INTRODUCTION:

Acute intestinal infections (AII) are one of the urgent health problems in all countries¹, including Kazakhstan. The ubiquity and high incidence of moderate and severe forms of complications, especially in infants, determine the need to find ways to optimize the tactics of treatment of this group of diseases. The most common cause of intestinal infections are pathogens such as Coli bacteria^{2, 3}, Salmonella^4,5, and under certain conditions Enterobacter⁶, Citrobacter⁷, Klebsiella⁸, Proteus⁹, yeast-like fungi¹⁰, and agents of viral nature¹¹. There are frequent cases of various inflammatory and septic processes caused by pathogenic cocci, including Staphylococcus aureus, Streptococcus pyogenes, Pneumococcus (Diplococcus) pneumoniae, etc.^12-15.

The complexity of the treatment of infectious diseases is associated with the massive irrational use of antibiotics and chemotherapeutic medications, which has led to the development of multi-medication resistance in pathogens¹⁶. In addition, antibiotics themselves often have side effects on the human body. Their most frequent side effects are toxic effects, allergic reactions, and dysbiosis. Dysbiosis occurs after prolonged treatment with broad-spectrum antibiotics. In this case, not only pathogenic microbes die, but also representatives of the normal microflora, which, first of all, stimulate the immune system, and normalize its functioning at different levels, including both local mucosal immunity and systemic humoral and/or cellular immunity. With dysbiosis, some groups of pathogens that are immune to the medication used and are no longer restrained by beneficial symbiont bacteria begin to actively multiply. In this case, an endogenous superinfection can occur, such as candidiasis. Thus, dysbiosis not only complicates an existing disease but also makes the body more susceptible to other diseases¹⁷.

The modern strategy of AII therapy gives priority to therapeutic measures aimed at correcting intestinal microbiocenosis to eliminate the focus of chronic infection localized in the intestine.

In this regard, in the last years, probiotics based on symbiont microorganisms of the gastrointestinal tract have become increasingly more popular globally instead of antibiotics for the treatment of intestinal and urogenital infections. Probiotics belong to the group of medical immunobiological medicines based on living bacteria that are antagonistically active against pathogenic and conditionally pathogenic microorganisms (pathogens of various infectious diseases) and do not adversely affect representatives of the normal human microflora.

According to the World Health Organization (WHO) definition, probiotics are living microorganisms that, when administered in an adequate amount, have a positive effect on the health of the host organism. In 2013 the International Scientific Association for Probiotics and Prebiotics (ISAPP) clarified this definition, stating that the word "probiotic" can be applied only to those medications that meet the following requirements: the presence of accurate information about the microorganisms that make up the preparations, indicating the strains; a sufficient number of viable bacteria is preserved by the end of the shelf life; studies have been conducted that have confirmed the safety and effectiveness of the included strains¹⁸.

Only a strain with a deciphered mechanism of action, the effectiveness of which has been proven in at least one randomized controlled trial (RCT), can be assigned to the probiotic group. The clinical effect and functional activity of bacteria of the same species, but of different strains, can be radically different¹⁹.

The main mechanisms of positive action of probiotics are antagonism to pathogenic and conditionally pathogenic bacteria of the microbiota, strengthening of the mucous barrier of the gastrointestinal tract, as well as the effect on the modulation of the immune response. As a result, a chain of mechanisms of immunological protection is triggered (an increase in the intensity of secretory immunoglobulin A (sIgA) production, cytokine production, and interferon production, which leads to an increase in the body's resistance to the effects of adverse factors^20-22. Besides that, the beneficial microflora inhabiting the gastrointestinal tract participates in the metabolism of proteins, carbohydrates, fats, and other substances entering the body or formed during digestion, synthesizes vitamins B, vitamins K, C, and some amino acids, and also destroys and removes toxic substances from the human body²³.

Most of the data on the use of probiotics was obtained in studies on their effectiveness in the treatment and prevention of a wide range of diseases of the gastrointestinal tract, such as infectious diarrhea²⁴, antibiotic-associated diarrhea²⁵, Clostridium difficile-associated²⁶ diarrhea, travelers' diarrhea²⁷, gastritis associated with Helicobacter pylori²⁸ infection, Crohn's disease, necrotic enterocolitis²⁹, as well as in prevention and/or treatment of allergic diseases^30-32 and prevention and/or reduction of infections of the urogenital tract³³.

Well-known therapeutic and prophylactic probiotics are not always effective against intestinal infections^{34, 35}. The reason for this is an insufficiently wide antimicrobial spectrum of action and that the antagonists to specific pathogens are not selected. In addition, the analysis of probiotics presented on the pharmaceutical market shows that the data indicated on their packages often do not correspond to reality³⁶, which indicates an insufficiently developed technology for the production of these probiotics.

