INTRODUCTION:

Acute intestinal infections (AII) are one of the urgent health problems in all countries¹.

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 multidrug resistance in pathogens².

The modern strategy of AII therapy gives priority to therapeutic measures aimed at correcting intestinal microbiocenosis to eliminate the foci of chronic infection localized in the intestine. In this regard, recently in the world for the treatment of intestinal and urogenital infections, probiotics based on microorganisms (symbionts of the gastrointestinal tract) are increasingly recommended instead of antibiotics^3-5. 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 attributed to the group of probiotics. The clinical effect and functional activity of bacteria of the same species, but of different strains, can be radically different. An example of this is the bacterial species Escherichia coli⁶. The infection with the 0157:H7 E. coli strain can cause the development of hemorrhagic diarrhea, kidney failure, and even death. At the same time, another strain of this species, E. coli Nissle 1917, has pronounced probiotic activity⁷.

The most useful microorganisms are lactic acid bacteria and bifidobacteria, the pathogenic potential of which is considered to be low enough for oral use in conventional dosages by the experts in this field^8,9. These medications quickly suppress pathogenic microflora, activating the body's immune system¹⁰.

Most of the information regarding the use of probiotics has been obtained in studies on their effectiveness in the treatment and prevention of diseases such as infectious diarrhea¹¹, antibiotic-associated diarrhea¹², Clostridium difficile-associated diarrhea¹³, traveler's diarrhea¹⁴, gastritis associated with Helicobacter pylori¹⁵, as well as in the prevention and/or treatment of allergic diseases^16-18 and infections of the urogenital tract¹⁹.

In addition, several studies have shown the effectiveness of probiotic strains from the standpoint of evidence-based medicine in diseases and conditions associated with metabolic disorders (diabetes mellitus, dyslipidemia, obesity), liver diseases (cirrhosis, non-alcoholic fatty liver disease, hepatic encephalopathy), pancreatic diseases (acute pancreatitis), etc.^20-22. However, unambiguous results have not always been obtained for these diseases, therefore further studies are required to clarify the effectiveness.

Symbiont microorganisms of the gastrointestinal tract, which are part of probiotics, have a positive effect on the body of animals and humans, expressed in the ability to colonize the surface of the intestinal mucosa, suppression of pathogenic and conditionally pathogenic microflora, and synthesis of biologically active substances, such as essential amino acids, B vitamins, and hydrolytic enzymes, thanks to which the regulation of metabolic processes in macroorganisms occurs²³. The presence not only of antagonists to mixed intestinal infections but also of producers of biologically active substances in the composition of probiotics allows for increasing the effectiveness of the preparations.

Currently, many probiotic preparations based on living microorganisms have appeared on the market^24,25.

At the same time, well-known therapeutic and prophylactic probiotics against intestinal infections are not always effective^10,26. The reason for this is an insufficiently wide antimicrobial spectrum of action and the fact that 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 reality²⁷, which indicates that the production technology of these probiotics is not sufficiently developed.

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

The study aimed to create associations of probiotic bacteria with a wide range of biological activity, resistance to antibiotics, and selection of the nutrient medium composition to increase the effectiveness of probiotics against intestinal infections in humans.

METHODS:

The object of the study was six associations of the most active strains of lactic acid and propionic acid bacteria isolated from healthy people.

Nine variants of nutrient media were used to cultivate bacterial associations. The cultivation of bacteria in liquid nutrient media was carried out for 18-20hours at a temperature of 37⁰C.

The resistance of bacteria to antibiotics was established by the method of standard disks impregnated with antibiotics, the determination of the number of microorganisms was carried out by a series of successive dilutions in sterile tap water and seeding them into the agarized nutrient medium De Man, Rogosa, and Sharpe agar (MRS), followed by counting the grown colonies, while the antagonistic activity was established by diffusion 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 pneumoniae ATCC 700603, Pasteurella multocida, Pseudomonas aeruginosa 835, Acinetobacter sp. 1522, Bacillus subtilis, Mycobacterium B₅)²⁸.

The enzymatic activity was determined by the hydrolysis zones of the substrate by seeding probiotic cultures on dense nutrient media with the addition of starch to the medium for the determination of the amylolytic activity, casein for the determination of the proteolytic activity, or pectin for the determination of the pectolytic activity.

