RU2429291C1 - Digestive agent of microbial enzymes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: digestive agent represents a complex enzyme preparation containing protease produced by Bacillus licheniformis bacteria, alpha amylase produced by Bacillus amyloliquefaciens bacteria and lipase produced by Yarrowia lipolytica yeast. The ration (activity unit) is specified to be equal to 0.055:0.85:1 respectively.

EFFECT: extended range of digestive agents of high pharmacological activity of microbial enzymes.

6 tbl, 8 ex

Description

The invention relates to microbiology and medicine, and relates to multienzyme drugs used to treat digestive disorders.

Significant interest in the practical use of enzymes in medicine is due to the important role they play in biological processes, in particular in digestion in humans and animals. Food breakdown occurs with the help of pancreatic enzymes with lipolytic (lipase), proteolytic (protease) and amylolytic (alpha-amylase) activities.

Digestive disorders of varying severity are found in almost all diseases of the gastrointestinal tract (GIT), primarily the pancreas (pancreas), the main source of digestive enzymes, as well as in disorders of the stomach, various intestines, gall bladder, and a number of thyroid diseases glands, aids, etc.

Enzyme preparations from various sources are widely used to compensate for the inherent insufficiency of digestive enzymes in humans (replacement therapy). The use of digestive enzyme preparations is not limited to compensating for secretory insufficiency. Such drugs are also indicated for healthy people to improve digestion.

Modern animal products based on pancreatin (a drug from the pancreas of pigs and cattle) contain protease, alpha-amylase and lipase and are microgranules or tablets coated with enteric coating. They are indicated for use in exocrine pancreatic insufficiency: chronic pancreatitis, cystic fibrosis; violation of food digestion to improve the digestion of food in people with normal gastrointestinal function in case of nutrition errors (eating fatty foods, large amounts of food, irregular nutrition) and with masticatory function disorders, a sedentary lifestyle, and prolonged immobilization.

Currently, such pancreatin-based drugs as Mezim, Panzim, Panzinorm, Pancreatinum (various manufacturers), Festal, Creon, etc. are widely used in medical applications.

The main drawback of all pancreatin-based drugs is their animal origin. The emergence of new diseases (prion and viral diseases), transmitted from farm animals to humans, has led to the fact that drugs based on pancreatin no longer meet the safety requirement. In addition, pancreatin contains a mixture of enzymes in a certain ratio, depending on the composition of the feedstock. However, for the treatment of various diseases, enzyme preparations with a different ratio of protease, alpha-amylase and lipase activity are necessary.

An alternative to pancreatin-based drugs is individual enzyme preparations derived from microorganisms. They allow you to create drugs with the necessary ratio of individual enzymes, optimal for the treatment of various cases of digestive disorders. But most importantly, the use of enzyme preparations of microbial origin excludes the possibility of infection with zoonoses for humans.

Known composition for the prevention and treatment of digestive disorders in humans, containing enzymes from fungi - lipase from Rhizopus delemar, a protease from Aspergillus melleus and amylase from Aspergillus oryzae (RF 2003124078).

To treat pancreatic insufficiency, it was proposed (RF 2007117728 - the closest analogue) to use a composition containing bacterial Pseudomonas or Burkholderia lipase, Aspergillus melleus fungal protease, and Aspergillus oryzae fungal amylase.

The objective of the invention is to expand the arsenal of digestive agents based on enzymes of microbial origin.

The problem is solved in that a digestive agent is proposed, which is a complex enzyme preparation, including a protease, alpha-amylase and lipase of microbial origin, characterized in that the protease used by the bacteria Bacillus licheniformis is used as a protease, amylase is used as alpha-amylase, produced by the bacteria Bacillus amyloliquefaciens, and the lipase used is the lipase produced by the yeast Yarrowia lipolytica, in the following ratio of components (unit of activity): protease: al a-amylase: lipase 0.055: 0.85: 1.

