PL235912B1 - Probiotic composition, applications of probiotic composition and the pharmaceutical product - Google Patents

Description

Description of the invention

The present invention relates to a probiotic composition, the use of a probiotic composition and a pharmaceutical product for the treatment and prophylaxis of gastrointestinal damage, necrotizing enteritis and sepsis in neonates.

The proper development of the newborn's digestive, immune systems and brain depends on the rapid colonization of the digestive tract with microbiota components that constitute pioneering organisms, enabling further gradual expansion of the composition of this microbiota in proportion to the expansion of the diet. Under normal conditions, a healthy newborn acquires such ingredients during delivery through the birth canal, i.e. the mother's vagina, and therefore they are mainly bacteria of the genera Lactobacillus and Bifidobacterium. On the other hand, the acquisition of abnormal, pathogenic components is associated both with disorders of the development of these organs, and in extreme cases with life-threatening infections; this is necrotizing enteritis (NEC for short) and infections of the bloodstream. Abnormal colonization mainly affects newborns with low birth weight. A much larger group are newborns with normal weight but born by caesarean section and newborns whose mothers received antibiotics in the perinatal period for the prevention of GBS carriage or prior to cesarean section (or for both). In such situations, the normal maternal vaginal microbiota is destroyed and newborns cannot acquire the correct components of the gastrointestinal microbiota.

Necrotizing enterocolitis is the most dangerous, inflammatory disease of the gastrointestinal tract of newborns, causing significant damage to the intestinal mucosa, requiring intensive treatment and sometimes even surgical intervention. This disease occurs primarily in premature babies. The causes include fetal hypoxia, ischemia, or infectious agents.

WO 01/83700 discloses a composition and method for treating and preventing damage to the digestive system. The composition comprises a combination of Gram (+) bacteria, in particular Lactobacillus and / or Bifidobacterium, and glutamine. The method includes oral or nasal administration of the composition. The composition according to the invention blocks the translocation of bacterial agents such as Gram (-) bacteria and other infectious agents. The composition is intended for the treatment of inflammation of the gastrointestinal tract, necrotic enteritis in newborns and sepsis.

The strains Lactobacillus salivarius ATCC 202196 and Lactobacillus plantarum ATCC 202195 are known from the US patent description US 6,132,710 to normalize the bacterial environment in the gastrointestinal tract. Lactobacillus strains prevent tissue damage from necrotizing enteritis in premature babies.

From the Polish patent description PL 212096, new strains of the species Bifidobacterium infantis capable of surviving in the intestinal tract and producing glutamine and arginine in vivo are known. The invention also relates to a pharmaceutical composition containing new strains of Bifidobacterium infantis and a carrier.

A new strain of Lactobacillus rhamnosus KL 53A is known from the Polish patent description PL 208422, which meets all the requirements for probiotic strains and is intended to supplement the physiological composition of the gastrointestinal flora. This strain cannot be used alone to colonize the digestive tract of newborns devoid of other bacteria.

A probiotic composition containing the Lactobacillus rhamnosus KL 53A strain and its use for the production of a preparation useful in the prevention and treatment of pathological conditions of the gastrointestinal tract is known from the Polish patent description PL 209430. The effective dose of the preparation contains 1x10 ¹⁰ bacteria. Such a large dose is not suitable for the treatment of neonates.

The object of the invention is to develop a probiotic composition containing strains with antioxidant properties, sealing the gut wall, antagonist of bacterial pathogens causing inflammation of the gut, adhering to the intestinal epithelial cells that survive the gastrointestinal passage well. The probiotic composition should contain strains corresponding to the mother's natural bacterial flora, and therefore contain bacteria of the genus Lactobacillus and Bifidobacterium, and its dose should be safe for newborns. The invention also aims to use the product and develop an effective method of colonizing the gastrointestinal tract of newborns.

The essence of the invention is a probiotic composition containing a mixture of Bifidobacterium breve PB 04 strains, deposited in the Polish Microorganism Collection in Wrocław, deposit number

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B / 00028 and Lactobacillus rhamnosus KL 53A, Deposit No. B / 00011, in the ratio of from 1: 1 to 1: 2 in an amount in the range from 1x105 to 1x10 ⁸ CFU.

The invention also relates use of a composition of the probiotic containing a mixture of strains of Bifidobacterium breve PB 04 and Lactobacillus rhamnosus KL 53A in a ratio of from 1: 1 to 1: 2, in an amount ranging from 1x105 to 1x10 ⁸ CFU for the manufacture of a pharmaceutical for the treatment of diseases associated with disturbances of the intestinal bacterial flora.

