RU2044772C1 - Process for preparing rennin - Google Patents

Abstract

FIELD: biotechnology, cheese making and medicine. SUBSTANCE: a process for preparing a rennin comprises grinding rennins of calves and lambs, extracting the rennin three times with a sodium chloride solution, separating the liquid phase, filter in the solution, concentrating the rennin, filtering the concentrate or drying. The raw material includes preserved rennins of calves and lambs which are treated with a 5-12% sodium chloride solution with the addition of 0.1-3% sodium benzoate, stirring the latter for 10-30 minutes every hour for 4 to 12 hours and lowering the pH of the solution to 4.0-5.8, extracting the rennin for 12 to 24 hours at 4-20 C. In this case the ratio of the raw material and the solution is 1:(6-30), repeating the extraction operation three times, the third extraction being carried out at the ratio of the raw material and the solution of 1:(3-15). After filtration operation the rennin is concentrated by ultrafiltration, the concentrate is then filtered and batched in the liquid state or dried. The ultrafiltrate is reused as an extracting liquid at an appropriate correction of the pH and a percentage of the preservative. EFFECT: more efficient preparation process. 2 cl, 1 tbl

Description

 The invention relates to methods for producing rennet from the abomasum of calves and dairy lambs and can be used in the production of milk-clotting preparations for cheese making in the dairy industry, as well as in medicine.

A method of producing powder from rennet Sychugov calves and lambs [1] comprising carrying out a triple estraktsii enzyme 10% solution of sodium chloride, dried shredded Sychugov calves, lambs at 2 ^to 4 ° C for 60-72 ^C. The extract was decanted, filtered through a suction filter, hydrochloric acid is added to a pH of 1.5-2.0, and when the salt is added to a concentration of 20%, the enzyme is salted out. After settling for 20-24 hours, the effluent is collected, excess moisture is pressed out and dried in a calorifer or freeze dryer. The dried enzyme is crushed, sieved, normalized and mixed with a filler (sodium chloride).

 The disadvantage of this method is: prolonged extraction of the enzyme for 60-72 hours, which can lead to undesirable development of psychrophilic microflora, and the salting out process is time-consuming and unstable, depends on the parameters of the process (temperature, pH, concentration of sodium chloride, etc.) and accompanied by a loss of enzyme. Losses are also observed when drying the enzyme efflorescence. It should also be noted that after salting out, the mother liquor remains, which is a strongly acidic 20% sodium chloride solution. This solution is not used further and is discharged into sewage, which is environmentally unsafe. To achieve the MPC level for chlorine ion, the necessary dilution factor is 125. When the rennet is salted out, not only its transition to an insoluble state is noted, but also activation (conversion of pepsinogen to pepsin, prorennin to rennin), accompanied by a partial loss of pepsin and rennin activity, since the outlines of these components of the rennet are different (the outflow of pepsin at pH 1.0, and rennin 4.6), and salting out occurs at pH 1.5-2.0.

 An object of the invention is to increase the yield of the enzyme and improve its quality.

To this end, the proposed method provides for the extraction of rennet from raw materials [crushed, dried or otherwise preserved abomasum of calves and dairy lambs (chilled, frozen, etc.)] 5-12% sodium chloride solution with the addition of a preservative of 0.1 -3.0% sodium benzoate for 4-12 hours with stirring for the first 10-30 minutes every hour, then lower the pH from 4.0 to 5.8 by adding hydrochloric acid, mix and continue extraction for 12-20 hours at 1-8 ^o C. The ratio of raw materials and liquids from 1: 6 to 1:30. The extraction is repeated three times, after each extraction, the liquid part is drained, filtered, separated, concentrated by ultrafiltration, the concentrate is purified by filtration and the liquid is packaged or dried.

 A comparative analysis of the proposed method with the prototype shows that the proposed method differs from the known one in that the extraction is carried out in a rennet favorable for stability of the rennet, consisting of two components (rennin and pepsin), pH 4.0-5.8. In this pH zone, the enzyme is activated during the extraction process (conversion of the precursors of these enzymes prorennin and pepsinogen to rennin and pepsin), which allows it to be concentrated by ultrafiltration immediately after purification.

 Another difference is that the process is conducted in the presence of a preservative (0.1-3.0% sodium benzoate), which inhibits the growth of psychrophilic microflora, and the extract, purified from most of the bacteria and ballast substances, is concentrated by ultrafiltration, ultrafiltrate is reused as extracting solution, and the concentrate after repeated filtration is packaged in a container in liquid form or dried.

 This set of features allows to increase the rennet yield by 10-20% to improve the quality of the enzyme (reduce the amount of insoluble residue and fat, reduce bacterial contamination of the product).

