PL150227B1 - A method of the industrial culture of microorganisms producing enzymes - Google Patents

Description

PATENT DESCRIPTION

150 227

REPUBLIC

POLAND czv · ćLNlA

Additional patent to patent no. Applied for: 86 06 01 / P. 260882 /

Priority

Patent Office

OFFICE

PATENT

RP

Application announced: θθ 04 28

The patent description was published: 90 07 31

Int. Cl. ⁵ C12N 1/14 C12N 1/20 C12N 9/62

Creators of the invention: Wiesław Rzędowski, Henryk Kluszczyk

The holder of the patent: Institute of Agricultural and Food Biotechnology, Warsaw; Kombinat Rolno-Przemysłowy Igloopol ", Zakłady Rolno-Przemysłowe Pektowin, Jasło / Poland /

THE METHOD OF INDUSTRIAL CULTIVATION OF ENZYME PRODUCING ENZYMES

The present invention relates to a method for the industrial cultivation of enzyme-producing microorganisms in solid media. Enzymes are produced by plants, animals and microbes. Enzyme producing microorganisms are fungi of the genera Aspergillus, Byssochlamys, Chaetomium, Chrysosporium, Cliocladium, Penicillium, Rhizopus, Trichoderms, Trichothecium; actinomycetes, in particular of the genera: Actinomyces, Micromonospora, Nocardia, Streptomyces, Thermoactinomyces, Thermonospora; and bacteria, in particular of the genera: Aeromonas, Arthobacter, Baclllus, Cellulobacillus, Cellulomonas, Cellvibrio, Corynebacterlum, Endothla, Mlcrococcus, Proteus, Pseudomonae, Sporocytophaga, Rumen.

Known and commonly used are tray methods of culturing microorganisms in solid media, in which their growth is carried out in media spread in a layer of several centimeters on trays or transporters located in the growth chamber.

In order to grow the microorganisms in this manner on an industrial scale, the moistened and inoculated medium is placed in a 2-5 cm layer on trays with perforated bottom, and then transported to the growth chambers and distributed on mobile or stationary storied shelves. During the intensive growth of microorganisms, large amounts of heat are removed by means of vigorous air exchange. The cultivation of microorganisms can also take place in vertically positioned cassettes with perforated walls. In these chambers there is a regulator of the amount and temperature of supplied air. The ventilation of the culture occurs through the side perforations of the cassettes. After growth, dry air at 35-42 ° C is passed between the cassettes to dry the culture. The process of obtaining enzyme preparations can also be carried out in a continuous system, for example using a mobile conveyor instead of trays and cassettes. The continuous lines, however, do not provide full containment and good performance.

150 227 * There are also methods of carrying out cultivation on a solid medium in rotary or drum-type agitator reactors. Compared to the tray method, they allow for a significant reduction in the breeding area. The efficiency of the enzyme production process depends on the type of the strain used, the method of preparing the medium and ensuring appropriate cultivation conditions throughout the process.

The object of the invention is to optimize the microbial cultivation process by increasing the thickness of the substrate layer.

Surprisingly, it turned out that the cultivation process of enzyme-producing microorganisms can be intensified by proper conditioning of the cultivation bed arranged in a layer with a thickness of more than 20.

The essence of the invention consists in the fact that the cultivation of enzyme-producing microorganisms is carried out in solid media in a thermally sterilized medium containing products of plant origin with the addition of mineral salts, which is moistened to the content of 50-75% water, the inoculum of the production strain is introduced into it and under full conditions sterility is incubated for several hours; then the substrate prepared in this way is transferred to the breeding chamber, in which it is evenly distributed in a layer with a thickness of more than 20 cm, and the culture of microorganisms is carried out by aerating it with varying intensity in alternating directions, while simultaneously regulating the temperature of the circulating air, its humidity and the content of CO in it ,, wherein the cultivation process is carried out until the peak phase of accumulation of metabolites in the medium is achieved, and an intermediate for the production of enzyme preparations is obtained.