Example 1. Obtaining a lipase using a yeast strain Yarrowia lipolytica VKPM Y-3260

1. Microbiological synthesis

Cells are grown at 30 ° C for 48 hours in test tubes on a YPD agarized beveled medium of the following composition (wt.%): Peptone - 1, yeast extract - 1, glucose - 2, agar - 2, water - the rest.

To prepare the seed, the culture is washed with sterile water at the rate of 10 ml per tube and inoculated into 750 ml Erlenmeyer flasks containing 50 ml of YPD liquid medium, at the rate of 2 ml of suspension per 1 flask and grown on a circular shaker with constant stirring (240 rpm) min) at 28-30 ° C, pH 6.5-6.8, for 48 hours

The fermentation process is carried out in an Applikon laboratory fermenter (Donau Trading AG, Switzerland) with a volume of 3 L with an initial medium content of 1.2 L for 120 hours, the aeration intensity is 1.5 rpm × min, and the stirring speed is 700 rpm. The amount of seed is 10%. The composition of the fermentation medium (wt.%): Peptone - 2; yeast extract - 2; glucose - 0.3; potassium phosphate monosubstituted - 1.32; ammonium sulfate - 0.5; potassium phosphate disubstituted - 0.23; magnesium sulfate - 0.05; calcium chloride - 0.01; sodium chloride - 0.01; olive oil - 5.0; Tween 40 detergent - 0.5 g; a solution of vitamins -1 ml / l; trace element solution - 1 ml / l; water - the rest, pH - 6.8-7.0. The composition of the solution of vitamins (in mg / 100 ml): para-aminobenzoic acid (PABA) - 20; biotin - 2; riboflavin - 20; thiamine - 40. The composition of the trace element solution (in mg / 100 ml): boric acid - 50; copper sulfate - 40; potassium iodide - 10; ferric chloride - 20; sodium molybdenum acid - 20; manganese sulfate - 40; zinc sulfate - 40. After 4.5 hours after inoculation, the fermenter is fed with a 50% glycerol solution. The feed rate until the end of the first day is 40 ml / h, then to the end of the cultivation process 27.5 ml / h.

Lipase activity is determined by the titrimetric method for the hydrolysis of olive oil [European Pharmacopoeia 6.0, 2007-2008, European Directorate for the Quality of Medicines & HealthCare]. The activity values are given in units of F.I.P.

By the end of fermentation (total time of 120 hours), the lipolytic activity of the culture fluid is 35,000 U / ml.

2. Isolation of lipase

After fermentation, the culture fluid is centrifuged at 6 ° C and 7000 rpm for 30 minutes and the supernatant is subjected to tangential type ultrafiltration on a membrane with a cut-off pore size of 30 kDa (the process is carried out at 4 ° C). A five-fold volume of a 3 mM calcium chloride solution is added to the resulting concentrate and again concentrated to the initial volume (100 ± 10) ml. This procedure is repeated once more, thus achieving a total washing degree of 25 times.

The concentrate is lyophilized dried and ground in a laboratory mill for 1.5 minutes until smooth. The specific activity of the obtained lipase substance is 2400 U / mg.

Table 1 shows the lipolytic activity of lipase at various pH values at a temperature of 37 ° C. From the presented results it follows that the maximum activity of lipase is at pH 7.0-8.0.

Table 1 pH Activity,% four 0.00 5 0.00 6 0.54 7 30.73 8 100.00 9 0.00

Example 2. Obtaining a lipase using a yeast strain Yarrowia lipolytica VKPM Y-3323

Obtaining a lipase using a yeast strain Yarrowia lipolytica VKPM Y-3323 and its isolation are carried out according to example 1.

The seed preparation time is 24 hours, the fermentation time is 72 hours, the lipolytic activity of the culture fluid at the end of the fermentation is 37,000 U / ml.

The specific activity of the obtained lipase substance is 2400 U / mg.

Example 3. Obtaining a protease using a strain of Bacillus licheniformis VKPM B-10288

1. Microbiological synthesis

Cells are grown at 30 ° C for 48 hours in test tubes on a LB agarized agar medium of the following composition (wt.%): Peptone - 1, yeast extract - 0.5, glucose - 0.2, agar - 1.5, water - rest.

