JPS6255080A - Production of pectinase preparation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a pectin-degrading enzyme preparation derived from a mutant of black mold. This preparation is used for extracts from the pulp of fruits and vegetables and to clarify their juices.

[Prior art and its problems]

The method for producing a pectin-degrading enzyme preparation obtained by culturing the black mold fungus Fantegun is well known, and a culture solution with an activity of 1500 U/male can be obtained (Bulgarian Patent No. 16862).

However, this method has the disadvantages that the activity level of the culture solution is insufficient and the optimum temperature for the reaction is too high at 45°C. The latter reason has the undesirable result of increased costs in manufacturing and using the formulation.

[Means for solving problems]

An object of this invention is to provide a method for producing a heat-stable pectinolytic enzyme. According to this method, large-scale cultivation is possible and products with high enzyme activity can be obtained. This purpose is
Black mold 8MX-41 (National Institute for Drug Control Kl)
K. (deposited as No. 8 on February 28, 1984) was cultured and fermented. This black mold MX-4I
Conidia of Mold 8M were naturally selected, irradiated with ultraviolet rays, hydrofluoric acidized under laboratory conditions, and sieved using existing enzyme activity measurement methods.

After culturing on agar containing malt for 72 hours, black mold JMX-
41 forms round colonies. The edge of the o=- is smooth, with a concave center and sharp radial folds. The diameter of the yellow-brown center is 1.73mm, and the width of the white part around it is 1.2mm. After 7 days the colony diameter reaches 9 cst K. The color is black. The center continues to be depressed, and the radial folds become more pronounced. The culture does not secrete pigment into its medium. The other side of the colony is a light cream color.

After culturing for 72 hours on potato agar, 8P black mold
/DC-41 forms spherical colonies. The central part is 0.5 cm in diameter and pale yellow, the next layer is 2.2 as wide and black, and the surface has three white layers. The diameter of the Habo7 caliber colony reaches 7 mm. The light colored center remains the same size, but the black layer increases by 2.5
The white layer on the surface becomes 0.53 cm.

On Chap*k-Dox agar, spherical colonies with a diameter of 1.1 cm are formed after culturing for 72 hours. The center is white, and the surface is lightly pigmented, giving it a pale cream color. The diameter of the 7-caliber colony reaches 3 to 411 m, with a white center surrounded by a 0.7 mm wide layer of yellow-green color. After that, 2
cI! A black layer of L and a peripheral white layer of 2 to 3 m grow.

In medium IIIK, conidia are formed 250 to 500 microns long and 2 to 3.5 microns thick. Its end is spherical, 1.5 to 2.2 microns in diameter, and dark brown to black in color.

On solid media, 8MX-41 forms colonies with well-bubbly air micelles and micelles made of putrid mycelia.

Cultures may also be grown on slanted agar plates containing 6-dry weight malt extract.

Comparing the culture product of Black Mold 1MX-4I with that of the conventional Black Mold 8M, there is a difference in that the enzyme activity of the former is several times higher. After 144 hours from the start of culture in the medium with the best composition, a culture with polygalacrodase activity of 4000 ± 300 U and pectin esterase activity of 20 ± 4 U accumulates in the fermentation solution ld in the former, but in the latter. Then they are 1500±20U and 4±0.5U, respectively.

After 24 passages, it is fermented in a medium with the following composition:

O Dried beet pieces 4.5 nai 5.5'no wheat bran 0.7 to 1.59 G0 Ammonium sulfate 0.35 to 0.75% Sodium dihydrogen phosphate 0.125 to 0.250 '40 Starch
0.5 to 1.5-0 magnesium sulfate
0.025 to 0.0540 Potassium Chloride 0
．． After removing 0.025 to 0.05% micelles, the fermentation liquid was filtered to remove impurities and ultrafiltrated to remove low molecular weight soluble mixtures. This resulted in a 7 to 9 fold reduction in volume and an increase in dry matter from 1.2 to 1.5 inches to 1.75 to 2 inches. Thereafter, vacuum J compression was repeated eight times at a temperature of 36°C or lower, and vacuum drying was performed at a temperature of 38°C or lower.

Comparing the current method and the method of this invention, the latter has
The formulation has the advantages of high thermal stability (optimum temperature 50°C), 3 times higher polygalacturodase activity, and 5 times higher pectin esterase activity. According to this method, the cost required for the manufacturing process is reduced, and a preparation with high purifying activity can be produced. As a result, the time required for R-detinylation is shortened even when the consumption of the formulation is low.

〔Example〕

Composition: Dried beet pieces 4.5 cm/Wheat bran
0.75tiammonium sulfate
0.35 Sodium dihydrogen phosphate 0.1
25%・Starch 0.5
%・Magnesium sulfate 0.025%・Potassium chloride 0.025% Fermentation medium was sterilized and cooled to 27℃, and black mold ljM
1.5j of the 24 hour load of X-41 was added. This inoculum was prepared in a medium identical to the fermentation medium composition described above. For fermentation in a continuous fermenter, the surface speed of the stirrer is 264
to 27.5±0. while stirring at 3.2 m/s.
The test was carried out at 5°C for 144±8 hours. At this time, 1 fraction of culture medium
! 0.8 to 1.1j of sterile air was pumped into the tank. At the end of the process, polygalactulodase activity 400
0±300U and pectin esterase activity 20±4
U has accumulated. After removing micelles and insoluble components in the medium, filter with fine filter paper and
A fermented liquid was obtained. Next, this was concentrated 8 times by ultrafiltration, then vacuum concentration was repeated 8 times at a temperature below 38°C, and finally vacuum drying was carried out at a temperature below 38°C. thus,
Polygalactulodase specific activity 400,000 U/II
, a preparation 300-350I with a pectin esterase specific activity of 2000 U/l was obtained. After commercialization with 4Sf lactulodase specific activity of 60,000 U/7 and pectin esterase specific activity of 3000/7, approximately 2F products were obtained.

Claims (2)

[Claims] (1) Black mold (Aspergillus niger)
8, dated February 28, 1984, to the National Agency for Pharmaceutical Control, regarding the method for producing enzyme preparations by mass culture using one strain of Van Tighen.
Pectin, which is characterized by culturing the black mold mutant 8MX-4I, deposited as No. A method for producing a degrading enzyme. (2) The method according to claim 1, wherein the fermentation medium comprises the following components:・Dried beet pieces 4.5 to 5.5% ・Wheat bran 0.7 to 1.5% ・Ammonium sulfate 0.35 to 0.75% ・Sodium dihydrogen phosphate 0.125 to 0.25
0% ・Starch 0.5 to 1.5% ・Magnesium sulfate 0.025 to 0.05% ・Potassium chloride 0.025 to 0.05%