FI76377B - MICROBIOLOGICAL FRAMSTAELLNINGSMETOD. - Google Patents

Description

76377

The present invention relates to a microbiological process for the preparation of xylitol from xylose-containing mixtures using the hii strain Candida guilliermondii VTT-C-71006.

Xylitol is usually prepared on a technical scale by hydrolyzing hemicellulose-containing materials, for example birch wood, to give a pentose-rich hydrolyzate. The xylose in the hydrolyzate can then be catalytically hydrogenated to xylitol.

Microbiological methods for preparing xylitol are also known. In H.Onishi & T.Suzuki, "The production of Xylitol, L-Arabinitol and Ribitol by Yeasts", Arg.Biol.Chem., Vol. 30, no. 11, pp. 1139-1144, 1966, describes the microbiological formation of xylitol from a xylose-containing solution in the yeast species Candida guilliermondii. The initial xylose content of the raw material solution was 10% by weight. The yield of xylitol was reported to be 40.3%. Strain Candida guilliermondii VTT-C-71006 has been deposited with the National Collection of Yeast Cultures (NCYC), Norwich, England, on 17 December 1986 under number NCYC-1644.

Candida guilliermondii VTT-C-71006 has the following morphological and physiological characteristics:

Morphological characteristics:

Macroscopic morphology: malt extract (3 g / l) yeast extract (3 g / l) -glucose (10 g / l) -peptone (5 g / l) agar (20/1) (MYGP (agar) soft , glossy, white / light yellow colonies 1-2 mm in diameter.

Microscopic morphology: cells in MYGP solution immobile, slightly oval, measuring 2-5 x 7-3 / μm. In some conditions, pseudomycel formation.

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Physiological properties:

Physiological properties were determined as described in N.J.W.Kreger-van Rij, "The Yeasts - a taxonomic study" 3rd edition. Elsevier Science Publishers B.V., Amsterdam 1984, pp. 76-.

Fermentation: glucose + sucrose + raffinose? galactose? maltose lactose xylose

Assimilation: glucose + sucrose + maltose + cellobiose + raffinose + xylose + lactose starch nitrate inositol

According to the method of the invention, the culture is carried out in such a way that the strain Candida guilliermondii VTT-C-71006 is preferably cultured under aerated conditions in a medium containing xylose with a xylose content of 5 to 50% by weight at 20 to 40 ° C for 20 hours to 10 days. . The xylitol formed in the culture after yeast removal can be recovered by any method known per se.

Suitable raw materials are any xylose-containing mixtures, for example xylose-rich mixtures obtained by hydrolysis of hemicellulose-containing material.

In the fermentation broth, 3 76377 different ammonium salts can be used as the N-source of yeast, as well as e.g. yeast extract, yeast produced in the process or plant-derived extract as a source of growth factors. It is essential to limit the oxygen supply to yeast. By adjusting the mixing and aeration conditions to the optimum, up to 78% xylitol is obtained. At very high initial concentrations of xylose, the process is slowed down. However, the process can be accelerated by up to 40% compared to the conventional batch process by moving to the so-called fed-batch culture, which means that nutrients are added to the fermenter during the process.

In our experiments, cultivation was performed in both fermentor and shake cultures with different xylose concentrations and different aeration efficiencies.

The composition of the culture medium was: xylose 40-300 g / l yeast extract 10 g / l

Bactopeptone 3 g / l

In our experiments, it was surprisingly found that the addition of furfural had a beneficial effect on the yield of xylitol. In the past, the presence of furfural in microbiological processes has been considered to be purely harmful, as furfural inhibits yeast growth. Inhibition of yeast growth is inherently desirable in such methods, as yeast growth consumes raw materials, which is reflected in a decrease in xylitol. Surprisingly, however, furfural did not completely inhibit yeast function but acted as a promoter of xylitol production. According to the literature, furfural generally interferes with both growth and the fermentation process (e.g., N. Banerjee et al., European J.Appi.Microbiol.Biotechnol. 1981, 11: 226-228).

In this method, it was found that the growth of yeast and the subsequent decrease in xylitol yield can be limited by adding furfural to the fermentation broth at 0.2-1.0 g / l.

The use of furfural to promote microbiological processes is not limited to the production of xylitol from xylose by the method described above. It is clear that this observation can also be applied to other microbiological processes, the purpose of which is not the production of the microbe itself but the production of substances independent of the growth of the microbe, for example in biotransformations.