The invention is also a pharmaceutical product, the dose comprises a mixture of strains of Bifidobacterium breve PB 04 and Lactobacillus rhamnosus KL 53A in a ratio of from 1: 1 to 1: 2 in an amount of from 1x105 CFU to 1x10 ^8, and a pharmaceutically acceptable carrier comprising maltodextrin and ascorbic acid.

The essence of the invention is based on the selection of two unique bacterial strains with outstanding probiotic properties, which, acting synergistically, exhibit a unique set of features particularly beneficial in terms of extinguishing intestinal inflammation and inhibiting the development of bacterial pathogens that contribute to the pathologies of the gastrointestinal tract occurring in the neonatal period, particularly important in low birth weight newborns (so-called premature babies), such as necrotizing enterocolitis, by administering one to two doses per day of the pharmaceutical product immediately after delivery, but no later than within 48 hours, and then during and after hospital stay for a period of 2 to 6 weeks.

The composition has the ability to adhere to intestinal epithelial cells, to survive in the gastrointestinal tract, to stimulate the production of occludin and zonulin leading to sealing of the intestinal wall, and has antioxidant and antagonistic properties against bacterial pathogens of intestinal inflammations.

The present invention is based on two strains: Bifidobacterium breve PB 04 and Lactobacillus rhamnosus KL 53 A.

The Bifidobacterium breve PB 04 strain has been deposited in accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent proceedings at the Polish Collection of Microorganisms (PCM) at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław (53-114 Wrocław, ul. Rudolfa Weigla 12) . The new strain Bifidobacterium breve PB04 was deposited on 04/06/2009 and received the number B / 00028. On March 1, 2013, the depository authority, the Polish Collection of Microorganisms, issued an attestation document regarding the announcement or amendment of the scientific description and / or the proposed taxonomic designation. The attestation carried out confirms the data contained in the deposit document of the Bifidobacterium breve PB 04 strain. The unique properties of this strain were disclosed in the patent description entitled "New strain of Bifidobacterium breve PB 04" P ..... 229020

The properties of the Lactobacillus rhamnosus KL 53A strain were disclosed in the Polish patent specification. "New Lactobacillus rhamnosus strain", Pat. 208422.

I. Investigation of the ability to produce antioxidant enzymes of anti-inflammatory nature.

Reactive oxygen species, including hydrogen peroxide, are responsible for the aging phenomena and disease processes. Most of the lactic acid bacteria in the natural flora of the gastrointestinal tract are able to reduce the risk of reactive oxygen species by inactivating them. However, in the organism in which the inflammatory process takes place, a surplus of reactive oxygen species may occur, in particular hydrogen peroxide, which is produced not only by the cells of the immune system, but also by selected bacteria of the genus Lactobacillus, Streptococus, which may worsen the process. inflammatory. On the other hand, some Lactobacillus strains have antioxidant properties due to their ability to break down hydrogen peroxide. Therefore, it is important to test selected strains for the production and inactivation of reactive oxygen species.

1. Measurement of hydrogen peroxide production by the mixture of probiotic strains.

In order to measure the production of hydrogen peroxide by the tested probiotic strains and their mixture, Merck test strips were used, which changed the intensity of the color,

PL235 912B1 depending on the amount of hydrogen peroxide released into the liquid cultures of these bacteria. The color was compared with the appropriate colorimetric scale allowing at the same time to calculate the approximate value of the produced hydrogen peroxide.

Table No. 1. Production of hydrogen peroxide (mg / l) by probiotic strains of the genus Lactobacillus and Bifidobacterium.

Test strains 4 hours 24 hours Lactobacillus rhamnosus KL53 A 0 0 Bifidobacterium breve PB04 0 0 mixture of Lactobacillus rhamnosus KL53A i Bifidobacterium breve PB04 0 0 negative control TSB broth 0 0

Based on the above results, it can be concluded that both the single strains and the tested mixture of probiotic strains did not exhibit extracellular production of hydrogen peroxide.

2. Measurement of the kinetics of hydrogen peroxide decomposition.

The measurement of the kinetics of hydrogen peroxide decomposition was carried out using two methods, i.e. based on the Merck test strip (colorimetric test changing the intensity of the color depending on the amount of H2O2) and using the Amplex® Red test (10-acetyl-3,7-dihydroxyphenoxazine) which in the presence of hydrogen peroxide turns into an oxidized form. The form of the dye oxidized under the influence of fluorescent light with a length of 590 nm. it releases light, the intensity of which is measured with an appropriate fluorescent reader.