To prepare the seed, the culture is washed off with sterile water at the rate of 10 ml per tube, seeded in 750 ml Erlenmeyer flasks containing 50 ml of liquid seed medium of the following composition (wt.%): Phytopepton - 1, yeast extract - 1, sodium chloride - 0 , 25, maltose - 2, water - the rest, at the rate of 2 ml of suspension per 1 flask and grown on a circular shaker with constant stirring (240 rpm) at 28-30 ° C, pH 6.5-6.8 for 48 h

The fermentation process is carried out in a laboratory fermenter Marubishi MDL150 (Marubishi Bioengineering Co. LTD, Japan) with a volume of 1 liter with an initial content of 400 ml for 44 hours, the cultivation temperature is 37 ° C, the aeration intensity is 1.0 rpm × min, speed stirring 700 rpm The seed volume is 5%.

The composition of the fermentation medium (wt.%): Phytopepton - 1, yeast extract - 1, corn flour - 2, soy flour - 2, sodium chloride - 5, calcium chloride - 1, glucose - 7, water - the rest, pH before sterilization 7 , 7-7.8 (adjusting the pH with a 10% NaOH solution), pH after sterilization of 6.8-6.9.

Protease activity is determined using the F.I.P method [European Pharmacopoeia 6.0, 2007-2008, European Directorate for the Quality of Medicines & HealthCare]. The activity values are given in units of F.I.P.

By the end of fermentation, the protease activity of the culture fluid is 120 U / ml.

2. Isolation of protease

After fermentation, the culture fluid obtained in example 3 is centrifuged at 6 ° C and 10,000 rpm for 30 minutes The QL centrifuge is subjected to microfiltration in a tangential flow through a 0.2 μm membrane cartridge, and then to a tangential type ultrafiltration using a membrane with a cut-off pore size of 10 kDa (the process is carried out at 4 ° C). The resulting concentrate is freeze-dried and ground in a laboratory mill for 1.5 minutes until smooth. The specific activity of the obtained protease is 4.5 U / mg.

Table 2 shows the proteolytic activity of the protease at various pH values at a temperature of 37 ° C. From the presented results it follows that the maximum activity of lipase is at pH 7.0-9.0.

table 2 pH Activity,% 4.0 twenty 5,0 40 6.0 70 7.0 80 8.0 one hundred 9.0 90

Example 4. Obtaining a protease using a strain of Bacillus licheniformis VKPM B-9608

Obtaining a protease using a strain of Bacillus licheniformis VKPM B-9608 and its isolation are carried out according to example 3.

The activity of the culture fluid at the end of the fermentation is 50 U / ml. The specific activity of the obtained substance of the enzyme is 4.5 U / mg.

Example 5. Obtaining alpha-amylase using a strain of bacteria Bacillus amyloliquefaciens VKPM B-5144

1. Microbiological synthesis

As a producer of alpha-amylase, a bacterial strain Bacillus amyloliquefaciens VKPM B-5144 is used.

Cells are grown at 37 ° C for 72 hours in test tubes on an agarized LB agar medium with erythromycin (pH 7.3) of the following composition (wt.%): Tryptone - 1.0, yeast extract - 0.5, sodium chloride - 0 5, erythromycin - 0.001, agar - 1.5, water - the rest.

To prepare the seed, the culture is first sown from the test tube using a microbiological loop on Petri dishes with a mowed agarized seed medium (pH 7.4) of the following composition (wt.%): Tryptone - 1.0, yeast extract - 1.0, sodium chloride - 0.25, erythromycin - 0.001, agar - 1.5, water - the rest, and grown at a temperature of 37 ° C for 20 hours. The resulting culture using a microbiological loop at the rate of two loops inoculate Erlenmeyer flasks with a volume of 750 ml, containing 100 ml of inoculum growth medium (pH 7.4) as follows its composition (wt.%): tryptone - 1.0, yeast extract - 0.5, sodium chloride - 0.25, D-maltose - 2.0, water - the rest, and grown on a circular shaker with constant stirring (240 rpm) at a temperature of 37 ° C for 8 hours