The need for standardization of methods used in the production of probiotics is also stated concerning the use of probiotics as antifungal agents³⁷. The optimal technology for the production of probiotics should ensure high output, stability, and the absence of specific allergens³⁸. To achieve such a production technology, optimization of output, cost, functionality, or stability is used by influencing gene expression, protein structure, or metabolic processes³⁹. Recently, many researchers have proposed the introduction of a microencapsulation technique, which consists in isolating a biologically active ingredient in an encapsulator or matrix, which allows for avoiding the stressful effects that occur during the production, storage, and consumption of probiotics^40,41.

To achieve the stability and functionality of probiotic strains during production, it is recommended to use cryoprotectors and lyoprotectors, i.e. substances that protect cells during freezing and lyophilization, respectively. To date, skimmed milk powder (SMP) is most often used as a protective agent⁴².

In connection with the above, research on improving the effectiveness of probiotic medications, expanding their spectrum of action due to the correctly selected microbial composition, and optimal technology of their production are relevant in AII control.

To create an effective probiotic therapeutic agent, we have selected two associations of lactic acid and propionic acid bacteria with a wide range of biological activity.

The study aimed to develop a technology for the production of a finished form of medicinal probiotic agents against human intestinal infections based on active strains of lactic acid and propionic acid bacteria with a wide range of biological activity and resistance to antibiotics.

MATERIALS AND METHODS:

The object of the study was two associations of the most active strains of lactic acid and propionic acid bacteria isolated from healthy people: No. 2 (L. plantarum 2v/A-6+L. brevis B-3/A-26+L. acidophilus 27w/60+P. shermanii 8) and No. 5 (L. plantarum 2v/A-6+L. cellobiosus 2/20+L. fermentum 27+P. shermanii 8+L. brevis B-3/A-26+L. plantarum 14d).

To obtain associations, the cultures included in their composition were grown together in an amount of 5-7% on two variants of nutrient media: No. 1(De Man, Rogosa, and Sharpe (MRS) medium with CoCl₂) and No. 4(yeast extract: 5.0g/l+glucose: 10.0g/l+CoCl₂: 0.01g/l). The cultivation of bacteria in liquid nutrient media was carried out for 24hours at a temperature of 30-32ºC.

One of the protective components was added to the liquid dosage form after cultivation to increase storage resistance:

1: Protective medium containing 7% sucrose and 1.5% gelatin;

2: Protective medium containing 7% sucrose, 1.5% gelatin, 0.08% ascorbic acid, 0.015% cysteine, and 0.17% table salt;

3: Control (without protective components).

The determination of the number of microorganisms was carried out by a series of successive dilutions in sterile tap water and seeding them into an MRS agar nutrient medium, followed by counting the grown colonies. The antagonistic activity was determined by diffusion of the bacteria in question into agar against various test cultures (Escherichia coli, Escherichia coli 8739, Salmonella gallinarum, Salmonella sp., Salmonella enteritidis 35382, Staphylococcus aureus 6538, Staphylococcus aureus 3616, Staphylococcus aureus 9, Candida albicans, Klebsiella pneumoniaeATCC 700603, Pasteurella multocida, Pseudomonas aeruginosa 835, Acinetobacter sp. 1522, Bacillus subtilis, Mycobacterium B₅)⁴³.

After that, the preparations were poured into sterile penicillin vials of 5ml and dried in a Liobeta-35 freeze dryer.

The accumulated experimental batches of liquid and dry forms of medications were stored in the refrigerator at a temperature from 2 to 8ºC. After that, liquid preparations were analyzed after 3, 4, 5, and 6 months of storage, and dry preparations were analyzed by an accelerated method after being warmed up at 60ºC for 15 minutes.

The experiments were carried out in three repetitions. Standard methods of finding average values and their average errors were used for the mathematical processing of the results. The statistical reliability of the results obtained was determined using Student's t-test. The differences were considered statistically significant at p <0.05.

Table 2: Antagonistic activity of dry variants of associations of probiotic bacteria with various protectors

Protectors

Diameter of the growth suppression zones of test cultures, mm

S. enteritidis 35382

E. coli 8739

K. pneumoniae АТСС 700603

S. aureus 6538

P. multocida

Mycobacterium B₅

S. aureus 3616

S. aureus 9

C. albicans

E. coli

S. gallinarum

B. subtilis

Salmonella sp.

