JPH0419836B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing sophorose using microorganisms. [Prior art] Production promoting substance for cellulase production by microorganisms,
Sophorose, which is useful as a cosmetic raw material, was first discovered as a sugar component of campherol glycosides in the fruit pods of Sophora japonica L., but it is also obtained in nature as a free disaccharide. is rare and only a small amount is known to exist in royal jelly. Regarding microbial sophorose production,
The bacterium Acetobacter was identified by Khan et al.
Nothing is known except that a small amount of sophorose was produced in studies of oligosaccharide production by microorganisms belonging to the genus [AWKhan et al., Nature, 183 , 682
(1959), TKWalker et al., Arch.Bio-Chem.
Biophys., 83 , 161 (1959)]. [Problems to be Solved by the Invention] Under these circumstances, an urgent search for sophorose-producing microorganisms has been desired. [Means for Solving the Problem] As a result of a wide search for microorganisms capable of producing sophorose, the present inventor discovered that some microorganisms belonging to the yeast genus Torulopsis have such an ability. , completed the invention. That is, the present invention provides a novel method for producing sophorose using a microorganism, which is characterized by culturing a sophorose-producing bacterium belonging to the yeast genus Torulopsis, producing and accumulating sophorose in a medium, and collecting the same. The microorganism used in the present invention belongs to the genus Torulopsis and has the ability to produce sophorose, such as Torulopsis bombicola.
bombicola) KSM-36, ATCC-22214, same
PRL123-64, NRRL/Y-1445N, etc., especially Trilopsis bombicola KSM
-36 is preferred. Torulopsis bombicola KSM-36 was isolated by the present inventor from a cabbage leaf in a cabbage field in Musashino City, Tokyo, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7586, and is described below. It has the mycological properties of (a) Growth status on each medium The growth status on wort medium, wort agar medium, potato-dextrose agar medium, and cornmeal agar medium is oval to oblong yeast, (0.75 to 3.0) × It had a size of 1.5 to 5.5) μ, multipolar budding, and no formation of true hyphae or pseudohyphae was observed. (b) Formation of ascospores As a result of examining the presence or absence of ascospore formation using a malt extract agar medium, no ascospore formation was observed. (c) Formation of extruded spores As a result of culturing on a wort agar medium and examining the presence or absence of extruded spores, no formation of extruded spores was observed. (d) Physiological properties Growth conditions PH3-8 (optimal 5-6) Temperature 7-38℃ (optimum 25-34℃) Assimilation of nitrate - Decomposition of fat + Decomposition of urea - Liquefaction of gelatin - Sodium chloride tolerance 6 ~8% (w/v) carotenoid production - organic acid production in Custer's medium
+ Production of starch-like substances − Requirement for vitamins + (requirement for biotin and thiamine) (e) Presence of assimilation and fermentability

【table】

[Effect]