The microbial growth medium includes products of plant origin such as beet pulp, apple pomace, shredded straw, cereal bran, extraction meal, lactose, starch, sawdust and wood shavings. The thoroughly mixed medium is subjected to the process of thermal sterilization, after which it is cooled down to the temperature of 28-40 ° C and the inoculum in the form of spores added in the amount of

Q at least 10 spores per 1 kg of substrate or the germinal biomass in the vegetative phase in an equivalent amount. Then, under the conditions of full sterility, the mixture is incubated for several hours, after which the medium is transferred to the culture chamber with a sieve grate, in which the cultivation process is carried out by aerating the perch with an adjustable flow rate and an adjustable frequency of its up and down changes. The temperature and humidity of the air recirculated in the process of cultivating the air is regulated by sprinkling it with sterile cold water or heating it with steam. After some time, when the permeability of the culture layer drops to a critical level, preventing further efficient thermoregulation of the process, the substrate mechanically crumbles. The following examples illustrate the invention in detail.

Example 1 1000 kg of sterilized medium with the composition in percent by weight; beet pulp - 17.0; wheat bran - 10.0; apple pomace - 2.6;

(NH) 2), B 0 R - 0.4; water - 70.0 was cooled to 37 ° C and inoculated with Aspergillus niger 46 spores added in an amount of at least 10 µ spores per 1 kg of medium, followed by 6 hours preincubation under sterile conditions without aeration at 35 ° C. During the incubation, the inoculated medium was mixed lightly every two hours, then transferred to the culture chamber and distributed in an even layer approximately 35 cm thick. In this chamber, the microorganism cultivation process was continued at 34 ° C, changing the air flow direction through the perch every 7 minutes / alternate aeration from the top and bottom /; CO concentration? in the chamber was kept at the level of about 3%, so that it did not exceed 5%, while before each opening of the chamber, the concentration of CO? in air was lowered to less than 1%.

In the initial stage of growth of filamentous fungi, which lasted 9 hours, aeration intensity was 0.2 - 0.7 m ^ air per 1 m ^{2 of} perches for 1 second; maximum growth phase, which lasted 20 hours, the intensity of aeration was 0.7 - 1.2 m ^ / m ² / sec .; while in the final period, which lasted 30 hours - 0.6-0.8 no / m ² / sec. The total cultivation time was 65 hours. From seven to 62 hours of breeding

150 227 the relative humidity in the growth chamber was maintained at 96%, while during the last four hours of the process at 60%. During the breeding process, the perch overgrown with biomass was subjected to mechanical crushing. The first mechanical crushing of the perch was performed when the substrate was fully covered with mycelium, which took place around the sixteenth hour of cultivation. When the perch re-fused into a compact body - around the 45th hour of breeding, the second meohanic crushing of the perch was carried out. The third crushing of the perch was carried out in the 65th, last hour of breeding, before cooling and drying the perch. By cultivating as described above, 680 kg of crude enzyme preparation with 50% moisture was obtained. This preparation had pectolltic-cellulolytic activity, which, in terms of dry substance, was 30,000 ° PM and 300 µCMC / g. From this crude preparation, the enzyme fraction was isolated by extraction, and the remainder was a high-grade feed additive.

Example II. 1000 kg of sterilized nutrient solution with the composition in percent by weight: wheat bran - 30.0; beet pulp - 12.0; straw chaff - 2.5;

SO. - 0.5; water - 55.0 cooled to 36 ° C and inoculated with AsperQ gillus awamori M-7 spores added in an amount of at least 10 spores per 1 kg of medium, and then preincubated for 8 hours under the same conditions as in the example

I. The substrate was placed in the cultivation chamber in a layer about 30 cm thick and the cultivation process was carried out at 32 ° C, changing the air flow direction every 5 minutes. The CO 2 concentration in the chamber was kept at 3%. In the initial phase of growth of filamentous fungi, which lasted 8 hours, the intensity of aeration was 0.2 -

cities in the last four hours of the process - at 65%.

During the cultivation process, the perch overgrown with biomass was subjected to three mechanical crushing - the first crushing at the 18th hour of cultivation, the second - at the 42nd and the third - at the last 60th hour. By cultivating as described above, 640 kg of crude enzyme preparation with a moisture of 45 $ was obtained. This preparation had a glucoamylase activity, based on dry substance, of 2000 µG / g. The enzyme fraction was isolated from the crude preparation by extraction, and the remainder constituted a high-grade feed additive.