The content of B vitamins was determined by diffusion into agar using the following vitamin-dependent microorganisms as test cultures: B₃: Saccaharamyces cerevisiae Meyen (Leningrad strain), B₅: Zygofabospora marxiana (Hansen), B₆: Debariomyces disporus VKMU-1034, B₈: Saccaharamyces carlsbergensis (Hansen). The quantitative content of vitamin B₁₂ was determined by the microbiological method according to the growth zones for the vitamin-dependent E. coli strain 133-3 and calculated according to the Dmitrieva table²⁹.

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-criterion. The differences were considered statistically significant at p < 0.05.

RESULTS AND DISCUSSION:

The following associations of probiotic bacteria were used for the study:

No. 2: L. plantarum 2b/A-6 + L. brevis B-3/A-26 + L. acidophilus 27w/60 + P. shermanii 8;

No. 4: L. plantarum 2b/A-6 + L. brevis B-3/A-26 + L. plantarum 14d + P. shermanii 8;

No. 5: L. plantarum 2b/A-6 + L. cellobiosus 2/20 + L. fermentum 27 + P. shermanii 8 + L. brevis B-3/A-26 + L. plantarum 14d/19;

No. 6: L. plantarum 2b/A-6 + L. brevis B-3/A-26 + L. plantarum 14d + L. acidophilus 27w/77 + P. shermanii 8;

No.8: L. plantarum 14d/19 + L. brevis B-3/A-26 + L. plantarum 14d/13 + P. shermanii 8;

No. 9: L. cellobiosus 2/20 + L. fermentum 27.

To obtain associations, their constituent cultures were grown together in an amount of 5-7%. To select the optimal composition of the nutrient medium that would ensure the high quality of the finished product, the cultivation of associations was carried out in the following nutrient media (g/l):

1. MRS medium with CoCl₂: 0.01;

2. Yeast extract: 5.0 + molasses: 20.0 + СоСl₂: 0.01;

3. Yeast water + molasses: 20.0 + СоСl₂: 0.01;

4. Yeast extract: 5.0 + glucose: 10.0 + CoCl₂: 0.01;

5. Whey + molasses: 10.0 + CoCl₂: 0.01;

6. Whey + yeast extract: 3.0 + СоСl₂: 0.01;

7. Yeast extract: 5.0 + glucose: 10.0 + ammonium citrate: 2.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + heptahydrate MgSO₄: way: 0.2 + MnSO₄: 0.05 + CoCl₂: 0.01;

8. Yeast extract: 5.0 + glucose: 10.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + MnSO₄: 0.05 + CoCl₂: 0.01;

9. Yeast water + glucose: 10.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + MnSO₄: 0.05 + CoCl₂: 0.01.

The pH for all media was 6.7-6.9.

The associations grown on the MRS nutrient medium had a high antagonistic potential against Salmonella sp., while the growth suppression zones of the test culture ranged from 30 to 35mm. The growth suppression zones for S. enteritidis 35382 were from 19 to 24mm, for S. gallinarum from 23 to 28mm, for E. coli 8739 from 17 to 22mm, for E. coli from 15 to 21mm, and for S. aureus 6538 from 19 to 33mm. The growth suppression zones for S. aureus 3616 were from 18 to 21mm, for S. aureus 9 from 12 to 30mm, for P. multocida from 14 to 21mm, for K. pneumoniae ATCC 700603 from 14 to 24mm, for C. albicans from 14 to 24mm, for Mycobacterium B₅ from 12 to 23mm, for B. subtilis from 18 to 19mm, for Acinetobacter sp. 1522 from 12 to 20mm, and for Ps.aeruginosa 835 from 19 to 24mm.

The most active associations for S. enteritidis 35382 were associations No. 2 (growth suppression zone equaling 24mm), 8 and 9 (21mm each), for S. gallinarum: No. 8 (28mm) and No. 9 (26mm), for E. coli 8739: No. 2 (22mm) and No. 4 (21mm), for E. coli: No. 5 (21mm), No. 2, 4 and 9 (18mm each), for S. aureus 6538: No. 2 (33mm), No. 8 and 9 (28mm each), for S. aureus 3616: No. 5 and 6 (21mm each), No.4 (20 mm), No.2, 8 and 9 (19mm each), for S. aureus 9: No. 5 (30 mm), for P. multocida: No. 5 (21mm), for K. pneumoniae ATCC 700603: No. 2 and 6 (24mm each), for C. albicans: No.2 (24mm), for Mycobacterium B₅: No. 8(23mm), No.2 (22mm), for Acinetobacte sp. 1522: No.2, 4, 5 and 8 (20mm each), and for Ps.aeruginosa 835: No. 4 (24mm), No. 8 (23mm), No. 2 and 9 (22mm each).