The strain belonging to the genus Torulopsis used in the present invention has the ability to produce sophorose as shown in Examples below. [Effects of the Invention] As mentioned above, only one example of the ability of microorganisms to produce sophorose is known, and only a small amount of sophorose production has been observed.
In contrast, the present invention is based on the new finding that bacterial strains belonging to the genus Torulopsis have a high ability to produce sophorose, and enables the production of sophorose using microorganisms. [Example] Next, the present invention will be described with reference to Reference Examples and Examples. Reference Example 1 () 10 g of cabbage leaves from a cabbage field on the outskirts of Musashino City, Tokyo were placed in a beaker containing 30 ml of sterile water and suspended and dispersed using a magnetic stirrer for 10 minutes. After standing still, 10 ml of the supernatant was collected and inoculated into a 500 ml Sakaguchi flask containing 100 ml of a liquid separation medium having the following composition, and cultured with shaking at 30° C. and 120 strokes/min for 7 days. This operation was repeated to perform enrichment culture four times. (Composition of liquid medium for separation) n-hexadecane 100ml Ammonium nitrate 25g Potassium dihydrogen phosphate 10g Magnesium nitrate heptahydrate 5.0g Potassium chloride 5.0g Yeast extract 1.0g Tap water 1000ml PH4.5 () Next, the accumulation of each stage Culture solution 0.5 from medium solution
ml was collected and applied uniformly with a Conradie stick to the surface of a separation agar medium prepared by the following method, then covered with paper impregnated with n-exadecane, sealed with sellotape, and cultured at 30°C. . (Preparation of agar medium for separation) Agar was added to the liquid medium for separation in an amount of 2.5% by weight based on the liquid medium, and then sterilized by steam. A fixed amount of this was dispensed into a sterilized petri dish while keeping it warm to prepare a flat agar medium. () Next, one platinum loop of the colony grown by the above culture was diluted 100 times with sterilized water, and 0.1 ml of this diluted solution was applied again to an agar medium with the same composition as the above-mentioned isolation agar medium, and similarly n- The cells were covered with hexadecane-impregnated paper and cultured at 30° C. for 3 days while supplying n-hexadecane. It was confirmed by macroscopic and microscopic observation that the multiple colonies produced were not different from each other. Furthermore, 10 of the above colonies were each inoculated onto a slanted agar medium having the same composition as the isolation agar medium, and cultured at 30°C for 3 days while supplying n-hexadecane using paper impregnated with n-hexadecane. . We reconfirmed that the bacterial strains on the 10 slanted media were macroscopically and microscopically the same strains, and also confirmed that the properties and physiological properties of these 10 bacterial strains on each medium were the same.
The properties and physiological properties of the above strains on each medium are as described above. As a result of the above test, it was found that each of the 10 cultured bacteria was a single strain isolated from nature. () Next, a loopful of the pure cultured bacterial strain on the slant medium is suspended in a cryopreservation vial containing a sterilized 10% glycerin aqueous solution (2 ml) and stored frozen at -80°C. Thus 3
After several months of cryopreservation, a loopful of the suspension obtained by rapid thawing was resuscitated on an ordinary agar medium, and the properties and physiological properties were examined on each medium under the same conditions as described above. No change was observed. Furthermore, as a result of similarly examining the properties and physiological properties on each culture medium of the strains subjected to the above-mentioned freezing and thawing process repeated five times every month, no changes were observed. Example 1 () 500 ml of seed medium (PH6.2) containing sterilized glucose 5% by weight/volume (hereinafter referred to as w/v%) and corn staple liquor 2w/v%
A 5-volume culture Erlenmeyer flask containing 500 ml of culture medium with the same composition was placed in a 500-ml Sakaguchi flask at 30°C.
50 ml of Torulopsis bombicola KSM-36 culture solution obtained by culturing for 48 hours was inoculated and cultured with shaking at 30°C for 48 hours. Next, 300ml of this culture solution was
Balm oil 15w/v% - Glucose 10w/v% -
This medium containing 0.5w/v% yeast extract (PH
5.6) Inoculate a 30 volume jar fermenter containing 15, temperature: 30℃, ventilation: 0.5VVM,