Acenetobacterium 1522

Ps. aerugenosa 835

Freeze-dried variants of association No. 2 grown on medium No. 1 with various protectors

Protector No. 1

25±0.2

20±0.3

22±0.3

25±0.2

18±0.3

21±0.3

22±0.2

18±0.3

20±0.3

21±0.3

22±0.3

25±0.2

19±0.3

20±0.3

Protector No. 2

28±0.1

22±0.3

25±0.2

30±0.1

20±0.3

25±0.2

23±0.2

21±0.3

22±0.3

23±0.2

23±0.3

26±0.2

22±0.3

23±0.2

Protector No. 3

25±0.2

20±0.3

17±0.3

18±0.3

15±0.3

20±0.3

17±0.3

15±0.6

15±0.3

19±0.3

24±0.2

20±0.3

18±0.3

21±0.3

Protector No. 4

25±0.1

18±0.3

22±0.3

17±0.3

16±0.5

23±0.2

20±0.3

13±0.5

16±0.3

20±0.3

18±0.3

13±0.5

15±0.6

16±0.6

Freeze-dried variants of association No. 2 grown on medium No. 4 with various protectors

Protector No. 1

19±0.3

15±0.3

18±0.3

20±0.3

14±0.3

19±0.3

18±0.3

20±0.3

14±0.7

15±0.3

17±0.3

20±0.3

18±0.3

19±0.3

Protector No. 2

28±0.1

18±0.3

27±0.2

25±0.1

18±0.6

24±0.2

15±0.3

17±0.3

18±0.3

12±0.6

19±0.6

19±0.3

18±0.3

20±0.3

Protector No. 3

20±0.3

18±0.3

23±0.3

18±0.3

15±0.3

17±0.3

15±0.5

15±0.3

12±0.6

13±0.5

15±0.3

18±0.3

20±0.3

18±0.3

Protector No. 4

20±0.3

16±0.3

20±0.3

13±0.6

14±0.5

12±0.6

18±0.3

13±0.5

12±0.6

18±0.3

16±0.3

18±0.3

17±0.3

Freeze-dried variants of association No. 5 grown on medium No. 1 with various protectors

Protector No. 1

20±0.3

16±0.3

18±0.3

15±0.6

14±0.6

21±0.3

20±0.3

15±0.3

17±0.3

18±0.3

16±0.3

25±0.2

17±0.3

18±0.3

20±0.3

Protector No. 2

25±0.2

19±0.3

21±0.3

29±0.1

15±0.3

19±0.3

22±0.3

18±0.3

15±0.3

18±0.3

20±0.3

16±0.3

18±0.3

20±0.3

Protector No. 3

21±0.3

15±0.3

18±0.3

15±0.3

18±0.3

14±0.6

15±0.3

14±0.3

20±0.3

18±0.3

17±0.3

15±0.6

12±0.6

17±0.3

20±0.3

Protector No. 4

12±0.6

11±0.7

15±0.5

12±0.6

11±0.7

10±0.7

13±0.7

10±0.7

15±0.7

14±0.7

Freeze-dried variants of association No. 5 grown on medium No. 4 with various protectors

Protector No. 1

15±0.3

20±0.3

15±0.3

16±0.3

18±0.3

10±0.7

15±0.3

18±0.3

16±0.3

13±0.5

Protector No. 2

20±0.3

16±0.3

20±0.3

27±0.2

13±0.5

15±0.3

20±0.3

15±0.3

12±0.6

11±0.7

15±0.3

20±0.3

18±0.3

17±0.3

Protector No. 3

13±0.5

14±0.3

17±0.3

14±0.3

13±0.5

12±0.6

13±0.5

15±0.3

11±0.7

15±0.3

14±0.3

15±0.3

12±0.6

Protector No. 4

14±0.3

13±0.5

12±0.6

15±0.6

14±0.3

13±0.5

14±0.3

13±0.5

11±0.7

17±0.3

12±0.6

14±0.3

12±0.6

16±0.3

Note: protectors: No. 1: 7% sucrose and 1.5% gelatin; No. 2: 7% sucrose and 1.5% gelatin+7% SMP; No. 3: 7% sucrose+10% SMP; No. 4: 10% yeast extract+10% SMP

Table 3: Antagonism and titer of bacteria of association No. 2 grown on medium No. 1 without a protector after storing in the refrigerator