Associations No. 2 and 5 had the greatest antagonism on this nutrient medium.

Associations grown on a nutrient medium containing yeast extract, molasses, and СоСl₂ had less high antagonistic activity against Salmonella sp., compared with the MRS medium, while the growth suppression zones of the test culture ranged from 19 to 28mm.

The growth suppression zones for S. enteritidis 35382 were from 11 to 16mm, for S. gallinarum: from 20 to 25 mm, for E. coli 8739: from 12 to 16mm, for E. coli: from 13 to 14mm, and for S. aureus 6538: from 12 to 19 mm. The growth suppression zones for S. aureus 3616 ranged from 14 to 17mm, for S. aureus 9: from 18 to 27 mm, for P. multocida: from 10 to 12mm, for K. pneumoniae ATCC 700603: from 11 to 30mm, for C. albicans: from 15 to 18mm, for Mycobacterium B₅: from 15 to 21mm, for B. subtilis: from 12 to 14 mm, for Acinetobacter sp.1522: from 19 to 29mm, and for Ps. aeruginosa 835: from 25 to 30mm.

The most active associations against Salmonella sp. were association No. 2 (growth suppression zone 28 mm), for S. enteritidis 35382: No. 6 (growth suppression zone 16mm), No. 4 and 5 (15mm each), for S. gallinarum: No. 9 (25mm), No. 4 and 8 (24mm each), for E. coli 8739: No. 6 (16mm), No. 2 and 4 (15 mm each), for E. coli all growth suppression zones equaled 13-14mm,for S. aureus 6538: No. 6 (19mm), No. 2, 5, and 8 (18mm each), for S. aureus 3616: No. 2 (17mm), No. 9 (16mm), for S. aureus 9: No. 4 (27mm), for P. multocida: No. 2 and 5 (12mm each), for K. pneumoniae ATCC 700603: No. 6 and 8 (30mm each), No. 9 (25 mm), for C. albicans: No. 4 (18mm), for Mycobacterium B₅: No. 4 (21mm), No. 2 (20mm), for Acinetobacter sp. 1522: No. 4 (29mm), No.2 (28mm), and for Ps.aeruginosa 835: No. 4, 5 and 8 (30mm each), No. 2 and 6 (28mm each).

A higher antagonistic activity to the majority of test cultures was revealed in association No. 2.

It was found that the growth suppression zones for the Salmonella sp test culture ranged from 19 to 24mm. The growth suppression zones for S. enteritidis 35382 ranged from 12 to 17mm, for S. gallinarum: from 21 to 28mm, for E. coli 8739: from 13 to 18mm, for E. coli: from 11 to 17mm, for S. aureus 6538: from 18 to 21mm. The growth suppression zones for S. aureus 3616 ranged from 16 to 22mm, for S. aureus 9: from 12 to 21mm, for P. multocida: from 14 to 19mm, for K. pneumoniae ATCC 700603: from 14 to 22mm, for C. albicans: from 12 to 15mm, for Mycobacterium B₅: from 20 to 24mm, for B. subtilis: from 16 to 20mm, for Acinetobacter sp. 1522: from 24 to 34mm, and for Ps. aeruginosa 835: from 17 to 27mm.

The most active associations for Salmonella sp. were associations No. 4 and 9 (growth suppression zones of 24mm each), for S. enteritidis 35382: No. 4 (16mm), No. 5 and 9 (15mm each), for S. gallinarum: No. 8 (28 mm) and No. 5(24 mm), for E. coli 8739: No. 4 (18mm) and No. 9(17mm), for E. coli: No. 6 and 8(17mm each), for S. aureus 6538: No. 8(21mm) and No. 4 and 9 (20 mm each), for S. aureus 3616: No. 2 and 9 (22mm), for S. aureus 9: No. 9 (21mm), for P. multocida: No. 8 (19 mm), for K. pneumoniae ATCC 700603: No. 9 (22mm), No. 4, 5, and 8 (20mm each), for C. albicans: No. 2 and 4 (15mm each), for Mycobacterium B₅: No. 8 and 9 (24 mm each), No. 6(23 mm), for B. subtilis: No.2 and 9 (20mm each), for Acinttobacter sp.1522: No. 2 (30mm) and No. 5(32mm), and for Ps.aeruginosa 835: No. 9 (23 mm) and No. 2 (22mm).

