LT4541B - Process for preparing medicinal preparate of acidophylic rod - Google Patents

Abstract

This invention relates to the production of bacterial preparations and their use in medicine for treatment purposes. Its aim is to simplify the production method of curative dry acidophilus stick preparations, improve preparation quality, prolong its expiry time and expand possibilities for its use in medicine. The curative dry acidophilus stick preparation production method provides for the preparation of nutritional medium, its cropping, biomass cultivation, chilling, mixing with a protective medium, pouring the liquid mixture into tanks, chilling and lyophilization. The novelty of this method is that to produce the preparation, three Lactobacillus acidophilus K1, K2, K4 stem cultures are used, characterized by antagonistic activity against pathogenic bacteria, cultivated in fat-free sterile milk with (2-3) % of yeast autolysis, the chilled biomass is mixed with the protective medium at a ratio of 2:1 respectively, and the protective medium consists of (7-9) % fat-free milk powder, (2.5-3) % sodium glutamate, (10-12) % glucose, (0.4-0.7)% sodium bicarbonate and distilled water, and the liquid preparation mixture is poured out into (3-5) cm<3> bottles or ampoules; the lyophilised preparation remains active for a period of (12-36) months and may be used in the event of a lack of bacterium treatment and for the prevention thereof.

Description

The present invention relates to the production of bacterial preparations and their use in medicine for therapeutic purposes.

Cultures of Lactobacillus acidophilus are used in the manufacture of therapeutic bacterial preparations for the treatment and prevention of dysbacteriosis.

In the world with Lactobacillus spp. bacterial preparations such as Azicur, Biolactyl, Bacid, Antibiophilus, Omniflora are known. These preparations are expensive and, therefore, in the difficult economic situation of the country, would be difficult to access for the majority of the Lithuanian population. In addition, the preparations should be registered with the Medicines Registration Bureau of the State Medicines Control Agency of the Ministry of Health of the Republic of Lithuania, and clinical trials of the proposed preparations should be performed.

The Lactobacillus acidophilus preparation of the present invention is already being tested in Lithuania. Clinical trials have demonstrated efficacy in the treatment of dysbacteriosis.

The closest to the present invention is the Authorization N1128890, 1980, USSR, A23C9 / 12, C12N1 / 20, A23C21 / 02, "Sposob Prigotoblenia bacterialnova preparation acidofilnich kultur" providing for the preparation of a nutrient medium from milk whey with the addition of a biostimulant and a sulfate salt. additives, medium inoculation with Lactobacillus acidophilus 12b strain culture, neutralization of the medium with gaseous ammonia and addition of glucose and manganese sulphate during biomass cultivation, separation of biomass, mixing with a protective medium consisting of sucrose, gelatin, water, calcium iodide, .

The disadvantages of this preparation method are that a number of chemical additives are used for the preparation of the bacterial preparation and a number of intermediate operations are performed during the cultivation of the biomass, such as nutrient addition, neutralization with gaseous ammonia, separation of biomass which complicates the preparation .

The aim of the invention is to simplify the method of production of the therapeutic dry acidophilic rod preparation by reducing the number of technological operations and selecting the composition of cultivation and protective media with the least amount of chemical additives, improving the quality of the preparation, extending its use. . This objective is achieved as follows.

The aforementioned known preparation method involves preparing a culture medium, seeding a culture, cultivating a biomass, chilling, mixing with a protective medium, pouring the liquid mixture into containers, freezing and lyophilization, and according to the present invention producing a nutrient medium in sterile skimmed milk containing (2-3)% of the total amount of sterile skim milk, and (7-9)% of skimmed milk powder, (2.5-3)% of sodium glutamate, (10-12)% of glucose, (0.4) -0.7)% sodium bicarbonate and mixes the cooled biomass with a protective medium at a ratio of 2: 1, respectively, and uses Lactobacillus acidophilus K1, K2, K4 strain cultures which exhibit antagonistic activity against pathogenic bacteria and inoculate the liquid mixture into low volume packs, for example, in vials or ampoules of (3-5) cm ³ . Each of the distinguishing features of the present invention is closely related to the object pursued.

According to the present invention, the nutrient medium is prepared from sterile skim milk by the addition of (2 to 3)% yeast autolysate. Compared to the prototype, our medium contains fewer components and the composition of the medium ensures the growth of the biomass and no additional technological operations such as nutrient addition, neutralization and biomass separation of the culture medium are required. All this helps to simplify the production method.