Test cultures	Test culture suppression zones, mm
	Shelf life
	Original version	3 months	4 months	5 months	6 months
S. enteritidis 35382	26±0.3	26±0.3	25.5±0.3	22±0.3	18±0.7
E. coli 8739	22±0.3	17±0.2	16.5±0.2	16±0.2	15±0.2
K. pneumoniae АТСС 700603	22±0,4	22±0,4	21.5±0,4	20±0.5	14±0.2
S. aureus 6538	30±0.3	30±0.5	18±0.2	15±0.2	13.5±0.2
P. multocida	18±0.2	18±0.2	15.5±0.2	15.5±0.3	15±0.2
Mycobacterium B₅	22±0.6	22±0.6	20.5±0.5	20±0.5	19±0.5
S. aureus 3316	22±0.3	22±0.3	20±0.5	20±0.5	18±0.7
S. aureus 9	17±0.2	17±0.2	15±0.2	15±0.3	15±0.2
C. albicans	21±0.6	22±0.3	21.5±0,4	20±0.5	20±0.5
E. coli	22±0.3	22±0.3	21±0.6	20±0.5	20±0.5
S. gallinarum	25±0.6	25±0.6	24.5±0.6	14.5±0.3	12±0.3
B. subtilis	18±0.2	18±0.2	18±0.2	18±0.2	18±0.2
Salmonella sp.	30±0.1	28±0.1	20±0.5	12±0.3	0±0.0
Acinetobacter sp. 1522	20±0.5	20±0.5	20±0.5	20±0.5	0±0.0
Ps. aeruginosa 835	21±0.5	22±0,4	22±0.5	21±0.5	20±0.5
Titer, CFU/ml	3.5±0.3×10⁹	3.6±0.3×10⁸	3.3±0.5×10⁸	3.2±0.6×10⁸	3.2±0.6×10⁸

When comparing the results obtained, it was found that the most active preparation from association No. 2 could be obtained by growing it on nutrient media No. 1 or No. 4 using protector No. 2 during freeze-drying. To obtain a dry preparation from association No. 5, the most favorable combination was obtained with nutrient medium No. 1 and freeze-drying protector No. 2.

The accumulated experimental batches of liquid and dry forms of medicines were stored in a refrigerator. The results of determining the antagonistic activity of association No. 2 grown on medium No. 1 without a protector after storing in a refrigerator are presented in Table 3.

As can be seen from the table below, association No. 2 grown on medium No. 1 without a protector (control), after 3 months of storage in the refrigerator, demonstrated almost complete preservation of antagonistic activity against all test cultures taken into the study. For six months, antagonism remained at the same level concerning test cultures of C. albicans (21-20 mm), E. coli (22-20mm), B. subtilis (18mm), and Ps. aeruginosa 835 (22-20mm). A significant decrease in antagonistic activity (up to 14-12mm) against K. pneumoniae ATCC 700603, S. aureus 6538, and S. gallinarum was noted after 5-6 months of storage. A slight decrease in activity was found for the remaining test cultures. By the sixth month of storage, association No. 2 had not shown antagonism to two of the 15 test cultures, namely Salmonella sp. and Acinetobacter sp. 1522.

The titer of bacteria of association No. 2 without a protector grown on medium No. 1 ad decreased by one degree after being stored for six months in the refrigerator (from 3.5±0.3×10⁹ to 4.2±0.6×10⁸ CFU/ml).

The antagonistic activity of association No. 2 with protector No. 1 grown on medium No. 1 was almost completely preserved for six months concerning all the included test cultures, while the diameters of the zones of suppression of their growth differed from the initial ones by 1-2mm.

The titer of bacteria of association No. 2 with protector No. 1 grown on medium No. 1 had decreased by one degree after six-month storage in the refrigerator (from 3.5±0.2×10⁹ to 6.1±0.6×10⁸ CFU/ml).

The antagonistic activity of association No. 2 with protector No. 2 grown on medium No. 1 was almost completely preserved for three months against all test cultures taken into the experiment, but by four months of storage, it had significantly decreased concerning P. multocida (from 18 to 12mm), the growth suppression zones shrinking after 6 months up to 9mm. The diameters of the growth suppression zones against S. aureus 6538, Salmonella sp., and S. gallinarum by six months of storage were significantly smaller than the initial ones (30±0.1 and 18±0.2; 28±0.1 and 20±0.5; 23±0.3 and 15±0.3mm, respectively).

The titer of bacteria of association No. 2 grown on medium No. 1 with protector No. 2 decreased after storage for six months from 7.1±0.3×10⁹ to 5.0±0.4×10⁸ CFU/ml.

According to the results of the study, association No. 2 on medium No. 1, in the case of using protector No. 1 (7% sucrose and 1.5% gelatin), after storage in the refrigerator for six months, retained antagonistic activity and the number of viable cells at a sufficiently high level compared to the control and with protector No. 2.

The antagonistic activity of association No. 2 without a protector (control) grown on medium No. 4 persisted for three months concerning all the included test cultures, differing from the initial parameters in the diameter of growth suppression zones mainly by 2 to 7mm. By four months of storage, this association did not show activity against E. coli. By five months of storage, antagonism was not detected against the following test cultures: S. aureus 6538, Salmonella sp., Acinetobacter sp. 1522, and after six months against Mycobacterium B_5. For the remaining nine test cultures, this association retained a certain activity by six months of storage.

The titer of bacteria of association No. 2 grown on medium No. 4 without a protector had decreased by one degree after storage for six months in the refrigerator (from 3.2±0.2×10⁹ to 1.5± 0.3×10⁸ CFU/ml).