The fermentation process is carried out in a laboratory fermenter Marubishi MDL 100 (Marubishi Bioengineering Co. Ltd) with a volume of 1 l, with an initial medium content of 380 ml, for 72 hours in the following mode: temperature - 37 ° C; aeration intensity - 1 r / (r × min); mixing - 700 rpm; pH 7.0-7.4; the oxygen content in the medium is not less than 10% of saturation. If necessary, adjust by increasing the speed of the mixer (up to 900 rpm). The volume of the inoculum is 5%. The composition of the fermentation medium (wt.%): Potato starch hydrolyzate - 10.0, corn extract - 1.25, disubstituted ammonium phosphate - 1.213, baked pressed yeast - 1.38, calcium chloride - 0.115, potassium sulfate - 0.43, magnesium sulphate heptahydrate - 0.022, copper pentahydrate - 0.00058, sodium chloride - 0.043, lactose - 0.087, water - the rest. Baked pressed yeast is pre-boiled in 70 ml of distilled water in a boiling water bath for 30 minutes and then added to the remaining components of the medium. Corn extract and potato starch hydrolyzate are prepared and sterilized separately from other components of the medium and added directly to the fermenter. In the fermentation process, an additional supply of a 50% starch hydrolyzate solution is carried out: 50 ml are added to the fermenter at 18 hours of cultivation, 30 ml at 20 hours and 80 ml are added at 23 hours.

Alpha amylase activity is determined using the iodometric titration method [European Pharmacopoeia 6.0, 2007-2008, European Directorate for the Quality of Medicines & HealthCare]. The activity values are given in units of F.I.P.

At the end of fermentation, the amylolytic activity of the culture fluid is 1200 U / ml.

2. Isolation of alpha-amylase

At the end of the fermentation, the culture fluid obtained in Example 5 is centrifuged at 6 ° C and 10,000 rpm for 30 minutes.

The supernatant is subjected to microfiltration in a tangential flow through a 0.2 μm membrane cartridge, and then to a tangential type ultrafiltration using a membrane with a cut-off pore size of 10 kDa (the process is carried out at 4 ° C). The resulting concentrate is freeze-dried and ground in a laboratory mill for 1.5 minutes until smooth.

The specific activity of the obtained alpha-amylase is 200 U / mg.

Table 3 shows the amylolytic activity of alpha-amylase at various pH values at a temperature of 37 ° C. From the presented results it follows that the maximum activity of lipase is at pH 6.5-6.7.

Table 3 pH Activity,% 4.0 0 5,0 twenty 6.0 90 6.5 one hundred 6.7 one hundred 7.0 90 8.0 thirty 9.0 twenty

Example 6. Obtaining alpha-amylase using a strain of bacteria Bacillus amyloliquefaciens VKPM B-10291

Obtaining alpha-amylase using a strain of Bacillus amyloliquefaciens VKPM B-10291 and its isolation are carried out according to example 5.

The activity of the culture fluid at the end of the fermentation is 1380 U / ml. The specific activity of the isolated enzyme is 200 PIECES / mg.

Example 7. Evaluation of the pharmacological activity of the claimed digestive agent in in vivo experiments on rats

The pharmacological activity of both individual enzymes and the entire composition was evaluated in in vivo experiments on non-linear white rats (weight 160-180 g, age 13-14 weeks).

To study the action of enzymes, models were selected that allow you to have a pronounced pharmacological effect of the action of enzymes, namely, the model of food load with carbohydrates, proteins and fats.

Studies were carried out with the enzymes obtained in examples 1, 3 and 5, made in the form of pellets, which are microspheres coated with an enteric coating.