Higher antagonistic activity to the majority of test cultures on this nutrient medium was revealed in associations No. 4 and 9.

Associations grown on a nutrient medium containing yeast extract (5.0) + glucose (10.0) + CoCl₂ (0.01) had antagonistic activity against Salmonella sp. with growth suppression zones of the test culture ranging from 19 to 24mm.

The growth suppression zones for S. enteritidis 35382 ranged from 13 to 20mm, for S. gallinarum: from 20 to 25mm, for E. coli 8739: from 11 to 19mm, for E. coli: from 13 to 24mm, and for S. aureus 6538: from 21 to 23mm. The growth suppression zones for S. aureus 3616 ranged from 13 to 18mm, for S. aureus 9: from 17 to 25mm, for P. multocida: from 16 to 22mm, for K. pneumoniae ATCC 700603: from 17 to 30mm, for C. albicans: from 15 to 25mm, for Mycobacterium B₅: from 22 to 28mm, for B. subtilis: from 13 to 20mm, for Acinetobacter sp.1522: from 21 to 32mm, and for Ps.aeruginosa 835: from 23 to 30mm.

The most active associations for Salmonella sp. were No. 6 (growth suppression zone 24mm) and No. 5 (23 mm), for S. enteritidis 35382: No.2 (20mm), for S. gallinarum: No. 4 (25mm), No. 6 (23mm), and No. 2, 5, and 9(21mm each), for E. coli 8739: No. 2 (19mm) and No. 6 and 9(17mm each), for E. coli: No. 6 (24mm), No. 4 and 5(20 mm each), and No. 2 (18 mm), for S. aureus 6538: No. 2 and 12(23mm each) and No. 5 and 6 (22mm each), for S. aureus 3616: No. 9(18mm) and No. 6 (17mm), for S. aureus 9: No. 2(25mm) and No. 4, 5, and 6(20mm each), for P. multocida: No. 4 (22 mm) and No. 5(21mm), for K. pneumoniae ATCC 700603: No. 6 (30mm), No. 9 (26 mm), and No. 2 and 8(24mm each), for C. albicans: No. 2(25mm), for Mycobacterium B₅: No. 4(28mm) and No. 2(25mm), for A cinetobacter sp. 1522: No. 8(32mm) and No. 2, 6, and 9(30mm each), and for Ps.aeruginosa 835: No. 2(30mm), No. 4 and 5 (28mm each), and No. 9(26mm).

The best results on antagonism in this nutrient medium were shown by associations No. 2, 4, 5, and 9.

Associations grown on a nutrient medium containing (g/l): whey + molasses: 10.0+CoCl₂: 0.01, had antagonistic activity against Salmonella sp. with growth suppression zones of the test culture ranging from 16 to 21 mm.

The growth suppression zones for S. enteritidis 35382 ranged from 11 to 17mm, for S. gallinarum: from 11 to 20mm, for E. coli 8739: from 11 to 19mm, for E. coli: from 12 to 19 mm, and for S. aureus 6538: from 11 to 21mm. The growth suppression zones for S. aureus 3616 ranged from 12 to 21mm, for S. aureus 9: from 11 to 18 mm, for P. multocida: from 11 to 15mm, for K. pneumoniae ATCC 700603: from 11 to 30mm, for C. albicans: from 11 to 20mm, for Mycobacterium B₅: from 19 to 29mm, for B. subtilis: from 12 to 20mm, for Acinetobacter sp. 1522: from 23 to 31mm, and for Ps. aeruginosa 835: from 26 to 30mm.

Associations No. 2 and 4 showed the lowest antagonistic activity against the studied test cultures, except for test cultures of Acinetobacter sp. 1522 (where the growth suppression zones were 26 and 28mm) and Ps. aeruginosa 835 (29 and 28mm). Higher activity was noted in associations No. 5 and 9.

The most active associations for Salmonella sp. were No. 9 (growth suppression zone equaling 21mm), for S. enteritidis 35382: No. 5 and 9 (17mm each), for S. gallinarum: No. 5 (20mm) and No. 9 (19mm), for E. coli 8739: No. 6 (19mm), for E. coli: No. 5 and 9 (19 mm each), for S. aureus 6538: No. 9 (21 mm) and No. 5 (20mm), for S. aureus 3616: No. 9 (21mm), for S. aureus 9: No. 5 and 8 (18 mm each) and No. 9 (17 mm), for P. multocida: No. 6 and 9 (15mm each) and No. 5 (14mm), for K. pneumoniae ATCC 700603: No. 5 (30 mm) and No. 6 (28 mm), for C. albicans: No. 9 (20 mm), for Mycobacterium B₅: No. 8 (29mm), for B. subtilis: No. 5 (20 mm), for Acinetobacter sp. 1522: No. 9 (31 mm) and No. 2 (29 mm), and for Ps. aeruginosa 835: No. 9 (30 mm) and No. 2 (29 mm).