After six months of storage, this association showed activity against all test cultures taken into the study. The diameters of the zones of growth suppression ranged from 12 to 24mm.

The titer of bacteria of association No. 2 grown on medium No. 4 with protector No. 1 had decreased by one degree after being stored for six months in the refrigerator (from 2.3±0.2×10⁹ to 2.1±0.2×10⁸ CFU/ml).

The antagonistic activity of association No. 2 with protector No. 2 grown on medium No. 4 persisted for five months against all test cultures with growth suppression zones from 10 to 20mm.

After six months of storage, antagonism in the studied association was observed against 13 out of 15 test cultures. The growth suppression zones of test cultures had decreased by an average of 3-9mm compared to the initial ones. Antagonistic activity against Salmonella sp. and S. gallinarum was not detected.

The titer of bacteria of association No. 2 grown on medium No. 4 with protector No. 2 had decreased after storage for five to six months from 2.6±0.2×10⁹ to 1.7±0.3×10⁸ CFU/ml.

Comparing the obtained results, we found that association No. 2, when grown on medium No. 1, showed a more complete preservation of production-valuable traits during storage compared with the results using medium No. 4. The advantage of protector No. 1 in comparison with protector No. 2 was also observed. In this regard, nutrient medium No. 1 with protector No. 1 (7% sucrose and 1.5% gelatin) is used to obtain active association No. 2 resistant to storage.

The results of determining the antagonistic activity of association No. 5 grown on medium No. 1 without a protector after storage in the refrigerator showed that it persisted for three months concerning all included test cultures, and the parameters differed from the initial ones (the diameters of growth suppression zones had shrunk mainly by 2 to 5mm).

After four months of storage, this association did not show antagonistic activity to S. aureus 6538, Mycobacterium B _5, B. subtilis, Acinetobacter sp. 1522, and after six months of storage to P. multocida.

As for the remaining ten test cultures, after six months of storage, this association had antagonistic activity against them, but with a rather significant decrease in the diameter of the growth suppression zones (up to 10 mm) compared to the initial parameters.

The titer of bacteria of association No. 5 grown on medium No. 1 without a protector had decreased after storage for six months in the refrigerator from 2.8±0.23×10⁹ to 2.4±0.2×10⁸ (CFU/ml).

The antagonistic activity of association No. 5 with protector No. 1 grown on medium No. 1 persisted for three months concerning all test cultures. The diameters of the growth suppression zones of test cultures differed from the initial parameters by 1 to 5mm. The antagonistic activity of association No. 5 after storage for 6 months differed from the initial values in the diameter of growth suppression zones by 2 to 10mm.

The titer of bacteria of association No. 5 grown on medium No. 1 with protector No. 1 had decreased after storage for six months in the refrigerator from 2.0±0.2×10⁹ to 1.2± 0.3×10⁸ (CFU/ml).

The antagonistic activity of association No. 5 with protector No. 2 grown on medium No. 1 persisted for three months after storage in the refrigerator for all test cultures, while the diameters of the zones of suppression of their growth were lower than the initial ones by 1 to 10mm. The antagonistic activity of association No. 5 persisted for all test cultures after 6 months, but it differed from the initial values by a lower diameter of growth suppression zones up to 11mm.

The titer of bacteria of association No. 5 grown on medium No. 1 with protector No. 2 had decreased after storage in the refrigerator after 3 months from 2.1±0.2×10⁹ to 1.8±0.3×10⁸ (CFU/ml), and after 6 months to 1.0±0.4×10⁸ (CFU/ml).

The antagonistic activity of association No. 5 without a protector (control) grown on medium No. 4 persisted for three months of storage in the refrigerator concerning all included test cultures, differing from the original values in diameter of growth suppression zones mainly by 3 to 10 mm. After four months of storage, this association showed no activity for 7 out of 15 test cultures: S. enteritidis 35382, E. coli 8739, K. pneumoniae ATCC 700603, S. aureus 6538, C. albicans, E. soli, S. gallinarum. After five and six months of storage, antagonistic activity was preserved only for three test cultures: P. multocida (12 mm), S. aureus 9 (15 mm), and Ps. aeruginosa 835 (12 mm).

The titer of bacteria of association No. 5 grown on medium No. 4 without a protector had decreased after storage in the refrigerator for three months from 2.3 ± 0.5× 10⁹ to 2.5± 0.5× 10⁸ (CFU/ml), and after six months to 2.0± 0.5× 10⁸.

The antagonistic activity of association No. 5 with protector No. 1 grown on medium No. 4 persisted after storage for three months in the refrigerator for all test cultures, but there was a decrease in the diameter of the growth suppression zones by 2 to 7 mm, and after 6 months the diameters of the growth suppression zones of test cultures had decreased by 3 to 18 mm.