Example III. 1000 kg of sterilized nutrient solution with the composition in percent by weight: wheat bran - 25.0; beet pulp - 10.0; wood trooins - 10.0; / NH ^^ SO ^ ”1.0; water - 54.0 was cooled to 40 ° C and inoculated with Aspergillus niger 7 spores added in the amount of at least 10 µ spores per kilogram of medium and for 10 hours subjected to preincubation in conditions analogous to the example

I. The substrate was placed in the rearing chamber in a layer 35 cm thick and the cultivation process was carried out at 32 ° C, changing the air flow direction through the perch every 5 minutes. The concentration _of C0 in the chamber was maintained at 3% · VN of the initial growth phase, which lasted 5 hours, nawietrzania intensity was 0.2 - 0.5 No / m / sec., Maximum growth phase, which lasted 35 hours, the intensity nawietrzania was

The total cultivation time was 72 hours. From the 11th to the 69th hour of cultivation, 7 / the relative humidity in the chamber was maintained at 95%, while during the last four hours of the process at 68%. During the cultivation process, the perch overgrown with biomass was subjected to mechanical crushing three times - the first crushing at the 22nd hour of cultivation, the second at the 50th hour, the third at the 72nd hour. By cultivating under the conditions described above, 670 kg of crude enzyme preparation with 45% moisture was obtained. This preparation had a cellulolytic activity, calculated as dry substance, amounting to 800 CMC / g. The enzyme fraction was isolated from the crude preparation by extraction, and the remainder constituted a high-grade feed additive.

150 227

Example IV: 1000 kg sterilized media with the composition in percent by weight: wheat bran - 35>0; beet pulp - 9.0; NaCl - 0.5; (NH 2) 2 SO 4 - 0.5; water - 55.0 cooled to 3β ° 0 and inoculated with Aspergillus nlger 41 spores added in the amount of at least 10θ spores per 1 kilogram of medium 1 for six hours was preincubated under the same conditions as in example 1. The medium was mixed in the culture chamber in a layer 30 cm 1 thick, the cultivation process was carried out at 35 ° C, changing the direction of air flow through the perch every 5 minutes.Concentration of C0 ₉ in the chamber was kept at 3%. 4 - 0.9 No / m / sec., the maximum increase in fez, which lasted 28 hours, nawletrzania intensity was 0.9 - 1.8 m? / m ² / eek., while in the final period, which lasted 10 hours - 0.7 -1.4 m ^ / m ² / sec. The total cultivation time lasted 50 hours. From the 7th to the 47th hour of cultivation, the relative humidity of the air in the cultivation chamber was kept at 98%, while in the last four hours of the process at 70%. During the process, the perch was mechanically crushed three times - first crushing at the 16th hour of cultivation, the second at the 42nd hour, the third at the 50th hour. Cultivation under the above-described conditions yielded 650 kg of crude enzyme preparation with 65% moisture. This preparation had a proteolytic activity, calculated as a dry substance of 40,000 µHb / g. The enzyme fraction was separated from the crude preparation by extraction, and the remainder was a high-value feed additive.

Example 5 · 1000 kg sterilized media with the composition in percent by weight: wheat bran - 20.0; beet pulp - 10.0; chaff from wheat straw - 9.8; /? 4/2? 4 water - 60.0; cooled to 35 ° C and seeded with spores

Trichoderma viride 50 in the amount of 10 ^ spores per 1 kg of medium 1 for 12 hours. were preincubated without aeration at 30 ° C. During the incubation period, the inoculated medium was slightly mixed every 3 hours and then transferred to the growth chamber where it was distributed in an even layer of 40 cm. The microbial cultivation process was continued in the chamber at 30 ° C, changing every 10 minutes. the direction of air flow through a · C0 concentration _of the chamber was maintained at 1%. In the initial growth phase, lasting 12 hours, the staggering intensity was 0.2 no / m2 / sec; in the phase of maximum growth lasting 48 hours, the staggering intensity was increased to * 3 0 OO

0.5 no / m / sec ·, while in the end period which lasted 36 hours. 0.5 no./m / sec. The total cultivation time was 96 hours. Air humidity was maintained at 97% throughout the cultivation period. During the cultivation period, the overgrown substrate was subjected to mechanical crushing three times, the first in 26 hours. breeding, the second in 42 and the third in 60 hours. breeding. When culturing as described above, 665 kg of crude enzyme preparation with 44% moisture was obtained. This preparation had a cellulolytic activity of 400 [mu] CMC / g. The enzyme fraction was isolated by extraction from the crude preparation.

