JPS5926277B2 - Method for producing D-fructose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing D-fructose by culturing bacteria in a medium containing methanol or ethanol as the main carbon source.

D-fructose is useful as a sweetener, an injectable infusion drug, and the like, and the establishment of an industrial-scale production method for it has been expected.

Recently, as a method for producing D-fructose, a method has been proposed in which glucose is converted into D-fructose using an enzyme glucose inmerase produced by a microorganism.

However, according to this method, the glucose added to the medium is expensive, so it is not necessarily an excellent method.

In view of the above-mentioned circumstances, the present inventors have devised D from an inexpensive carbon source.
- As a result of intensive research aimed at producing fructose, the present invention was completed.

In other words, the present invention is directed to Pseudomonas, Acetobacter, Aeromonas, Gluconobacterium, Brevibacterium, Bacillus, Serratia, Arthrobacter, Agrobacterium, Chromobacterium, Sartina, Sporo I Sartina microorganisms that belong to the genus Micrococcus, Corynebacterium, Cellulomonas, Flavobacterium, or alkaline earth metals and have the ability to assimilate methanol or ethanol and produce D-fructose. Cultured in a medium with carbon source, D in the medium
- A method for producing D-fructose, which is characterized by accumulating fructose and collecting it.

Specific examples of microorganisms that can be used in the present invention include, for example, Pseudomonas aeruginosa (Pseudomonas aeruginosa).
domonas aeruginosa ) (1FO-
13275).

Acetobacter aurantium (Ace-tobac
-ter aurantium) (IFO-324
8) , Aeromonas °liquefaciens (Aero
monas 1equefac-11ens ) (I
FO-12981), Gluconobacter 1ndustrius
ius) (IFO-3261), Brevibacterium fuscum (Brevibacterium fu)
scum) (IFO-12127).

Bacillus brevi
s) (IFO; -12335), Serratia
Marsesc (5err-atia marcesc)
ens) (FERM-P43421. IFO-1
2626), Arthrobacter passens (Art
hrobacter pascens ) (IFO
-12139), Agro-bacterium tumefaciens (Agro-bacterium tumefaciens)
(IFO-12667), Chromobacterium diantinum (IFO-12667)
terium janthinum) (IFO-373
9) Sarcina ″′ Luginata (Sarcina m.
arginata) (IFO-3066), Sporosarcina urea (Sporosarcina -na
ureae) (IFO-12698), Micrococcus caseolytyphi (Micrococcus c
aseolyticus) (1FO-3760), Corynebacterium faciens (Corynebac.
terium fascians ) (IFO-12
155), Cellulomonas flavigena (Cel 1 u
l om-onas f lavigena ) (I
FO-3775), Flavobacterium suaveolens (F1avobacterium su-
aveolens) (IFO-3752), Alcaligenes faecalis (Alcal igenes f.
aecal is) (IFO-12669).

Among the microorganisms listed above, those with ■FO numbers are
Bacteria that have been deposited with the Fermentation Research Institute, and are not marked with an asterisk, are included in the List of Cultures, 1962, 3rd edition, published by the Institute, and the 1975 Supplement to the Fermentation Edition ( In5titude Fo
r Fermentation, 0saka, Li5
tof Cultures 1962 Th1rd E
dition, 1972 Fi-fth Edition
n, 1975 Supplement to the
Fifth Edition).

Those with FERM-P numbers have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Among the above microorganisms, those not listed (
The mycological properties of those marked with an asterisk (*) are listed below.

Mycological properties of Serratia marsetuscens FO-12626 Morphological observation Image short culm-like motile Yes, periflagellated sporulation No Durham staining Negative physiological properties Optimum culture temperature 25-30°C Oxygen requirement Aerobic lidmus milk Acidic Gelatin decomposition Yes Hydrogen sulfide production No Indole production No Nitrate reduction Yes Oxidase No Catalase Yes Urease No VP reaction Positive sugar fermentability Arabinose No Glucose Yes Inositol Yes Glycerol Yes Cellobiose Yes Trehalose Yes Sucrose Yes Dulcitol No Mannitol Yes Maltose Yes Lactose None Assimilation of related carbon sources Citric acid Yes Gluconic acid Acetic acid Yes When culturing microorganisms in the method of the present invention, among the nutrients added to the medium, methanol or ethanol is used as the main carbon source. It will be done.