Culture was carried out for 5 days under the conditions of internal pressure: 0.75 Kg/cm ² and stirring: 350 rpm. () Pour about 1.5 mL of the culture solution into a phase separation tube with an inner diameter of 15 cm and a length of 100 cm and a heat-insulating jacket with a conical tip, and stir while blowing air out from the bottom.
After heating at 70°C for 30 minutes, ventilation was stopped and the culture solution was allowed to settle for 30 minutes. At this time, the culture solution is separated into four phases. After the lowest sedimentation phase and the bacterial cell phase above it were gently removed from the lower nozzle, the aqueous phase corresponding to the upper phase (the second phase from the top at the time of phase separation) was gently collected. () Next, take the collected aqueous phase 1 and perform an extraction operation with 500 ml of a chloroform-methanol mixture (2:1) in a separating funnel if necessary to remove chloroform-methanol soluble substances in the aqueous phase. . Then, the aqueous phase of this extraction treatment was heated at 5°C.
Centrifugation was performed at 3500 rpm for 30 minutes to obtain a supernatant. This supernatant liquid was concentrated under reduced pressure using a rotary evaporator on a 35°C hot bath to remove mixed methanol and the like. Furthermore, in order to remove ionic substances from about 50 ml of the obtained concentrate, a cation exchange resin and an anion exchange resin were added thereto, and after stirring for 2 hours, the ion exchange resin was washed away. () A total of 320 ml of this deionized water phase was packed into an activated carbon column. Note that the activated carbon column has an inner diameter of
Prepared by filling a 2.5 cm x 60 cm long glass column with 180 ml of activated carbon (activated carbon for chromatography, manufactured by Wako Pure Chemical Industries, Ltd.), pouring 1000 ml of a 4N hydrochloric acid aqueous solution, and then pouring 400 ml of distilled water. I used the one I made. In the separation operation using this activated carbon column, 1000 ml of distilled water was first run as the eluent.
Next, 2500 ml of 5% ethanol aqueous solution, 1000 ml of 10% ethanol aqueous solution, 1000 ml of 20% ethanol aqueous solution
Stepwise elution was carried out in the order of ml, and finally, 1000 ml of a 50% ethanol aqueous solution was applied to complete the elution. The eluate was fractionated in a 100 g weight fraction/fraction using a large flank collector. The separated fractions were analyzed using thin layer chromatography, gas chromatography, gas chromatography (GC-MS), etc., and were separated into fractions No. 11 to No. 40. The substance was confirmed to be sophorose (Figure 1). The amount of sophorose obtained under the above fermentation conditions was about 30 g per fermentation liquid. Example 2 Into test tubes containing 10 ml of seed medium (glucose 5 w/v % - yeast extract 0.5 w/v %), one platinum loopful of each of fresh Torulopsis bombicola species (Table 1) grown on YM slant was added. After each inoculation, the cells were cultured at 30°C for 48 hours. Next, 1 ml of this culture solution was inoculated into a Sakaguchi flask containing 50 ml of this medium containing 10 w/v% glucose, 10 w/v% safflower oil, and 0.5 w/v% yeast extract.
Fermentation was carried out for 10 days at 120 strokes/min. 5 ml of this fermented liquid was collected, centrifuged and filtered to obtain a clear fermented liquid, the entire amount was put into a 20 ml sample bottle, and freeze-vacuum dried to form a powder.
The weight was measured. Next, 10 mg of this powder was accurately weighed into a 5 ml vial, 3 mg of trifluoroacetic acid and 0.5 ml of formamide were added thereto, and dissolved uniformly.Finally, trifluoroacetic anhydride was added and the vial was tightly stoppered. It was left at room temperature for a minute. A certain amount of this reaction solution was subjected to gas chromatography analysis to quantitatively analyze sophorose. Gas chromatography analysis: 3%
The test was carried out using Silicone SE-52-Chromosorb W80/100 mesh (manufactured by Shimadzu Corporation) packed in a 2 mmφ x 4 feet glass column. The column temperature was maintained at 100°C for the first 5 minutes.
Then, after increasing the temperature at a rate of 10℃/min,
It was maintained at ℃ for 2 minutes. Helium was used as the carrier gas, and the flow rate was 30 ml/min. A hydrogen flame detector was used for detection. The sophorose production amount of each Torulopsis bombicola species is shown in Table 1.

【table】 [Brief explanation of drawings]

FIG. 1 is an elution curve showing the results of column chromatography in Example 1.

Claims (1)

[Claims] 1. A method for producing sophorose, which comprises culturing a sophorose-producing bacterium belonging to the genus Torulopsis to produce and accumulate sophorose, and collecting the same.