Associations No. 5 and 9 showed the best results on this nutrient medium.

Associations grown on a nutrient medium containing (g/l): whey + yeast extract (3.0) + CoCl₂ (0.01), had antagonistic activity against Salmonella sp. with growth suppression zones of test culture ranging from 15 to 26 mm.

The growth suppression zones for S. enteritidis 35382 ranged from 11 to 21 mm, for S. gallinarum: from 15 to 20 mm, for E. coli 8739: from 11 to 20 mm, for E. coli: from 11 to 20 mm, and for S. aureus 6538: from 11 to 21 mm. The growth suppression zones for S. aureus 3616 ranged from 12 to 20 mm, for S. aureus 9: from 12 to 23 mm, for P. multocida: from 11 to 21 mm, for K. pneumoniae ATCC 700603: from 18 to 22 mm, for C. albicans: from 11 to 20 mm, for Mycobacterium B₅: from 12 to 15 mm, for B. subtilis: from 11 to 17 mm, for Acinetobacter sp. 1522: from 20 to 32 mm, and for Ps.aeruginosa 835: from 20 to 38 mm.

Associations No. 2, 4, 6, and 8 showed the lowest antagonistic activity against the studied test cultures, except for the test cultures K. pneumoniae ATCC 700603, Acinetobacter sp. 1522, and Ps.aeruginosa 835.

The most active associations for Salmonella sp. were No. 9 (growth suppression zones equaling 26 mm), for S. enteritidis 35382: No. 9 (21 mm) and No. 5 (16 mm), for S. gallinarum: No. 9 (20 mm), for E. coli 8739: No. 9 (20 mm), for E. coli: No.5 (20 mm), for S. aureus 6538: No. 9 (21 mm) and No. 5 (20 mm), for S. aureus 3616: No. 5 (20 mm) and No. 9 (19 mm), for S. aureus 9: No. 5 (23 mm) and No. 9 (21 mm), for P. multocida: No. 5 (21 mm), No. 9 (17 mm), and No. 12 (15 mm), for K. pneumoniae ATCC 700603: all associations had growth suppression zones from 18 to 22 mm, for C. albicans: No. 12 (24 mm) and No. 9 (20 mm), for Mycobacterium B₅: No. 5 and 9 (15 mm each), for B. subtilis: No. 12 (20 mm) and No. 9 (17 mm), for Acinetobacter sp. 1522: No. 5 (32 mm), No. 6 (30 mm), No. 4 (28 mm), and No. 2 (25 mm), and for Ps.aeruginosa 835: No. 5 (38 mm), No. 6 and 9 (34 mm each), and No. 2 (32 mm).

Associations No. 5 and 9 showed higher activity against the majority of test cultures.

Antagonism of associations of probiotic bacteria when grown on a nutrient medium containing (g/l): yeast extract: 5.0 + glucose: 10.0 + ammonium citrate: 2.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + heptahydrate MgSO₄: 0.2 + MnSO₄: 0.05 + CoCl₂: 0.01.

It was found that the associations grown on this nutrient medium had antagonistic activity against E. coli 8739: from 20 to 26 mm, E. coli: from 12 to 22 mm, and S. aureus 6538: from 18 to 25 mm. The growth suppression zones for S. aureus 3616 ranged from 19 to 25 mm, for S. aureus 9: from 18 to 29 mm, for P. multocida: from 14 to 22 mm, for K. pneumoniae ATCC 700603: from 22 to 32 mm, for C. albicans: from 12 to 24 mm, for Mycobacterium B₅: from 23 to 28 mm, for B. subtilis: from 19 to 25 mm, for Acinetobacter sp. 1522: from 25 to 38 mm, for Ps. aeruginosa 835: from 24 to 38 mm, and for Salmonella sp.:from 25 to 27 mm. The growth suppression zones for S. enteritidis 35382 ranged from 20 to 22 mm, and for S. gallinarum: from 20 to 27 mm.