The addition of (2-3)% yeast autolysate to sterile skim milk sufficiently enriches the culture medium with group B vitamins and amino acids, which activate bacterial cell development and biomass growth and activity.

Less than 2% of the yeast autolysate supplement, for example 1%, will be insufficient to allow bacterial cells to develop vigorously and remain viable for long periods (see Table 1).

Addition of yeast autolysate more than 3%, for example 4%, is inappropriate, since optimal bacterial cell development is achieved by the addition of (2-3)% yeast autolysate and its higher content does not alter cellular activity and additionally increases the cost of the preparation.

The use of a protective medium according to the present invention comprising (7-9)% skimmed milk powder, (2.5-3)% sodium glutamate, (10-12)% glucose, (0.4-0.7)% sodium bicarbonate provides optimal dry matter content. The content of the preparation helps to maintain a good consistency, facilitates the lyophilization process and results in a good quality preparation that ensures the long-term viability and activity of living Lactobacillus acidophilus cells (see Tables 2, 3).

Less than 7% skimmed milk powder, for example 5%, will not provide sufficient dry matter in the preparation, which complicates the lyophilization process and degrades the consistency and quality of the preparation.

More than 9% of skimmed milk powder, for example 11%, is unsuitable for the manufacture of the preparation, because during autoclaving the protective medium coagulates and is unsuitable for use.

Less than 2.5% sodium glutamate, such as 1%, does not guarantee bacterial cell long-term viability, and greater than 3%, such as 4%, is not appropriate because the optimal effect of increasing cell resistance to lyophilization, injured cell repair and viability is obtained by adding 3% glutamate sodium, and its higher content would increase the cost of the preparation.

Glucose levels below 10%, such as 7%, do not ensure long-term cell survival in the anabiotic state, reduce dry matter content, degrade appearance and consistency. The use of more than 12% glucose, such as 15%, is inappropriate as optimal formulations have already been achieved and higher glucose levels would only increase the cost of preparation.

Using less than 0.4% sodium hydrogencarbonate, for example 0.25%, does not achieve medium neutralization, does not provide the required pH value, and in acidic medium bacterial cells die faster. The use of more than 0.7% sodium bicarbonate, such as 0.9%, would be inappropriate since an excessively high pH value would be obtained and the best pH to maintain the viability of Lactobacillus acidophilus cells is a neutral or slightly acidic pH (see Table 4).

Compared to the prototype, the production of our protective medium is simpler because the use of gelatin in the prototype protective medium complicates the preparation of the medium and the lyophilization process.

According to the present invention, the bacterial biomass is mixed with a protective medium in a ratio of 2: 1, respectively. The above quantitative and qualitative composition of the components of the protective medium, together with the dilution ratio of 2: 1 used, ensures optimal dry matter content, helps to obtain a good homogeneous consistency of the preparation, and ensures long-term activity of the preparation. Mixing the biomass with a protective medium in a ratio of 1: 1 results in excessive dilution, reduces the amount of live bacterial cells per 1g, and can produce an inhomogeneous dry biomass.

Mixing the biomass with a protective medium in a ratio of 3: 1 results in inadequate dry matter content in the preparation and does not ensure long-term cell viability.

When mixed with a protective medium, the 3: 1 biomass remains too viscous, which makes it difficult to dispense a liquid formulation.

In accordance with the present invention, strains of Lactobacitlus acidophilus K1, K2, K4, which exhibit antagonistic activity against the pathogenic bacteria Shigella sonnei, S. flexneri 2a, Salmonella enteritidis, Escherichia coli 018, Staphylococcus aureus and relatively pathogenic bacteria, are used in the production of the present invention. which, together with other distinguishing features of the prototype, helps to extend the application of the product in the treatment of dysbacteriosis of various origins.

Compared to the prototype, the liquid formulation according to the present invention is packaged in low-volume packaging, for example, in vials or ampoules of (3-5) cm ³ . Compared to the prototype, dispensing the liquid formulation into vials avoids the risk of foreign bacterial contamination before and after lyophilization, ensures product quality, and extends shelf life (see Table 5).

Filling less than 3 cm ³ , for example 2 cm ³ , is not beneficial as it results in too little preparation for single use.

It is not expedient to pour more than 5 cm ³ , for example 7 cm ³ , since larger capacities are required and the process of lyophilization of the preparation is complicated and lengthened.