The titer of bacteria of association No. 5 grown on medium No. 4 with protector No. 1 after storage for three months in the refrigerator had decreased from 1.3± 0.3×10⁹ to 9.6± 0.3× 10⁸, and after six months to 1.0± 0.4×10⁸ CFU/ml.

The antagonistic activity of association No. 5 with protector No. 2 grown on medium No. 4 persisted for three months against all the used test cultures, while the zones of suppression of their growth decreased by 1 to 5 mm. Antagonism remained at the same level concerning the following test cultures: S. aureus 6538 (20 mm), S. aureus 3316 (15 mm), S. aureus 9 (22 mm), and B. subtilis (15 mm). After 4 months of storage, no antagonistic activity was detected against K. pneumoniae ATCC 700603, and after 5 months against S. aureus 3316 and E. coli. For the remaining test cultures, the growth suppression zones ranged from 9 to 15 mm. The titer of bacteria of association No. 5 grown on medium No. 4 with protector No. 2 after storage for six months in the refrigerator had decreased from 1.1±0.4×10⁹ to 1.0±0.4×10⁸ (CFU/ml).

Thus, association No. 5 grown on medium No. 1 showed a more complete preservation of production-valuable characteristics compared to the results with the use of medium No. 4, while the use of protector No. 1 was more optimal. In this regard, nutrient medium No. 1 and protector No. 1 should be used for the production of medications from association No. 5. The most active one in terms of bacterial titer, antagonistic activity, and stability during storage was the preparation obtained by growing association No. 2 on nutrient medium No. 1 using 7% sucrose and 1.5% gelatin as a protector.

To test the stability during the storage of dry forms of probiotic preparations, an accelerated method was used by warming them up for 15 minutes at 60°C. The antagonistic activity and the number of viable cells after warming up of dry variants of association No. 2 grown on medium No. 1 and dried with various protectors are presented in Table 4.

The initial titer of bacteria of association No. 2 grown on medium No. 1 and dried with protectors was equal to 3.5±0.3×10⁹ CFU/g. After warming up in the variant with protector No. 1, the titer of bacteria decreased to 8.3×10⁸ CFU/g, and antagonistic activity was detected only for five test cultures out of 15, namely: K. pneumoniae ATCC 700603, Mycobacterium B ₅, S. aureus 3316, E. soli and B. subtilis.

In the variant with protector No. 3, the titer of bacteria decreased to 1.9×10⁸ CFU/g, and antagonistic activity was detected for 11 test cultures with growth suppression zones from 10 to 16mm, while no activity was detected for S. aureus 6538, P. multocida, C. albicans, and Salmonella sp. The lowest preservation of the number of viable cells after warming up was found in the variant of association with protector No. 4 (2.9×10⁷ CFU/g), while antagonistic activity was detected for eight test cultures with a diameter of growth suppression zones from 10 to 15mm. No activity was established for S. aureus 6538, P. multocida, C. albicans, S. aureus 9, Salmonella sp., Acenetobacterium 1522, and Ps. aerugenosa 835.

The best preservation of viable bacterial cells was observed in the variant with protector No. 2 (7% sucrose and 1.5% gelatin+7% SMP), equaling 1.2×10⁹ CFU/g. Antagonistic activity in this variant of the association was established concerning all test cultures taken into the study with zones of suppression of their growth from 10 to 24mm (Table 4).

The initial titer of dried association No. 2 grown on medium No. 4 was 3.2±0.2×10⁹ CFU/g. After warming up in the variant of association No. 2 with protector No. 1, the titer of bacteria decreased to 5.8×10⁷± 0.3 CFU/g, and antagonistic activity was detected for nine test cultures out of 15, namely: S. aureus 6538, Mycobacterium B ₅, S. aureus 9, E. coli, S. gallinarum, Salmonella sp., Acenetobacterium 1522 and Ps. aerugenosa 835. The titer of the bacteria of the studied dried association with protector No. 3 after warming up was 2.0±0.2×10⁷ compared to the initial 3.2±0.2×10⁹ CFU/g, while antagonistic activity was detected in 12 of the 15 test cultures. Activity against Mycobacterium B5, S. aureus 3616, and C. albicans was not detected. In association No. 2 on medium No. 4 with protector No. 4, the titer of bacteria was 2.0±0.2×10⁷ compared to the initial 3.2±0.2×10⁹ CFU/g, antagonistic activity was not detected for four of the 15 test cultures. The best result in preserving the number of viable cells and antagonistic activity was obtained in association No. 2 with protector No. 2 (7% sucrose and 1.5% gelatin+7% SMP), where, after warming up, the titer of bacteria was equal to 3.5± 0.3× 10⁸ compared to the initial 3.5±0.2×10⁹ CFU.