 PRI me R 1. Chilled or defrosted after freezing abomasum of calves or dairy lambs weighing 10 kg is ground in a spinning top with a lattice diameter of 5 mm

The resulting raw material is poured into 60 l of 10% sodium chloride (1: 6), 900-1000 g (1.5%) of sodium benzoate are added, stirred for the first 10 minutes of each hour for 6 hours, then concentrated hydrochloric acid is added with stirring (density 1.19), reducing the pH to 5.0-5.5, continue extraction without stirring for 18 hours at a temperature of the extractant -4 ^about C.

 After this, the extract is poured off, the same procedure is repeated twice with the remaining raw materials, and the third extraction is carried out by halving the amount of extracting solution (30 l of 10% sodium chloride).

 The liquid transparent part of the extracts is drained by siphoning, separated from suspended particles by separation at 5-10 thousand rpm, then the extract is filtered through special filtering materials, fed to a filter press and filtered through cardboard of the KO brand or filter plates of the F-1, KFO type and etc.

 The purified extract is fed by a feed pump to an ultrafiltration unit (a flat membrane module using polysulfone membranes on dacron and polypropylene substrates with a pore diameter of 200-300 angstroms or separation apparatus on hollow fibers of the type AP-2.0, etc.) and is concentrated in 30-200 time. After all the extract is fed into the installation, the concentrate is displaced into the last circuit by ultrafiltrate. The resulting concentrate is normalized to a predetermined activity, a preservative is added, it is fed to a filter press and filtered through filter plates of the brands F-1, KFO, SF-1, etc., packaged in liquid form into 450-500 ml bottles. , cork stoppers and metal caps.

To carry out the lyophilization in the concentrate is added to 25% sodium chloride to the volume dispensed on a layer of 6-15 mm trays, frozen frozen layer to a temperature of from -40 to ^-25 ° C and the dry film is not higher than 40 ^o C to achieve a different points of the product temperature from 30 to 35 ^about C. The dry concentrate is crushed in a ball mill and sieved through a sieve.

 Normalization (bringing to standard activity) of liquid rennet is carried out by adding concentrate, and dry mixing with a filler dry, crushed, sifted salt in a mixer.

Collected during the ultrafiltration to the vessel with a water jacket (water temperature ^about 4-20 C) the filtrate is recycled. For this purpose, a filtrate (about 36 L) is added to the abomasum for the second extraction, and the remaining part (14 L) is made up with 12% NaCl with the addition of 1.5% preservative; during the third extraction, these volumes are half as much. The consumption of abomasum per 1 liter of liquid rennet (activity for coagulation of milk 100 thousand units) amounted to 16 pcs.

PRI me R 2. Dry abomasum of calves in the amount of 10 pcs (400 g) is crushed on a plane with a width of 8 mm, pour 12 l of a 12% solution of sodium chloride (1: 6) (pH 6.3), add 180 g of sodium benzoate (1.5%), stirred for the first 12 hours, add concentrated hydrochloric acid (density 1.19) to a pH of 4.0, then stand for 10-12 hours, the process is carried out at a temperature of 20 ^about The clear extract is siphoned and the same procedure is repeated twice. Further, the process is carried out as in example 1. An increase in the concentration of sodium chloride of more than 12% does not increase the efficiency of extraction.

 Abomasum consumption per 1 liter of liquid rennet (milk coagulation activity 100 thousand units) 19 dried abomasums.

PRI me R 3. Chilled abomasum of milkweed calves are ground in a spinning top (raw material weight 5.0 kg). The resulting feed was poured into 30 L of 8% sodium chloride (1: 6), 0.1% sodium benzoate (300 g) was added, stirred for 20 minutes every hour for 4 hours, then the pH was reduced by adding concentrated hydrochloric acid to pH 5.0, allowed to stand for 20 hours. by adding a preservative in an amount of less than 0.1% is marked growth of microflora in the extraction temperature from 4 to 20 ° ^C. Further, the resulting extract was treated as in example 1.

 The consumption of chilled calves abomasum per 1 liter of liquid rennet (100 thousand activity) is 18 pcs per 1 kg of dry (100 thousand units) 18.5 pcs.

 PRI me R 4. Frozen and crushed abomasum of calves are crushed in a top (1 kg). The resulting raw material is poured into 6 l of 6% sodium chloride (1: 6) and add (600 g) 3% sodium benzoate, and then the extract is processed as in example 3. Adding a preservative in an amount of more than 3% is economically disadvantageous.

 The consumption of frozen calves abomasum per 1 liter of liquid rennet (act. 100 TUE) is 16 pcs., 16.5 pcs. Per 1 kg of dry rennet.

PRI me R 5. Frozen and chilled abomasum calves are crushed in a top (1 kg). The resulting raw material is poured with 10 L of 8% sodium chloride (1: 10) and 3% of sodium benzoate is added, stirred for the first 30 minutes of each hour for 12 hours, then the pH is reduced to 5.8 and settled for 12 hours. The extraction is carried out at 6 ^about C, the procedure is repeated twice. Next, the extract is processed as in example 1.