The most active associations for E. coli were No. 2 (22 mm), for P. multocida: No. 5 (22 mm) and No. 8 (21 mm), and for C. albicans: No. 2 (24 mm). For the rest of the test cultures, all associations had sufficient antagonistic activity.

Associations grown on a nutrient medium containing (g/l) yeast extract: 5.0 + glucose: 10.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + MnSO₄: 0.05 + CoCl₂: 0.01 had antagonistic activity against Salmonella sp. with growth suppression zones of the test culture ranging from 18 to 27 mm.

The growth suppression zones for S. enteritidis 35382 ranged from 14 to 17 mm, for S. gallinarum: from 24 to 32 mm, for E. coli 8739: from 15 to 20 mm, for E. coli: from 16 to 21 mm, and for S. aureus 6538: from 17 to 29 mm. The growth suppression zones for S. aureus 3616 ranged from 19 to 24 mm, for S. aureus 9: from 20 to 27 mm, for P. multocida: from 12 to 22 mm, for K. pneumoniae ATCC 700603: from 16 to 22 mm, for C. albicans: from 10 to 12 mm, for Mycobacterium B₅: from 20 to 27 mm, for B. subtilis: from 19 to 28 mm, for Acinetobacter sp. 1522: from 20 to 38 mm, and for Ps. aeruginosa 835: from 15 to 30 mm.

The most active associations for Salmonella sp. were No. 8 (growth suppression zone 27 mm) and No. 2 (24 mm), for S. enteritidis 35382: No. 2, 5, 6, and 8 (17 mm each), for S. gallinarum: No. 8 (32 mm), No. 4 (31 mm), and No. 6 (28 mm), for E. coli 8739: No. 9 (20 mm) and No. 8 (19 mm), for E. coli: No. 5 (21 mm), for S. aureus 6538: No. 6 (29 mm) and No. 9 (25 mm), for S. aureus 3616: No. 4 (24 mm), for S. aureus 9: No. 6 (27 mm), for P. multocida: No. 2 (22 mm), for K. pneumoniae ATCC 700603: No. 4 (22 mm) and No. 5 and 6 (20 mm each), for Mycobacterium B₅: No. 9 (27 mm), for B. subtilis: No. 4 (28 mm) and No. 2 and 5 (24 mm each), for Acinetobacter sp. 1522: No. 2, 5, 6, 8, and 9 (30-36 mm), and for Ps. aeruginosa 835: No. 6 (30 mm) and No. 2 (27 mm).

Associations No. 2 and 5 had the greatest antagonism on this nutrient medium.

Associations grown on a nutrient medium containing (g/l): yeast water + glucose: 10.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + MnSO₄: 0.05 + CoCl₂: 0.01, had antagonistic activity to all tested test cultures.

At the same time, for Salmonella sp., the growth suppression zones of the test culture ranged from 19 to 29 mm. The growth suppression zones for S. enteritidis 35382 ranged from 21 to 25 mm, for S. gallinarum: from 19 to 21 mm, for E. coli 8739: from 20 to 23 mm, for E. coli: from 16 to 20 mm, and for S. aureus 6538: from 23 to 30 mm. The growth suppression zones for S. aureus 3616 ranged from 19 to 21 mm, for S. aureus 9: from 20 to 29 mm, for P. multocida: from 12 to 20 mm, for K. pneumoniae ATCC 700603: from 25 to 28 mm, for C. albicans: from 12 to 16 mm, for Mycobacterium B₅: from 12 to 25 mm, for B. subtilis: from 19 to 29 mm, for Acinetobacter sp. 1522: from 32 to 34 mm, and for Ps. aeruginosa 835: from 28 to 40 mm.

The most active associations for Salmonella sp. were No. 4 (growth suppression zone 29 mm) and No. 2 and 9 (25 mm each), for S. enteritidis 35382: associations No. 2 and 6 (24 mm each) and No. 4 (25 mm), for E. coli: No. 2, 5, 8, and 9 (20 mm each), for S. aureus 6538: No. 4 (30 mm) and No. 9 (28 mm), for S. aureus 9: No. 2 and 4 (29 mm each), for P. multocida: No. 4 (20 mm) and No. 8 (19 mm), for Mycobacterium B₅: No. 9 (25 mm), and for B. subtilis: No. 4 (29 mm) and No. 2 and 9 (25 mm each). For the test cultures of S. gallinarum, E. coli 8739, K. pneumoniae ATCC 700603, Acinetobacter sp. 1522, and Ps. aeruginosa 835, all associations showed antagonism at a sufficient level.