In this case, a mixture of methanol and ethanol may be used.

Various mixing ratios from 1:9 to 9:1 are used.

The amount of methanol and/or ethanol added is 0.1 to 10, more preferably 0.5 to 50
It is 7o.

If the growth of bacteria is inhibited by adding desired methanol and/or ethanol to the medium at once at the start of culture, it is desirable to add methanol and/or ethanol to the medium as appropriate as the bacteria grow.

In the present invention, when culturing a bacterial strain, methanol or ethanol is used as a carbon source added to the medium.

Nitrogen sources include organic nitrogen sources such as corn steep liquor, cottonseed meal, yeast extract, dried yeast, fishmeal, meat extract, peptone, and casamino acids, as well as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate,
Ammonium phosphate, sodium nitrate, aqueous ammonia,
Inorganic nitrogen sources such as ammonia gas are used.

In addition to these carbon sources and nitrogen sources, various metal ions, vitamins, amino acids, etc. necessary for the growth of the microorganisms used are added as appropriate.

Cultivation is carried out under aerobic conditions such as shaking or submerged culture with aeration and stirring.

The culture temperature is usually selected from a range of 20°C to 45°C that is suitable for the growth of the strain used.

Moreover, the pH of the medium is preferably in the range of 5 to 9.

Hydrochloric acid, sulfuric acid, aqueous ammonia, ammonia gas, sodium hydroxide, calcium carbonate, slaked lime, etc. may be added as appropriate to maintain the pH during culturing in the optimum pH range.

D-fructose is usually accumulated in the medium after culturing for about 2 to 5 days.

In order to collect the D-fructose accumulated in this way, commonly used sugar purification methods are used, such as filtration or centrifugation of the culture solution to remove bacterial cells, followed by treatment with activated carbon or ion exchange resin. D-fructose can be easily collected by methods such as removing impurities, concentrating it, adding a hydrophilic organic solvent such as ethanol, and crystallizing it.

The present invention will be explained in more detail below using Examples, but the content of the present invention is not limited thereby.

Example 1 Dispense sterilized sorbitol 0.5%, peptone 1.0%, yeast extract 0.2 parts into a 200 ml Erlenmeyer flask,
One platinum loop of Pseudomonas aeruginosa I F 013275 cells was inoculated into 10 mJ of a medium containing 0.2% sodium chloride, cultured at 28'C for 1 day, and 1 ml of this was dispensed into a 2001111 Erlenmeyer flask with sterilized ethanol. 1.0% dry yeast, 2.0% dry yeast, 0.5% ammonium sulfate, and 2.0% calcium carbonate. When the cells were cultured for an additional 24 hours, 5.3 μ/ml of D-fructose was accumulated.

The cultured solution 11 was filtered to remove bacterial cells, the tear fluid was applied to a cation and anion exchange resin column, and the passed solution was decolorized with activated carbon, concentrated, and ethanol was added to obtain 2.9 g of crystalline D-fructose.

Example 2 Acetobacter aurantium IFO-3248 was cultured in the same manner as in Example 1 except that methanol was used instead of ethanol.
/IIIA? of D-fructose accumulated.

This cultured solution 11 was treated in the same manner as in Example 1 to obtain crystals D.
- Obtained 2.7 g of fructose.

Example 3 When the strains shown in Table 1 were cultured in the same manner as in Examples 1 and 2, the accumulated amounts of D-fructose shown in Table 1 were observed in each strain.

Claims (1)

[Claims] 1 Pseudomonas, Acetobacter, Aeromonas, Gluconobacter, Brevibacterium, Bacillus, Serratia, Arthrobacter, Agrobacterium, Chromobacterium, Sartina, Sporosarcina, Micrococcus, Coryne Culture microorganisms belonging to the genus Bacterium, Cellulomonas, Flavobacterium, or alkaline earth metals that have the ability to assimilate methanol or ethanol and produce D-fructose in a medium containing methanol or ethanol as the main carbon source. and accumulate D-fructose in the medium,
A method for producing D-fructose, which comprises collecting the D-fructose.