydrolyzate of corncob of 0.8-1.2 in pH was neutralized with NaOH to adjust the pH to around 7.0 and then concentrated by dry drying 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 hydrolyzate of corncob obtained by the above method was adjusted to 20-30% concentration, and then passed through a strong acid cation exchange resin in the form of 3-4% divinyl benzene-crosslinked sulfated polystyrene. Concentrated to 95%.

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

50 ml of strain Candida paracylosis KFCC-10875 in frozen storage (-70 ° C) in YM medium (consisting of 20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract) Inoculated into a 250 mL flask containing a shaker was incubated at 240 rpm, 30 ℃ until the cell concentration increased 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 5 g / L, ammonium sulfate 5 g / L, potassium diphosphate 5 g / L, ammonium sulfate). 0.2 g / L) was inoculated into a 10 L fermenter containing 6 L, the culture temperature was 30 ℃ and cultured until the cell concentration was about 10 g / L and centrifuged.

Cells concentrated at about 20 g / L were inoculated into a 5 L fermenter containing 30 L 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, and the final culture volume was 3L (final concentration of xylose was 300 fed-batch cultivation to make g / L). The pH was adjusted to 4.5-5.5 during the whole fermentation process, the temperature was 28-32 ° C., the aeration was 0.8-1.2 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 xylose was consumed completely.

Xylose concentration of xylitol was measured using HLPC (Shimadzu C-R7A, Japan) equipped with Sugar-Pak I column, using solvent as water, temperature at 90 ° C, and flow rate at 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 as follows.

Example 1

The mass balance of the process for obtaining high concentration of xylose from corn cobs is shown in FIG. Acid hydrolysis of corn cob was performed by stirring at 145 ° C. for 20 minutes using a 25 L stainless steel reactor. 70 g of concentrated sulfuric acid was added to 1000 g of corncob, and then 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 the pulp. The hydrolyzate of corncob of pH 0.90 was neutralized with NaOH to adjust the pH to around 7.0 and then concentrated to dry drying 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, 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 parasilosis strain, 50 g / L for reagent, 5 g / L of yeast extract, ammonium sulfate In a 5 L fermenter with 5 g / L, 3 g medium containing 5 g / L potassium diphosphate, 0.2 g / L ammonium sulfate, on the other hand 50 g / L xylose obtained from corncob hydrolysates, yeast extract 5 g / L, 5 g / L ammonium sulfate, 5 g / L potassium diphosphate, 0.2 g / L ammonium sulfate in a 5 L fermenter containing 3 L medium at 30 ° C., 350-380 rpm, pH 4.5-5.0 Table 2 shows the xylitol production yield and xylitol productivity from xylose when cultured.

Example 4

Under the experimental conditions described in Example 3 above, 300 g / L xylose was added by the fed-batch method, cultured using 20 g / L concentrated cells, and then centrifuged at the point where all the xylose was consumed. The culture was removed and cells were added into a 5 L 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 a KCDA-10875 cell which is concentrated to 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.