Associations No. 2, 4, and 9 had the greatest antagonism on this nutrient medium.

The following associations were selected for further research:

No. 2: L. plantarum 2B/A-6 + L. brevis B-3/A-26 + L. acidophilus 27w/60 + P. shermanii 8;

No. 5: L. plantarum 2b/A-6 + L. cellobiosus 2/20 + L. fermentum 27 + P. shermanii 8. + L. brevis B-3/A-26 + L. plantarum 14d/19;

No. 9: L. cellobiosus 2/20 + L. fermentum 27.

These associations were grown on nutrient media of the following composition (g/l):

1 MRS Medium with CoCl₂: 0.01;

4 Yeast extract: 5.0 + glucose: 10.0 + CoCl₂: 0.01;

8 Yeast extract: 5.0 + glucose: 10.0 + sodium acetate: 5.0 + K₂HPO₄: 2.0 + MnSO₄: 0.05 + CoCl₂: 0.01.

The tested associations of probiotic bacteria showed activity against all test cultures taken in the experiment. Associations No. 2 on media 1 and 4, No. 5 on media 1 and 8, and No. 9 on media 1 and 8 showed the greatest activity for most test cultures.

The largest number of probiotic bacteria was found in association No. 2 on nutrient medium 1 (8.5×10¹⁰ colony-forming units (CFU)/ml). Associations in other nutrient media had a bacterial titer of 2.2-3.7x10¹⁰ CFU/ml (Table 1).

Table 1. Titer of bacteria in associations

Medium/ Association	Titer, CFU/ml
1/2	8.5±0.6×10¹⁰
4/2	3.3±0.2×10¹⁰
8/2	3.0±0.2×10¹⁰
1/5	2.6±0.2×10¹⁰
4/5	2.3±0.2×10¹⁰
8/5	2.2±0.4×10¹⁰
1/12	3.7±0.3×10¹⁰
4/12	2.9±0.3×10¹⁰
8/12	2.5±0.3×10¹⁰

The enzymatic activity of the selected associations of probiotic bacteria is presented in Table 2.

Table 4. Resistance of associations of lactic acid and propionic acid bacteria to antibiotics

No. of the item	No. of the medium	No. of the assoc iation	Zones of inhibition of bacterial association growth by antibiotics, mm
No. of the item	No. of the medium	No. of the assoc iation	1	2	3	4	5	6	7	8	9	10	11
1	1	2	7	7	0	0	0	5i	0	0	0	0	9i
2	4	2	8	7	0	0	0	7i	0	0	0	0	10i
3	8	2	8	7	0	0	0	5i	0	0	0	0	9i
4	1	5	12	5i	5	0	0	8i	0	0	0	0	6i
5	4	5	10i	9i	6	0	0	7i	0	0	0	0	6i
6	8	5	10i	6i	5	0	0	0	0	0	0	0	6i
7	1	9	13i	9i	0	0	0	13	0	0	0	0	7i
8	4	9	12i	9i	5i	0	0	14	0	0	0	0	5i
9	8	9	13i	10i	5i	0	0	13	0	0	0	0	7i

Note: 1: ciprofloxacin 30 µg, 2: chloramphenicol 30 µg, 3: ofloxacin 5 µg, 4: dioxicillin hydrochloride 30 µg, 5: ceftriaxone 30 µg, 6: tetracycline 30 µg, 7: trimethoprim 30 µg, 8: cotrimoxazole 25 µg, 9: kanamycin 30 µg, 10: streptomycin 10 µg, 11: gentamicin 30 µg, i: growth inhibition

As can be seen from the table above, the studied bacterial associations were slightly sensitive to ciprofloxacin (30µg), chloramphenicol (30µg), ofloxacin (5 µg), except for association No. 2, tetracycline (30 µg), and gentamicin (30µg).

Dioxicillin hydrochloride (30µg), ceftriaxone (30µg), trimethoprim (30µg), cotrimoxazole (25µg), kanamycin (30 µg), and streptomycin (10µg) did not affect the growth of the studied associations of probiotic bacteria. The obtained results can be used if the complex use of probiotics with antibiotics is necessary^30-32.