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During the culture, the yeast concentration was determined by turbidity measurements, the xylose content by determining the content of reducing compounds and the formation of xylitol qualitatively by thin-layer chromatography and quantitatively by liquid chromatography or enzymatic method.

The method according to the invention has many advantages over known methods. These include, for example: high xylitol yield, 50-78%, depending on the xylose content. The prior art process mentioned at the beginning gave a yield of xylitol of only 40.3%, although the starting content of xylose was only 10%. Applying our method, a yield of 74% was obtained with the corresponding initial xylose content.

- It is possible to use high initial xylose concentrations. This fact is of great technical and economic importance. With an initial xylose content of the raw material of 300 g / l, a xylitol yield of up to 50% was obtained.

- The xylitol produced by this method is pure and thus quite suitable for food use. Xylitol produced by catalytic hydrogenation may contain residues of the toxic catalyst. The process according to the invention is also more economical, since the catalysts are expensive and wear out in the process.

- This method is also very specific, as the yeast strain Candida guilliermondii VTT-C-71006 only converts xylose to xylitol. In addition to xylitol, by-products can be obtained in catalytic hydrogenation, which make purification difficult.

The following examples describe in more detail the method according to the invention.

Example 1;

Aseptically grow Candida guilliermondii strain VTT-C-71006 as a shake culture in a 100 ml solution volume in a 250 ml vessel. The growth temperature is 28 ° C. The medium contains 80 g / l xylose, 10 g / l yeast and 3 g / l Bactopeptone. The pH of the medium was adjusted to 5.0. 1 ml of overnight MYGP medium was used as inoculum. After two days of culture, the xylose 5 76377 is depleted and the xylitol content is 50 g / l. Xylose was determined as reducing blinds and xylitol was determined by liquid chromatography. The xylitol yield was thus 62.5%. Example 2:

The procedure described in Example 1 was repeated, with an alkyloyl content of 300 g / l. After six days, the xylose was consumed and the xylitol content was 150 g / l, corresponding to a yield of 50%.

Example 3;

The procedure described in Example 1 was repeated, but furfural was added to the medium to a concentration of 0.6 g / l. The xylitol content after 53 hours of growth was 61 g / l (yield 76%).

Example 4:

The strain mentioned in Example 1 is aseptically grown in a fermentor on a medium containing 100 g / l of xylose, 10 g / l of yeast extract and 3 g / l of Bactopeptone. The pH of the medium was adjusted to 5.0 and the stirring speed to 400 rpm and aeration to about 0.5 g / min. After 50 hours of culture, the xylose was depleted. The xylitol content was 74 g / l (yield 74%).

Example 5;

The procedure described in Example 4 was repeated except that the alkucylose content was 250 g / l. After 160 hours of growth, the xylose was depleted and the xylitol content was 138 g / l (yield 55%).

Example 6:

The strain mentioned in the previous example is grown in a fermentor as fed-batch culture. The initial volume of the culture medium was 60% of the final volume and the composition was 100 g / l xylose, 10 g / l yeast extract and 3 g / l Bactopeptone. The culture was started as in e.g. 4, but when the xylose content had dropped to 40 g / l, the feed of the xylose solution at a concentration of 350 g / l was started. With the feed, the xylose content in the fermentor was kept at 40 g / l. When the final volume was reached, the feed was stopped. After 120 hours of growth, the xylose was depleted and the xylitol content was 164 g / l, corresponding to a xylitol yield of 78%.

Claims (4)

6 76377 A process for the preparation of xylitol from xylose-containing mixtures, characterized in that the yeast strain Candida guilliermondii VTT-C-71006, accession number NCYC 1644, is preferably cultured under limited aerated conditions in a xylose-containing medium at a temperature of 20-40 ° C for up to 20 hours. the xylitol formed is recovered. Process according to Claim 1, characterized in that 0.2 to 1.0 g / l of furfural is added to the medium in order to limit the growth of the yeast and thus to improve the yield of xylitol. Process according to Claim 1 or 2, characterized in that the xylose-containing pentose-rich sugar mixture obtained in the hydrolysis of hemicellulosic materials is used as the growth medium. Process according to one of the preceding claims, characterized in that the fed-batch process is used in the fermentation.