In accordance with the present invention, all distinctive features provide Lactobacillus acidophilus cell activity, minimal cell death, reduce the frequency of deformed and injured cells, stimulate the survival and cell repair of sublethally damaged cells, and ensure purity and homogeneity of the lyophilized biomass resulting in long-term bacterial cell viability 'activity.

Repeated long-term laboratory studies confirmed that the dry acidophilic rod preparation remains viable and active for 12-36 months with a live and active cell content of 100g per 10 ⁹ 10 ¹² .

Clinical trials conducted at the Gastroenterology and Dietetics Clinic of Vilnius University Santariskiu Hospital and the Infectious Disease Clinic of KMA confirmed that the therapeutic dry acidophilic rod preparation of the present invention can be used to treat functional and infectious diseases of the intestine, biocenosis of the natural microflora of the gastrointestinal tract. and regulation.

The results of the clinical trials of the preparation lead to the following conclusions: dry acidophilic rod preparation is well tolerated, has no side effects; no clinically relevant evidence of overdose at higher doses; in patients with dysbiosis with lactic acid bacterial deficiency, bacterial recovery occurs very rapidly in parallel with a change in clinical symptoms; In the case of invasive dysbiosis, the improvement of the condition of the patients, the decrease of complaints, is not always lasting; an increase in bifidobacteria has also been observed with lactic acid bacterial agents; decreased frequency and depth of iatrogenic dysbiosis when administered with appropriate antibiotics; clinical improvement in appetite, decrease in bloating, improvement in intestinal transit.

Based on the clinical-bacteriological examination, doctors recommended dry acidophilic rod preparation for the treatment of dysbiosis, adjustment of antibiotic therapy, treatment of functional bowel diseases as a prophylactic treatment.

Thus, each distinctive feature of the present invention is necessary, essential, and, taken together with the general essential features of the prototype, is sufficient to accomplish its purpose.

The Buddha is used as follows:

example

5 L of sterile milk containing 100 cm ^{3 of} yeast autolysate (2%) is inoculated with 25 cm ^{3 of} Lactobacillus acidophilus K1, K2, K4 cultures. Mix well and cultivate at 37 ° C for 18h. The biomass is cooled to 8 ° C and mixed with 2.55 l of sterile protective medium (2: 1) made up of 2.5 g of distilled water with 175 g of skim milk powder (7%), 65 g of sodium glutamate (2.6%), 250 g of glucose (10). %), 10g of sodium bicarbonate (0.4%). PH of the prepared liquid mixture (mix biomass with protective medium 2: 1, respectively) pH 4.2. Dispense 3 cm ³ into vials, freeze at -45 ° C for at least 24 hours, then lyophilize to + 26 ° C with a residual moisture of

3.5%. Aseptically, the vials containing the dry acidophilic rod are sealed.

The vial lyophilized preparation is a volatile, homogeneous, amorphous or crystalline, white or cream-colored biomass containing 3x10 ¹² - 3x10 ⁹ live and active Lactobacillus acidophilus cells, retained at least 3x10 ⁷ / vial after 12 months storage.

example

5 L of sterile skimmed milk containing 150 cm ^{3 of} yeast autolysate (3%) is inoculated with 25 cm ^{3 of} Lactobacillus acidophilus K1, K2, K4 cultures. Mix well and cultivate at 37 ° C for 18h. The biomass is cooled to 8 ° C and mixed with 2.55 L of sterile protective medium (2: 1) prepared by adding 225g of skimmed milk powder (9%), 75g of glutamate sodium (3%), 300g of glucose to 2.5 L of distilled water. %), 17.5g of sodium bicarbonate (0.7%). The reconstituted liquid product pH 5. Pour in 5 cm ³ in flasks, frozen in -45 ° C which is maintained for at least 24 hours, and lyophilized until the product temperature reaches + 26 ° C, and the residual moisture of 3.5%. Aseptically, the vials are sealed.

The vial lyophilized preparation is a fluffy, homogeneous, amorphous or crystalline, white to white-creamy biomass containing 5x10 ¹² - 5x10 ⁹ live and active Lactobacillus acidophilus cells, retained at least 5x10 ⁷ / vial after 12 months storage.