 The consumption of frozen calf's abomasum per 1 liter of liquid rennet (100 TUU) is 16 pcs. per 1 kg of dry 20 pcs.

 PRI me R 6. Obtaining rennet is carried out in accordance with example 1, but without stirring during the extraction with 12% sodium chloride.

 A sharp deterioration in the extraction process was noted and the abomasum consumption amounted to 30 pcs. per 1 liter of liquid rennet (activity 100 TUE).

 PRI me R 7. Obtaining rennet is carried out in accordance with example 1, but the extraction is carried out four times, and the fourth extraction is carried out as a third (30 l of 10% sodium chloride).

 It was found that the fourth extraction does not significantly affect the yield. The consumption of abomasum per 1 liter of liquid enzyme was 16 pcs.

 Technical characteristics of rennet obtained by the proposed and known methods are shown in the table.

 The proposed method for producing rennet allows improving the quality of the finished product by reducing the amount of insoluble residue to 0.2-0.5% in the finished product and increasing the yield by 5-25% compared to the traditional one.

 An analysis of the results of the experiments in examples 1-5 shows that a decrease in pH to 4-5.8 affects the increase in extraction efficiency, as a result of which, on the one hand, partial denaturation of ballast substances extracted from the abomasum occurs, on the other hand, native enzymes of abomasum tissues are activated (cathepsin and other tissue proteinases), while conditions are created for the transition of the precursors of the components of the rennet preparation (pepsinogen and rennin) from the tissues to the solution. When the abomasum is extracted with stirring for less than 4 hours, the extraction efficiency decreases, and the increase in exposure for more than 12 hours does not lead to a significant increase in the number of proenzymes in the extract.

 Lowering the extraction pH to 4-5.8 allows not only the further extraction process in the stability zone of the enzyme, but also creates the conditions for the gradual conversion of proenzymes (prorennin and pepsinogen) into active enzymes (rennin and pepsin). This is especially true for rennin, since its content is 60-90% in rennet.

 With a decrease in pH less than 4.0, rennin formed from prorennin enters the unstable zone and activity is partially lost; at pH more than 5.8, proenzymes do not transform into their active forms.

 When the ratio of raw materials and extracting liquid 1: (6-30), the loss of the enzyme is reduced. With a ratio of raw materials and liquid less than 1: 6, losses can be 10-15%, and with a ratio of more than 1: 3, losses during further processing of raw materials during separation, filtration, and ultrafiltration significantly increase due to the continuous processing of large volumes of extract. In addition, when the ratio of raw materials to liquid is less than 1: 6, the phase separation process slows down, and with a decrease of 1:30, the separation time increases.

Adding preservative (sodium benzoate) in an amount of from 0.1 to 3% by volume of the extract allows the process at a temperature of from 4 to 20 ^C. When the concentration of sodium benzoate is less than 0.1% an increase of undesirable bacterial growth in the extract, and increasing concentrations of a preservative of more than 3% does not significantly suppress the growth of microflora and is economically disadvantageous.

 In order to improve the quality of the enzyme and increase the yield after separation of the extract from the residues of the abomasum, it is purified by separation and filtration, concentrated further by ultrafiltration, the purified concentrate is used in liquid form or dried in a freeze dryer.

 The proposed method allows to reduce the consumption of sodium chloride by 2 times and the acid consumption by 5-10 times, solves environmental issues, since the filtrate obtained at the ultrafiltration stage is reused as extracting liquid.

 The method for producing rennet from the abomasum of calves and lambs was tested under experimental conditions of NPO Kompleks.

Claims (2)

1. A METHOD FOR PRODUCING COLUMN ENZYME, which involves grinding the abomasum of calves and lamb milkers, extracting the enzyme three times with a solution of sodium chloride, separating the liquid phase, filtering the solution, concentrating the enzyme, characterized in that canned calves and lamb milkers are used as raw materials. treated with a 5-12% solution of sodium chloride with the addition of 0.1-3% sodium benzoate, stirring the mixture for 10-30 minutes every hour for 4-12 hours, and then reduce the pH of the solution to 4.0-5.8, extract the enzyme for 12-24 hours at 4-20 ^o C, while the ratio of raw material and solution is 1: 6-30, the extraction is repeated three times, and the third extraction is carried out at a ratio of raw material and solution 1: 3-15, after filtration, the solution is concentrated by ultrafiltration , then the concentrate is filtered and packaged in liquid or dried.  2. The method according to claim 1, characterized in that the ultrafiltrate is reused as extracting liquid with an appropriate correction of pH and the percentage of preservative.

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