CS245213B1 - Process for producing microbial rennet - Google Patents

Abstract

The invention solves the problem of a method of producing microbial rennet for the needs of the dairy industry, the essence of which is the use of the mold strain Rhizomucor sp. MX CCF 1863 (Mezinár. federation of collections No. 182), which is stored in the Collection of Mold and Fungal Cultures, Department of Botany of Lower Plants, Charles University, Prague 2, Benátská 2, and at the same time a method of isolating the enzyme. The higher technical effect lies in the replacement of the deficient classical rennet from calf stomachs.

Description

The invention relates to a method for producing microbial rennet produced by the newly isolated and described fungal strain Rhizomucor sp. MX (CCP 1863).

The lack of natural sources of classical rennets, especially of the chymosin type (EC 3.4.23.4), has led to the search for a suitable substitute among proteolytic enzymes produced by various microorganisms. So far, industrial production of microbial rennets has been carried out most often using the fungi Rhizoma miehei (Cooney and Emerson) Schipper, Endothia parasitica and Rhizomucor pusillus (Lindt) Schipper.

The disadvantage of rennet from the fungus Endothia parasitica is its relatively low thermal stability, since at room temperature there is a 40% loss of enzyme activity within 10 hours, the low stability range of the enzyme in relation to pH and the difficulty of purification due to the simultaneously produced toxins and antibiotics in the fermentation medium. The disadvantage of using the fungus Rhizomucor pusillus for rennet production lies in the low production of the enzyme under the conditions of conventional submerged cultivation in a stirred fermenter or in culture flasks on a shaking machine. M.A.A. SCHIPPER (Studies in Mycology, No 17, p.61, Centraalbureau voor Schimmelcultures, Baam, Dec. 1978) with reference to other authors states that the yield of rennet enzyme from Rhizomucor pusillus is at most 4% of the yields achieved when using the fungus Rhizomucor miehei.

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The above-mentioned shortcomings are eliminated by the method according to the invention, the essence of which is the discovery that using the newly isolated and below described strain of the fungus Rhizomucor sp.MX No. CCP 1863, a large amount of rennet enzyme is obtained, which has a relatively low non-specific proteolytic and lipolytic activity in addition to a high curdling (coagulating) activity, see Table 1., whereby the production strain according to the invention is cultivated on a suitable nutrient medium

- 2,245,213 environment and the produced enzyme is isolated.

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Tab·1 Enzyme activity of the fungal rennet according to the invention in comparison with the commercial preparation Renniías NOVO

Production strain

Rhizomucor sp. MX No.GGP 1863

Rhizomucor miehei

Preparation designation

MX batch 3

Rennie's

NEW

Curd (coagulation) activity ^x SJ.ml"'

45,714

58,400

Nonspecific proteolytic activity «J.ml ^T

290

310

Political activity

154

286 ^xxx J.ml- ¹

Soxhlet units. The measure of curdling (coagulating) activity, given in Tables 1 and 3, is the amount of enzyme that _coagulates 1 ml of milk at a temperature of 35 °C in 40 minutes. _π -1 M (ml) 2400 (s) , _n n

SJ· ¹ “ ¹ = B· rice) * ¹⁰⁰⁰

M = ml of milk; E = amount of enzyme; T = time of onset of milk coagulation azocasein unit = 1 jig of digested azocasein. h”’' at 37 °C units relative to milliequivalents of digested substrate (tributyrin)

The fungus strain Rhizomucor sp. MX was isolated from potato tubers infected with rot and deposited in the Collection of Fungal and Fungal Cultures (Inventory of the Federation of Collections No. 182), Department of Botany of Lower Plants, Charles University Prague 2, Benátská 2, under No. GGP 18£3.

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The strain Rhizomucor sp. MX CCF 1863 used can be characterized as follows:

when cultivated on wort agar at 37 °C, colonies 1<<3 πΛ· high are formed, initially gray-blue, later brown-gray to dark gray. Rhizoids poorly developed, sporangiophores branched,

5.7 to 9.0 µm thick. Sporangia (45-50 µm) with encrusted wall cracking at maturity, columella obovate, sometimes pear-shaped (30-35 µm), sporangiospores broadly oval, mostly almost spherical, 2.9 to 3.9 µm in diameter. Zygospores not seen, stolons 10 to 20 µm thick. Azygospores not found either. Grows well and sporulates at 30 G, growth is rapid at 37 G. The strain used according to the invention differs from the type culture Rhizomucor pusillus (Lindt) Schipper in the amount of production and thermal stability of rennet enzyme, hydrolysis of casein and size of sporangia. Other features, such as the absence of zygospores, azygospores, growth independence from thlamin, width of stolons, size of culmela, growth on sucrose, prevent the strain according to the invention from being classified in the group of the species Rhizomucor miehei (Cooney et Emerson) Schipper. A summary of the features is given in Table 2.