The animals were prescribed a diet saturated with fats, carbohydrates and proteins. Experimental animals consumed a homogeneous feed of the following composition:

casein 35% sunflower oil mixed with pork fat 40% starch fifteen% agar 2% salts, vitamins four% cholic acid one% thiouracil one% cholesterol 2%

In addition, rats were additionally intragastrically injected with a 10% oil emulsion of pork fat at a dose of 100 mg / kg and minced raw beef at a dose of 500 mg / kg. Similar feeding was carried out for three days.

Compared complex enzyme preparation and comparison product "Creon" was administered intragastrically at a dose of 100 mg / kg on the fourth day one hour before the introduction of pork fat and minced meat on the background of a food load. In 1 gram the pellet of the used comparison drug Creon 10000, the protease content is 2750 IU, amylase - 42710 IU, lipase - 50,060 IU. With a pellet mass of 250 mg in the capsule of the drug, 150 mg per pancreatin content. The remaining 100 mg in pellets, dosed in 1 capsule of Creon 10000, constitute excipients. Thus, a dose of 100 mg / kg Creon pellet corresponds to the administration of 67 mg / kg of pancreatin to animals. In terms of the activity of individual enzymes, this dose is 275 IU / kg of protease, 4271 IU / kg of amylase and 5006 IU / kg of lipase.

For animal testing, the pellets of the test enzymes of the lipase, protease, and alpha-amylase obtained in Examples 1, 3, and 5, respectively, were mixed in order to obtain a preparation with a similar composition in terms of the enzyme activity ratio: protease 275 U / kg, amylase - 4271 U / kg, lipases - 5006 U / kg (ratio of components (unit of activity): protease: alpha-amylase: lipase 0.055: 0.85: 1). In this case, the dose for administration to animals was 144 mg / kg per total weight and 60.1% per weight of enzymes.

The intact group of rats received the usual vivar diet. The control group of rats was administered solvent. Each group included 10 animals of the same sex (males weighing 180 g).

During the fourth day of the experiment, the experimental and control animals were individually landed in the exchange cages of the Italian company Tecniplast Gazzada for 24 hours to collect excrement (feces). Moreover, in feces, the amount of neutral fat, the presence of fatty acids, soaps, the amount of protein, the presence of fibers and pH were determined by conventional methods. The experimental results are presented in table 4.

As can be seen from table 4, in the group of control intact animals that received the usual vivar nutrition, there is a daily excretion of feces at the level of 8.4 g. The excretion of fat is at the level of 1.6 mg / t. Products of incomplete digestion of fats, such as fatty acids, and soaps are absent or their quantities are at the limit of sensitivity of analysis methods. The reaction of the stool pH is neutral - 7.2.

Table 4 The effect of the compared drugs on the coprogram of white rats Experimental group The total amount of feces, g The content of neutral fat in feces, mg / g The presence of fatty acids, ± The presence of soap, ± The content of soluble protein in feces, mg / g Muscle fibers, qty in sight The reaction, unit pH Intact 8.4 ± 0.1 1.6 ± 0.2 - - 27 ± 6 2 ± 1 7.1 ± 0.1 Control without treatment 13.2 ± 0.2 44 ± 11 +++ +++ 136 ± 15 27 ± 7 8.2 ± 0.1 The claimed treatment 9.2 ± 0.3 1.9 ± 0.2 - + 36 ± 8 6 ± 1 7.2 ± 0.1 Treatment with Creon 8.9 ± 0.2 1.4 ± 0.1 - + 28 ± 6 4 ± 1 7.1 ± 0.1

The group of control animals with a food load of fats and proteins has pronounced differences with a 27-fold increase in the concentration of fats and 5-fold protein in feces and an increase in their volume, and the pH of the medium rises. Thus, a pronounced digestion is observed due to the lack of digesting activity of the secrets of the stomach and intestines.

When animals are prescribed a complex drug and a Creon comparison drug in equally effective doses, digestion indicators return to their original state. The differences between animals treated with the preparations and intact animals are within the statistical error and are not significant. A certain increase in the content of salts of fatty acids (soap) at the level of one plus, apparently, can be considered as a failure of the absorption process under conditions of overloading by products of fat digestion.