CONCLUSION:

We compared six associations of the most active strains of lactic acid and propionic acid bacteria isolated from healthy people. The bacterial strains had antagonistic activity against test cultures of Escherichia coli, Escherichia coli 8739, Salmonella gallinarum, Salmonella sp., Salmonella enteritidis 35382, Staphylococcus aureus 6538, Staphylococcus aureus 3616, Staphylococcus aureus 9, Candida albicans, Klebsiella pneumoniae ATCC 700603, Pasteurella multocida, Pseudomonas aeruginosa 835, Acinetobacter sp. 1522, Bacillus subtilis, and Mycobacterium B₅ and also produced hydrolytic enzymes and B vitamins. Nine variants of nutrient media were used to cultivate bacterial associations.

We found that the tested associations of probiotic bacteria exhibited sufficient antagonistic activity to all test cultures taken in the experiment. The associations showing the highest activity against most test cultures were association No. 2 (L. plantarum 2b/A-6 + L. brevis B-3/A-26 + L. acidophilus 27w/60 + P. shermanii 8) on media 1 (MRS medium with CoCl₂: 0.01g/l), No. 4 (yeast extract: 5.0 g/l + glucose: 10.0g/l + CoCl₂: 0.01 g/l), No. 5 (L. plantarum 2b/A-6 + L. cellobiosus 2/20 + L. fermentum 27 + P. shermanii 8 + L. brevis B-3/A-26 + L. plantarum 14d/19) on media 1 and 8 (yeast extract: 5.0 g/l + glucose: 10.0g/l + sodium acetate: 5.0 g/l + K₂HPO₄: 2.0 g/l + MnSO₄: 0.05g/l + CoCl₂: 0.01 g/l), and No. 9 (L. cellobiosus 2/20 + L. fermentum 27) on media 1 and 8. These associations had a bacterial titer of nx10¹⁰CFU /ml and contained proteolytic, amylolytic, and pectolytic enzymes, as well as B vitamins such as B₃, B₅, B₆, B₈, and B_12. We studied the resistance of selected associations of lactic acid and propionic acid bacteria to the antibiotics used, which will allow them to be used in complex therapy if necessary.

Selected nutrient media and active associations of probiotic bacteria with a wide range of biological activity and antibiotic resistance can be used to create effective targeted medicinal probiotic agents.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 15.08.2022 Modified on 06.10.2022

Research J. Pharm. and Tech 2023; 16(5):2427-2435.

DOI: 10.52711/0974-360X.2023.00400

Item No.	No. of the nutritional medium	No. of the association	Proteolytic activity (casein)	Amylolytic activity (starch)	Pectolytic activity (pectin)
1	1	2	19.0±0.5	20.0±0.5	20.0±0.6
2	4	2	18.0±0.4	30.0±0.6	19.0±0.7
3	8	2	19.0±0.2	36.0±0.4	17.0±0.2
4	1	5	18.0±0.3	22.0±0.5	17.0±0.4
5	4	5	19.0±0.5	35.0±0.7	20.0±0.7
6	8	5	18.0±0.4	20.0±0.5	18.0±0.4
7	1	9	19.0±0.4	18.0±0.2	19.0±0.7
8	4	9	18.0±0.3	17.0±0.7	16.0±0.4
9	8	9	19.0±0.5	18.0±0.4	18.0±0.3

No. of the item	No. of the medium	No. of the association	Growth zone diameter for the vitamin-dependent test cultures, mm				Vitamin B₁₂ content, µg/ml
No. of the item	No. of the medium	No. of the association	B₃ (pantothenic acid)	B₅ (nicotinic acid)	В₆ (pyridoxine)	В₈ (inositol)	Vitamin B₁₂ content, µg/ml
1	1	2	19.0±0.4	17.0±0.3	17.0±0.3	19.0±0.5	0.91±0.01
2	4	2	18.0±0.5	15.0±0.4	15.0±0.4	18.0±0.4	0.87±0.03
3	8	2	20.0±0.5	18.0±0.3	16.0±0.5	19.0±0.3	0.90±0.04
4	1	5	20.0±0.6	17.0±0.4	15.0±0.3	19.0±0.2	0.70±0.03
5	4	5	17.0±0.4	16.0±0.2	16.0±0.4	16.0±0.4	0.60±0.04
6	8	5	19.0±0.5	17.0±0.5	16.0±0.5	14.0±0.3	0.65±0.05
7	1	9	18.0±0.4	19.0±0.5	12.0±0.3	19.0±0.5	0.25±0.04
8	4	9	17.0±0.5	17.0±0.3	12.0±0.4	19.0±0.4	0.20±0.03
9	8	9	18.0±0.3	18.0±0.4	11.0±0.3	18.0±0.5	0.25±0.05