The method according to the invention is further illustrated in the following examples of fermentation and isolation of the novel microbial rennet, without limiting the scope of protection by these examples.

The advantages of the method according to the invention lie in the simplicity of implementation, while obtaining an enzyme preparation of unusual purity, with a good yield of rennet (coagulation) activity - see Tab. 3.

Example of storing a production strain.

The culture of Rhizomucor sp. MX (CCF 1863) is maintained on a slant of wort agar or in tubes with sterile potato wedges, filled with 2-3 ml of liquid Czapek-Dox medium. The culture is allowed to grow and sporulate at 30-37 °C, then stored in a refrigerator at a temperature of + 5 °C. Re-inoculation is carried out after 3-6 months.

- 4 Tab.2 Morphological and physiological characteristics of type cultures

Rhizomucor pusillus and Rhizorauoor miehei in relation to the characteristics of the culture of Rhizomucor sp. MX (COP 1863)

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Morphological and physiological characteristics Rhizomucor pusillus (Lindt) Schipper Rhizomucor miehei (Cooney and Emer- son) Schipper Rhizomucor sp.MX CCF 1863 Colony Height 1 (<2) mm 1mm 1 3mm stolon width •until 5pm until 9 pm 1Qrf20 pm rhizoids poorly developed poorly developed poorly developed sporangiophores (size) until 11nx(l5)pm until 7 pm 5.7«9.0 pm sporangia (diameter) up to 80«a((100)pm until 5O(4'6o)pm 45^50 pm columella up to 45x38 um rarely larger up to 28x25 pm rarely larger 30x35 cm sporangia spores 3»4 years 3c£4pm 2.9áÉ3.9 pm zygospores - + - thiamine-dependence - + - +/casein hydrolysis ( + ) + +/sucrose as C-source + + Formation of rennet enzyme under standard/.' cultivation /KUNITZ Rennet Units.l“'/ USA Pat. 1 ,108.287 ^{out of} 300 * 10000 11.428 I i Thermal stability of rennet enzyme 4· + ^ Q 15 /65 C 10% activity 15*/65°C 50% activity i 15,765 °C 53% activity

+ H.J. SCHOLLER, E. MÜLLER : Path.Microbiol. £2 (1966) 730<i741

M.A.A. SCHIPPER.: Studies in Mycology, No 17-, Dec. 1978, Baam, pp. 53<7O ++ G.A. SOMKUTI, F.J. BABEL: J.Bacteriol. £9 (1968) 1407«1414

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Example of inoculum preparation 1

The inoculum for the 1st stage of submerged fermentation (100 l) is prepared by placing potato cubes, prepared from carefully washed, peeled potatoes, into 1 l wide-necked Erlenmeyer flasks and moistening them with Czapek-Dox medium.

Sterilization 30 min. at 121 °C in an autoclave. After sterilization, the flasks are inoculated with a spore suspension and cultured for 34 5 days at 3Q«37 ^O C.

Example of inoculum preparation 2

Preparation of inoculum by submerged cultivation on a rotary shaker in 500 ml boiling flasks containing 100 ml of nutrient medium, with the following composition:

soy flour 1% potato flour 1% barley groats 1% molasses pellets (60% dry matter) 1%

KH ₂ P0 ₄ 0.5%

CaCO^ 0.5%

For preparation, tap water about 50 °C warm is used, in which 0.1% bacterial amylase (B.subtilis) is dissolved. After about 30-45 min. hydrolysis, the nutrient medium is filled and sterilized for 30 min at 121 C in an autoclave. After inoculation of the cooled flasks with a spore suspension and cultivation for 108 hours, the content of rennet enzyme — 1 in the culture medium increased to 3636 SJ.ml