Example 8. Evaluation of the pharmacological activity of the claimed digestive agent in in vivo experiments on dogs

To assess the pharmacological activity used peletirovanny complex preparation consisting of pellet enzymes obtained in examples 2, 4 and 6.

In the experiments used 6 dogs weighing 18-22 kg (2 dogs were in the experimental group and 4 in the control group) to simulate pancreatitis. Pancreatitis was caused by the administration of a trypsin solution at a dose of 0.2 mg / kg body weight into the pancreatic duct and subsequent ligation of the duct. Pelletized complex preparation was prescribed in capsules at a dose of 600 PIECES of protease, 8000 PIECES of amylase and 10000 PIECES of lipase (ratio of components (unit of activity): protease: alpha-amylase: lipase 0.060: 0.8: 1) twice a day before meals and after 4 hours. The feces were collected, weighed, dried in an oven at 60 ° C for a day. Then it was ground and sieved in order to free it from hair, sawdust and sand. The protein content in feces was determined by the Kjeldahl method. This method is widely used to evaluate food products with the calculation of the amount of protein according to the content of amine nitrogen in materials. The presence in the stool of neutral fat, fatty acids and starch was determined by conventional methods.

In the postoperative period and during the experiment, the body weight of the dogs remained virtually unchanged. All animals completely ate the food they left. The nature of the chair was fairly constant. Feces are usually dense, decorated. Macroscopically undigested muscle fibers were determined only in the control group during the first 2 weeks. The experimental results are presented in table 5.

Table 5 The effect of the drug on the coprogram in control and experimental dogs with subacute pancreatitis Study time, weeks Protein in feces (M ± m) The presence of starch, ± The presence of neutral fat, ± The presence of fatty acids, ± Counter. Experience Counter. Experience Counter. Experience Counter. Experience Before surgery 265.7 ± 13.8 254.0 ± 11.4 - - - - - - one 335.4 ± 14.8 265.6 ± 18.8 ++ - +++ - +++ - 2 332.4 ± 48.8 225.2 ± 14.1 ++ - +++ - +++ - 3 291.6 ± 16.8 200.0 ± 10.7 + - ++ - ++ - four 225.0 ± 12.3 218.7 ± 20.7 - - ++ - ++ -

As can be seen from the table, in the control group of dogs there was a significant increase in the amount of protein in the feces (~ 30% relative to the background) during the first 2 weeks. Stool, fatty acids and neutral fat were also tested in feces. Then the protein and starch content decreased, approaching the initial level. The presence of neutral fat and fatty acids was tested in the feces of the control group of dogs throughout the experiment. During the entire experiment, the amount of protein in the experimental group did not exceed the initial level (except for a statistically unreliable increase in the first week) and was even lower than the background figures. When comparing the experimental and control groups, it is seen that within 1, 2 and 3 weeks there are significant differences in the protein content in the feces. In the control, the amount of protein exceeded its level in the experimental group by 30-48%. Also, in the experimental group throughout the experiment, starch and products of incomplete digestion of fats, neutral fat and fatty acids were absent or their quantity was at the limit of sensitivity of analysis methods. A histological examination confirmed the presence of subacute pancreatitis in all operated dogs.

The enzyme-compensating effect of the claimed drug is clearly shown in case of digestive disorders and deficiency of pancreatic enzymes.

Thus, the claimed composition based on enzymes of microbial origin in pharmacological activity is comparable with the well-known animal drug "Creon". The optimum action of the enzymes included in the composition is in the region of neutral pH values.

Claims (1)

A digestive agent, which is a complex enzyme preparation, including a protease, alpha-amylase and lipase of microbial origin, characterized in that it contains a protease produced by the bacteria Bacillus licheniformis, and as an alpha-amylase, the amylase produced by the bacteria Bacillus amensoliquef as a lipase, the lipase produced by the yeast Yarrowia lipolytica in the following ratio of components (unit of activity): protease: amylase: lipase = 0.055: 0.85: 1.