Influence of yeast autolysate additive on culture medium for the development of Lactobacillus acidophilus

Yesterday No. Composition of culture medium Total bacterial count / Ig biomass Acidity of biomass after 16 h, ° T 1. Sterile skim milk (SP) '9,410 * * · 148 2. SP + 1% yeast autolysate (ma) 2.6-10 ⁸ 452 3. SP + 2% ma 4.2-10 ⁹ 178 4. SP + 3% ma 4.5-10 ⁹ 166 5. SP + 4% ma 4.8-10 ⁹ 172 6th SP + 2% ma + 1% sucrose 3.1-10 ⁹ 176 7th SP + 2% ma + l% sucrose + + 0.01% ZnSO ₄ 7.9-10 ⁸ 156

Note: Microscopic image. In L.acidophus cultured in medium containing (2-4)% yeast autolysate, cells were brighter, more stained, and more adherent to 2-3-4-5-6 cells than control (SP).

Influence of protective media on dry acidophilic rod biomass index (culture medium - sterile skim milk + 2% yeast autolysate; mixing ratio 2: 1)

Come on No. Protective media composition External preparation view Notes 1. K (without guard media) Homogeneous or sheet structure, biomass solid, surface crooked, bounces badly from the wall of the vial Complicated preparation pouring on a large viscosity; transient activity of the preparation 2. H2O + 2.5% glucose + 1.5% Na acetate + 1% gelatin + 0.25% sodium glutamate Homogeneous, friable, slightly uplifting, soft biomass, crushes easily Biomass is difficult to dissolve; inadequate activity storage period 3. H2Ū + 7% skimmed milk powder + 10% glucose + 3% sodium glutamate + 0.4% NaHCO ₃ Puri, homogeneous, amorphous or crystalline, white, white creamy colors lyophilized biomass All indicators characterized by high quality dry acidophilic sticks preparation 4. H2O + 9% skimmed milk powder + 12% glucose + 2.5% glutamate sodium + 0.7% NaHCO ₃ Puri, homogeneous, amorphous or crystalline, white, white creamy colors lyophilized biomass All indicators characterized by high quality dry acidophilic sticks preparation 5. H2O + 11% skimmed milk powder + 12% glucose + 4% glutamate sodium + 0.6% NaHCO ₃ Not suitable for production Autoclave sterilization during which the medium coagulates, improper preparation for production

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Influence of NaHCO3 additive on pH change of protective medium

Composition of protective medium Addition of NaHCCb concentration,% PH of the protective medium (± 0.05) H2O + 7% skim milk 0 6.75 powder + 10% 0.25- 6.9 glucose + 3% Na 0.4 7.1 glutamate + X% 0.7. 7.2 NaHCO ₃ 0.9 7.3 1.0 7.55 1.1 7.8 1.2 7.9 1.3 8.1

Note: By mixing L.acidophilus biomass with a protective medium containing (0.4-0.7)% NaHCCb in a ratio of 2: 1, respectively, the acidity of the dry preparation is reached at pH 4-5.5, which is suitable for the survival of L.acidophilus cells.

table

Influence of Quantitative Ratio of Biomass Mixing with Protective Medium on Dry Acidophilic Rod Preparation Indicators

Biomass and protective media mixing ratio Lyophilized preparation characteristic Number of live L.acidophilus cells lg during storage, month 1 3 6th 9th 1: 1 The biomass is spongy, inhomogeneous, common bed structure, in places biomass crumbles, ugly 10 ⁹ 10 ⁹ 10 ⁸ 10 ⁷ 2: 1 Puri, homogeneous, amorphous or crystalline, white, white cream-colored biomass 10 ¹² 10 ¹¹ 1O ¹⁰ 10 ⁹ 3: 1 Hard, uneven, in places cracked or peeled- decaying biomass, bad bounce off the vial wall 10 ^u 10 ⁹ 10 ⁸ 10 ⁷

Claims (1)

Definition of the Invention A method of producing a medicated dry acidophilic rod preparation comprising preparing a nutrient medium, inoculating a culture, cultivating a biomass, chilling, mixing with a protective medium, dispensing, freezing and lyophilizing a liquid mixture, characterized in that the nutrient medium is prepared in sterile which adds 2 to 3% of the total amount of sterile skim milk and, when making the protective medium, adds 7 to 9% of skimmed milk powder, 2.5 to 3% of glutamate sodium to distilled water, 10-12% glucose, 0.4-0.7% sodium bicarbonate, and mixes the cooled biomass with a protective medium in a 2: 1 ratio, respectively, and uses Lactobacillus acidophilus K1, K2, K4 strains for inoculation of the culture medium, which exhibit antagonistic activity against pathogenic bacteria. the liquid preparation mixture is blown into small containers (for example vials or ampoules) of 3-5 cm ³ .