Example of fermentation in a volume of 100 l. The fermentation is prepared by adding about 100 g of bacterial amylase (B.subtilis-BOLAIÍYLASA - Slovak starch, n.p, Trnava) to 100 l of tap water at 100 °C in a fermenter and adding additional nutrients while stirring:

kg soy flour kg oat flour kg _t malt extract with dry matter 65%

0.5 kg floated CaCO-j

0.3 kg KH ₂ P0 ₄

After 60 minutes of hydrolysis, 25-50 ml of soybean oil is added and the medium is sterilized for 1 hour at 121°C. After cooling the fermenter to 37-40°C, inoculation is carried out with the MX culture on raspberry cubes according to the example above. 500 ml of sterile water is poured into 6 flasks with a well-grown culture in such a way as to prevent dusting of spores. The contents of the 6 flasks are mixed aseptically and transferred to the fermenter. After inoculation, intensive aeration of the medium is set (0.05 min) and, if necessary, defoaming fat is added. The fermentation temperature is maintained at 35°C.

Once maximum enzyme production is reached, fermentation is terminated.

In 1 liter of fermented medium from a laboratory fermenter, the content of rennet enzyme was found to be in the range of 4700 to 6480 SJ _with ffll ¹ and

Rennet enzyme isolation example 1

To isolate the rennet (coagulation) enzyme from the fermentation medium, a batch method of ion exchange with a weak cation exchange resin OSTI0MM (OTICHEM, np Ústí n.lj (Amberlit XE 64), 0.315 mesh, prepared in the H ⁺ cycle is used. 500 ml of wet ion exchanger is added to 2400 ml of the filtrate of the fermentation medium, freed from mycelium.

After 30 min. of stirring the mixture, the pH drops from 5.4 to 3.5 and sorption is terminated. The mother liquor is filtered or removed by decantation, washed once with distilled water and 250 ml of 0.5 mol NaD solution is poured onto the sorbent. The pH is adjusted from 3.0 to 5.5 to 6.0 with a 30% NaOH solution and the mixture is stirred for 30 min. The cation exchange resin is separated by filtration through a filter. Since the enzyme is stable in a wide pH range (3.0 to 7.0), the eluate is adjusted only by adding 0.05 mol CaCl and the pH is adjusted to 4.0 to 4.2 using HG1. The enzyme concentrate is preserved with 10% NaCl and 0.1 to 0.2% sodium benzoate. The results are shown in Table 3.

Rennet enzyme isolation example 2

After the fermentation of the fungus, the nutrient medium is freed from suspensions with mycelium by filtration or centrifugation. While stirring, the pH of the filtrate is reduced from 5.5 to 4.0 using dilute hydrochloric acid. 3 g of cross-linked polyacrylic acid, e.g. Carbopol 934 (GOODRICH), are added to 2400 ml of filtrate (5217 SJ.ml) and stirring is continued. After 30 minutes, the precipitate formed is centrifuged and the supernatant is decanted. The active sediment is resuspended245 213

- 8 is dissolved in 200 ml of distilled water. After complete homogenization, 30 g of floated CaGO^ is added to the solution and the mixture is stirred for another 30 minutes. The formed complex of insoluble calcium salt of polyacrylic acid is removed by filtration or centrifugation. The sour, concentrated enzyme solution is preserved by adding 10% table salt and 0.25% Na-benzoate after reducing the pH value to 4.0 to 4.5 with diluted hydrochloric acid. The resulting concentrate contained 86% of the original curdling (coagulant) activity.

The enzyme can also be separated from the fermentation medium using a DEAE-cellulose sorbent. However, in this case, a larger proportion of the pigment binds to the ion exchanger, so the resulting concentrate contains more impurities. Also, losses due to irreversible binding to DEAE-cellulose are higher than on the Ostion KM sorbent.

Claims (2)

Object of the invention. 245 213 Process for the production of microbial rennet, characterized in that on a nutrient medium containing as a C source 0.5 to 6.0% W, sugars, preferably hydrolyzed starch, 0.2 to 3.1% Ittwt. a source of organic nitrogen substances, preferably malt extract and soy flour, is cultivated by submerged or surface method at 25 to 40 ° C and pH 4.8 to 7.2 of the rhizomes of Rhizomucor sp. MX CG? 1863, and the enzyme produced is isolated. 2. A process according to claim 1, wherein the rennet enzyme is separated from the fermentation broth by ion exchange sorption (exclusion) on a weak cation exchange resin from the ion exchange resin in a batch process or by column chromatography.