Antagonistic activity in this variant of the association was present against all test cultures taken in the study.

The initial titer of association No. 5 grown on medium No. 1 and dried with protectors was 2.8±0.2×10⁹ CFU/ml. After warming up association No. 5 grown on medium No. 1 and dried with protector No. 1, the titer of bacteria decreased to 5.6±0.3×10⁸ CFU/g, and antagonistic activity was detected for seven test cultures out of 15, namely: K. pneumoniae ATCC 700603, Mycobacterium B ₅, S. aureus 3316, S. aureus 9, E. soli; B. subtilis, Salmonella sp; in the variant of association with protector No. 2, the titer of bacteria after warming up was equal to 1.1±0.2×10⁸ CFU/g, and antagonistic activity was not detected against eight test cultures. In the association dried with protector No. 3, after warming up, the titer of bacteria decreased to 2.0±0.2×10⁸ CFU/g, while antagonistic activity was detected for 13 out of 15 test cultures (there was no antagonism to Mycobacterium _B5, and S. gallinarum). The lowest preservation of the number of viable cells after warming up was found in association variant No. 5 on medium No. 1 dried with protector No. 4, equaling 2.0×10⁷ CFU/ml, and antagonistic activity was not detected for three of the 15 test cultures (antagonism to P. multocida, Mycobacterium _B5 and S. gallinarum was not observed).

The initial titer of bacteria of association No. 5 grown on medium No. 4 and dried with various protectors was 2.3±0.2×10⁹ CFU/g, and after warming up in the variant of association No. 5 on medium No. 4 with protector No. 1, the titer of bacteria decreased to 2.0±0.2×10⁷ CFU/g. The antagonistic activity was observed against nine test cultures out of 15, namely: S. aureus 6538, Mycobacterium B ₅, S. aureus 9, C. albicans, E. coli, S. gallinarum, B. subtilis, Salmonella sp., Acenetobacterium 1522. In the variant of association No. 5 with protector No. 3 after warming up, the titer of bacteria was 2.0± 0.2×10⁷ CFU/g, while antagonistic activity was detected for 12 out of 15 test cultures (antagonism to E. coli 8739, P. multocida, E. coli was not established). In the variant of association No. 5 with protector No. 4, the titer of bacteria after warming up was 2.0 ±0.2×10⁷ CFU/g, and antagonistic activity was not detected for eight of the 15 test cultures (it was absent for K. pneumoniae ATCC 700603, S. aureus 653, P. multocida, C. albicans, E. coli B. subtilis, Salmonella sp., and Acenetobacterium 1522). The greatest preservation of cell viability was observed in association variant No. 5 on medium No. 4 with protector No. 2 (7% sucrose and 1.5% gelatin+7% SMP), equaling 2.0±0.2×10⁸ CFU/g. Antagonistic activity in this variant of the association was observed against all test cultures taken in the study.

Thus, after warming up, the best preservation of viable bacterial cells was observed in association No. 2 on nutrient media No. 1 and No. 4 and association No. 5 on medium No. 4 dried with protector No. 2 (7% sucrose and 1.5% gelatin+7% SMP), while the titer of bacteria was equal to 1.2×10⁹, 3.5×10⁸, and 2.0±0.2×10⁸ CFU/g, respectively. Antagonistic activity in these association variants was observed against all test cultures taken into the study with zones of suppression of their growth ranging from 10 to 24 mm.

DISCUSSION:

The technology for the production of dry and liquid forms of probiotic medication has been developed.

For the preparation of liquid and dry forms of the medication, the following associations are used: associations No. 2 (L. plantarum 2v/A-6+L. brevis B-3/A-26+L. acidophilus 27w/60+P. shermanii 8) and No. 5 (L. plantarum 2v/A-6+L. cellobiosus 2/20+L. fermentum 27)+P. shermanii 8+L. brevis B-3/A-26+L. plantarum 14d) grown on nutrient media No. 1 (MRS medium with CoCl₂) and No. 4 (yeast extract: 5.0 g/l+glucose: 10.0g/l+CoCl₂: 0.01g/l). To obtain these associations, the cultures included in their composition are grown together in an amount of 5-7% at a temperature of 37°C for 24hours.

One of the protective components is added to the liquid dosage form after cultivation to increase storage resistance:

1: a protective medium containing 7% sucrose and 1.5% gelatin.

2: a protective medium containing 7% sucrose, 1.5% gelatin, 0.08% ascorbic acid, 0.015% cysteine, and 0.17% table salt.

The titer of bacteria in liquid preparations from association No. 2 on medium No. 1 is equal to 7.5x10⁹ CFU/ml, on medium No. 4 3.5x10⁹ CFU/ml; from association No. 5 on medium No. 1 2.8x10⁹ CFU/ml, and medium No. 4 2.3x10⁹ CFU/ml.