Example VI. 1000 kg of sterilized nutrient solution with the composition in percent by weight: wheat bran - 30.0; beet pulp - 10.0; water - 60.0; were cooled to 30 ° C and inoculated with the 2% addition of crushed seed culture Trichothecium roseum 51 and for 12 hours subjected to pre-incubation without aeration and agitation at 28-30 ° C. The inoculated substrate was then transferred to the growth chamber and distributed in an even layer of 30 cm. In this chamber, the cultivation process was continued at 28 ° C, changing every 10 minutes. air flow direction through the layer. The CO 2 concentration and changes in the intensity of replication of the culture were kept at the same intervals as in Example V. The total cultivation time was 96 hours. Air humidity was maintained in the range of 96-98% throughout the cultivation period. Mechanical crushing of the medium overgrown with biomass was carried out twice, i.e. at 36 and 60 hours of cultivation. By carrying out the control as described above, 620 kg of crude enzyme preparation was obtained with a moisture content of 42% and a cellulose activity of 380 [mu] C / g. The enzyme fraction was isolated by extraction from the crude preparation.

Example VII. 1000 kg of sterilized nutrient solution with the composition in percent by weight: beet pulp - 20.0; soybean meal - 2.0; lactose - 4.0; starch - 2.0;

150 227 water - 72.0 cooled to 40 ° C and inoculated with 1% addition of a liquid inocular culture of Bacillus subtilis 2 strain containing more than 10 ¹⁰ viable cells in 1 cm 2 1 for 5 hours. was preincubated without aeration at 36 ° C.

During the incubation, the medium was mixed twice and then transferred to the growth chamber, where it was evenly distributed in the 30 cm layer. The microbial cultivation process was continued in the chamber at 36 ° C, changing the direction of air flow through the layer every 6 minutes. The CO 2 concentration was kept at 2%. During the cultivation period, the cultivation layer was moistened with sterile water every 5-6 hours in order to keep the humidity in the cultivation layer above 70%. During the cultivation layer is ventilated at a constant rate of 0.4 m ^ / m ² / sec. using 3 times mechanical mixing of the perch in 12, 18 and 22 hours. breeding. The culture was stopped after 28 hours. obtaining 920 kg of the preparation with a humidity of 70%. This preparation had an acylolytic activity of 58 [mu] AS / g, and a proteolytic activity of 28,000 [mu] Hb / g. Enzymatic fractions were separated from the crude preparation by extraction.

The method according to the invention results in significant savings in raw materials, labor and electricity. The consumption of the medium for the production of 1 kg of enzyme preparation is about 30%, labor by about 50%, and electricity is about 10% less than in the traditional tray method.

The method of industrial cultivation of microorganisms according to the invention can be successfully used for the production of enzymes from fungi, actinomycetes and bacteria.

The method can also be used for the biosynthesis of other metabolites such as organic acids, proteins, amino acids, vitamins, antibiotics, or for the production of microorganisms for the production of biological plant protection products and animals.

Claims (6)

Patent claims 1. The method of industrial cultivation of enzyme-producing microorganisms in a solid medium containing products of plant origin with the addition of mineral salts, characterized in that the thermally sterilized medium moistened to the content of 50-75% water with the introduced inoculum of the production strain is incubated under full sterility conditions for several hours, then it is transferred to the breeding chamber, where it is evenly distributed in a layer with a thickness of more than 20 cm, and the culture of enzyme-producing microorganisms is carried out by aerating the substrate with variable intensity and in alternating directions, while simultaneously regulating the temperature of the circulating air, its humidity and its CQ content ₂ »and this process is carried out until reaching the peak phase of accumulation of metabolites in the medium. 2. The method according to p. The method of claim 1, characterized in that the aeration process is carried out with an adjustable flow rate through the layer and an adjustable frequency of its upward and downward changes. 3. The method according to p. The method of claim 1, wherein the permeability of the culture layer is reduced to a critical level that prevents further effective thermoregulation of the process, the substrate is crushed and mechanically mixed. 4 · The method according to p. The process as claimed in claim 1, characterized in that the temperature and humidity of the air recirculated in the process are controlled by sprinkling it with sterile cold water or heating it with steam. 5. The method according to Eastrz. The process of claim 1, wherein in the event of excess CO 2 in the recirculated air, more fresh air is automatically supplied to the air-conditioning system. 6. The method according to p. A method as claimed in claim 1, characterized in that the medium comprises such products of vegetable origin as beet pulp, apple pomace, milled straw, grain bran, extraction meal, lactose, starch, sawdust and wood shavings.