JPS6035113B2 - Method for producing sorbitol using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing sorbitol from glucose by a microbial method. It relates to an efficient manufacturing method.

Sorbitol has a unique sweet taste, has low reactivity, is harmless, and has a moisturizing effect, so it is used as an additive in foods, toothpaste, cosmetics, etc., and is also used as an intermediate raw material in the production of vitamin C and surfactants. It is widely used as

Sorbitol is currently produced industrially by catalytic reduction of glucose using an Nj catalyst such as Raney nickel, but such a method inevitably requires high temperature (
16,000), high pressure (170k9/average), which consumes a lot of energy, requires a pressure-resistant container, and has the inherent danger of explosion due to the handling of hydrogen. The present inventors differed from the above-mentioned methods and attempted a method for producing sorbitol under mild conditions by microbiologically reducing glucose.

The mechanism for reducing fudu sugar to sorbitol has been demonstrated in animal cell systems in the spermatozoa of sheep (Japanese G. Hem Biochem.
Bioph pine. Acta37 (1960) 127-13
8) Or in a lens (M.lou Biochem.
BiophyS. Acta 141 (1967) 547
Its existence is known in 1559), but there is no example of microorganisms producing sorbitol, and only a few
No. 4834 and No. 46-23038 only disclose a method for aerobically fermenting and reducing xylose to xylitol. Further, even if the strain disclosed in the above invention was cultured in a glucose medium, sorbitol could not be obtained. Therefore, the present inventors conducted tests on many types of microorganisms, and found that cultures, cells, or We have completed the present invention by discovering that bacterial cell extracts and freeze-dried bacterial cells reduce the substrate glucose to sorbitol in high yield.

Any microorganism used in the present invention may be a commercially available strain, and examples of the Azotobacter genus include, for example, Azotobacter pinella landii (Azotobacter pinelandii).
ndii), among which Azotobacter vinelandii AMI078 is preferred.

The genus Beijerinckia includes Beijerinckia indica (Mr. jerinckiaindica), among which Beijerinckia indica lA
MII95 is preferred. The genus Mycobacterium is Mycobacterium freyi (Mycocterimph).
lei), among which Mycobacterium freyi AM12064 is preferred.

In growing Azotobacter and Beijerinchia bacteria according to the present invention, 2 to 15% carbon source is used.
○ Contains pentose such as xylose, D-arabinose, D-ribose, and also K2HP04, CaC03, Mg
A nitrogen-free medium containing only inorganic salts such as S04, NaCl, FeS04, Na2Moo4, etc. is used.

In addition, when growing Mycobacterium bacteria, 2 to 15% of pentose such as D-xylose, D-arabinose, and D-ribose are used as a carbon source, and yeast extract or corn stay plica is added. Use the added liquid medium. Cultivation is carried out at 25-30 oo for 2-10 days.
Any culture method can be adopted as long as it is under aerobic conditions, such as aeration culture, shaking moat culture, and rotating drum method. The growth rate can also be increased by adding various vitamins, minerals, and appropriate organic substances to the medium in addition to the above-mentioned components. Next, sorbitol can be obtained by contacting the culture thus obtained with glucose, but a certain degree of effectiveness can be observed even if the live bacteria obtained by washing the cells are used as they are.

However, it is much more effective to use treated bacterial cells such as freeze-dried bacterial cells, freeze-thawed bacterial cells, and treated bacterial cells with organic solvents such as acetone and ether. Furthermore, crushed bacterial cells and bacterial cell extracts prepared by ultrasonication or using a crusher such as a pibrogen cell mill can also be used. To reduce glucose, add the treated bacterial cells, crushed bacterial cells, bacterial cell extracts isolated therefrom, or mixtures thereof obtained in the above method to a glucose solution with a concentration of 10 to 60%.
The reaction is carried out at oC, preferably 25 to 35 qo.

At this time, reduced nicotinamide and adenine are used as coenzymes.
Dinucleotide, phosphate (hereinafter referred to as "NADPH")
That's what it means. ) can dramatically improve the productivity of sorbitol. NADP
The amount of H added varies greatly depending on the method of preparing the bacterial cells used or the extraction procedure for the bacterial cells, and is 0.1 mmol to 20 mol, preferably 0.1 mol to 10 mol, per mol of glucose. The time required for the reaction also varies greatly depending on the conditions.
For example, when a cocoon extract is used, the reaction is completed in 30 minutes to 14 days. After the reaction is completed, sorbitol is separated from the mother liquor by using a combination of known methods such as centrifugation, ultra-ditching, and ion exchange.

Hereinafter, the present invention will be explained in detail with reference to Examples.

The product was confirmed by thin layer chromatography, high performance liquid chromatography, and gas chromatography. Quantification was performed using high performance liquid chromatography. Example 1 Fifty centimeters of a sterilized medium having the composition shown in Table 1 were placed in a 500-liter digestion flask. VinelandmAMI07
Eight beads were inoculated into one platinum loop and cultured for 30 days for 6 days.

The cultured bacterial cells were washed twice with phosphate buffer and suspended in phosphate buffer so that the turbidity at 610n skin was 25.
This cell suspension was sonicated for 20 minutes, centrifuged at 14,000 yen for 20 minutes, and the supernatant fraction was used for the production of sorbitol. Using the supernatant fraction obtained in the above procedure, a reaction solution having the composition shown in Table 2 was prepared, the pH was adjusted to 7.5, and 30 qo
The mixture was allowed to react for 24 hours.

0.5 sorbitol was obtained in the reaction solution. Table I Table 2 Example 2 B. The cells were cultured in the same manner as in Example 1 except that indicaI AMII95 strain was used.
After washing twice, it was freeze-dried.

A reaction composition solution shown in Table 3 was prepared using the obtained freeze-dried bacterial cells, and reacted at 30oo for 24 hours.

1.5 ml of sorbitol was obtained in the reaction solution. Table 3
Example 3 50 liters of medium with the composition shown in Table 4 was placed in a 500' capacity digestion flask.
Sterilize it with M. The phlcil AM12064 beads were plated and cultured for 30 qo for 6 days.

The cultured cells were washed twice with phosphate buffer and then freeze-dried. A reaction composition solution shown in Table 3 was prepared using the obtained freeze-dried bacterial cells, and the reaction mixture was reacted at 30°C for 2 hours.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Azotobacter, Beisierinchia, or Mycobacterium and capable of producing sorbitol is cultured in a medium containing pentose as the main carbon source, and then the resulting culture is brought into contact with glucose. A method for producing sorbitol using a microorganism, characterized by producing sorbitol.