Associations No. 2 and No. 5 showed the greatest antagonistic activity to the tested test cultures, mainly when grown on nutrient medium No. 1(MRS with CoCl₂). For the accumulation of bacterial cells, association No. 2 on the same nutrient medium showed the best results.

For freeze-drying, one of the protective components was added to the liquid associations (1:7% sucrose and 1.5% gelatin; 2:7% sucrose and 1.5% gelatin+7% SMP; 3:7% sucrose+10% SMP; 4:10% yeast extract and 10% SMP). For both associations of bacteria, the tested protective media showed a good result when dried. The titer of bacteria in dry preparations was nx10⁹ CFU/g.

The most active preparation from association No. 2 was obtained when it had been grown on nutrient media No. 1 or No. 4 using a protector No. 2 during freeze-drying. To obtain a dry preparation from association No. 5, the most favorable combination was obtained with nutrient medium No. 1 and freeze-drying protector No. 2.

The accumulated experimental batches of liquid medications were stored in the refrigerator at a temperature from 2 to 8°C.

It was found that the most active preparation in terms of bacterial titer and antagonistic activity and the most stable one during storage for 6 months was the liquid preparation obtained by growing association No. 2 (L. plantarum 2v/A-6+L. brevis B-3/A-26+L. acidophilus 27w/60+P. shermanii 8) on nutrient medium No. 1 (MRS with CoCl₂) using 7% sucrose and 1.5% gelatin as a protector. The liquid preparation from association No. 5 grown on medium No. 1 showed a more complete preservation of production-valuable signs during storage compared to the results of using nutrient medium No. 4, while the use of protector No. 1 was more optimal.

To test the stability during the storage of dry forms of preparations, an accelerated method was used by warming them up for 15 minutes at 60^°C. It was found that after warming up, the best preservation of viable bacterial cells was observed in association No. 2 on nutrient media No. 1 and No. 4 and association No. 5 on medium No. 4 dried with protector No. 2 (7% sucrose and 1.5% gelatin+7% SMP), while the titer of bacteria was equal to 1.2×10⁹; 3.5×10⁸ and 2.0±0.2×10⁸ CFU/g, respectively. Antagonistic activity in these association variants was observed against all test cultures taken into the study with zones of suppression of their growth ranging from 10 to 24mm.

Thus, the selection of the optimal composition of the association of microorganisms, nutrient medium, and protectors that increase the shelf life of finished forms of medications is an important point in the development of technology for the production of probiotic medications. The results obtained by us are consistent with the data from literary sources. Thus, it has been found that the composition of the nutrient medium for growing probiotic association affects the antimicrobial activity of the probiotic⁴⁴.

According to the results of a study by C. Romyasamit et al., the optimal cryoprotector was identified to preserve the stability of the probiotic strain of Enterococcus faecalis during storage (SMP at a concentration of 6-10%). After storing the lyophilized strain of E. faecalis for 30 days at a temperature of 4°C, its antagonistic activity against C. difficile ATCC 43255 and C. difficile ATCC 630 was proved. Moreover, lyophilized E. faecalis demonstrated more significant inhibitory activity against C. difficile ATCC 630 than the strain that had not been subjected to lyophilization and storage⁴².

Data from a similar paper by Jawan et al. showed that L. lactis Gh1, after lyophilization with a cryoprotector consisting of a mixture of 10% (m/v) galactose and trehalose, at 30°C and stored for 120 days, could inhibit L. monocytogenes ATCC 15313⁴⁵.

According to a study in which the viability and specific functions of Lactobacillus plantarum TISTR 2075 were studied, the use of protectors based on rice proteins contributed to the preservation of the antagonistic activity of the lyophilized strain against E. coli O157: H7 DMST 12743 and S. typhimurium ATCC 13311⁴⁶.

CONCLUSION:

The developed technology for the production of a new Kazakh-made probiotic to improve the effectiveness of the treatment of human intestinal infections with further clinical research and organization of production will contribute to the development of the pharmaceutical market of Kazakhstan. The production of a new Kazakh-made complex probiotic medication can be organized in Kazakhstan for internal use, as well as for export to other countries.

CONFLICT OF INTEREST:

The authors have no conflict of interest to declare.

ACKNOWLEDGMENTS:

The publication of this paper was paid for from the budget funds under the grant financing of project AR09259195 "Development and preclinical trials of a new made-in-Kazakhstan therapeutic probiotic agent against human intestinal infections".

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Received on 28.02.2023 Modified on 24.04.2023

Research J. Pharm. and Tech 2023; 16(9):4093-4104.

DOI: 10.52711/0974-360X.2023.00670