Table no. 2. Determination of the kinetics of hydrogen peroxide decomposition by the tested probiotic strains and their mixture based on the strip method.

Test strains 0 min. thirty min. 60 min. 90 min. 120 min. 150 min. 180 min. 210 min, 24 hours Amount of hydrogen peroxide (mg / l) Lactobacillus rhamnosus KL53A thirty 10 10 3 0 0 0 0 0 Bifidobacterum breve PB04 thirty thirty thirty thirty thirty thirty thirty 10 0 a mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 thirty thirty thirty thirty 10 10 10 0 0 MRS control thirty thirty thirty thirty thirty thirty thirty thirty thirty BL control thirty thirty thirty thirty thirty thirty thirty 10 0

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Table no. 3. Measurement of the kinetics of H2O2 decomposition by the tested probiotic strains and their mixture using the Amplex Red test.

Tested strains - results of fluorescence measurement Kinetics of H2O2 decomposition (fluorescence intensity at 590nm) 1 hour 4 hours 20 h mixture of Lactobacillus rhamnosus KL53A i Bifidobacterium breve PB04 41254 45 223 39 909 Lactobacillus rhmnosus K153A 24 233 48 213 34132 Bifidobacterium breve PB04 25 171 44 688 39 126

Based on the measurements of the kinetics of hydrogen peroxide decomposition based on the strip test and with the use of the Amplex® Red Catalase Assay Kit by Molecular Probes, it can be concluded that the ability to decompose H2O2 by the tested mixture of probiotic strains is comparable to the level of H2O2 decomposition by individual strains.

II. Research on the impact on the improvement of the intestinal barrier function by stimulating the production of tight junctions, in particular occludin and zonulin (ZO-1).

The tight junction proteins control the process of penetration through the mucosa barrier. Increasing the amount of tight junction proteins, in particular zonulin and occludin, seals the epithelium.

Materials and methods.

Single probiotic strains and their mixture were selected for the study. Lactobacillus rhamnosus GG and the enteropathogenic strain of Escherichia coli were used as controls (courtesy of the National Institute of Health - National Institute of Hygiene in Warsaw). Based on the literature data, especially the enteropathogenic strains of Escherichia coli (EPEC) have a very strong effect preventing the formation of tight junction proteins between cells, therefore the EPEC strain was used as a negative control for this study.

By creating mixtures of probiotic strains, bacteria of the genus Lactobacillus and Bifidobacterium were combined in a 1: 1 volume ratio, keeping the proportion of 10: 1, respectively, i.e. 10 ⁷ cfu of Bifidobacterium per 10 ⁸ cfu of Lactobacillus.

The intensity of the illumination was observed in a fluorescence microscope at the wavelength: wzb. 494nm - emi. 520 nm., Under 400x magnification, and the intensity of light was assessed on a semi-quantitative scale. The test results are presented in Tables 4 and 5.

Table 4. Summary of the results of the interaction of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 strains on the stimulation of the production of occludin and zonulin tight junction proteins (ZO-1) produced between cells of the Caco-2 and HT-29 tissue lines.

Test strains Caco-2 tissue HT-29 tissue Occludin Zonulin ZO-1 Occludin Zonulin ZO-1 L. rhamnosus KL53A +++ ++ / +++ +++ + B. breve PB04 +++ ++ ++ - Control Enteropathogenic strain of E. coli (EPEC) L. rhamnosus GG +++ + / ++ ++ -

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Table 5. Summary of the results of the impact of a probiotic mixture with a density of 10 ⁵ cfu on the stimulation of the production of occludin and zonulin tight junction proteins (ZO-1) produced between cells of the Caco-2 tissue line.

Test strains Strain Density Caco-2 tissue Occludin ZonulinaZO-1 a mixture of Lactobacillus rhamnosus KL53A and Bifidobactenum breve PB04 2, lxl0 ⁵ +++ ++ Control: enteropathogenic strain Escherichia coli (EPEC) 2.8x10 ⁵ - Control Lactobacillus rhamnosus GG 3.2x105 +++ ++

Table 6. Summary of the results of the impact of a probiotic mixture with a density of 10 ⁸ cfu on the stimulation of the production of occludin and zonulin tight junction proteins (ZO-1) produced between cells of the Caco-2 tissue line.

Test strains Strain Density Caco-2 tissue Occludin ZonulinaZO-1 a mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 3.1x10 " - -

- no light (no tight junction proteins forming between cells) + low light (low tight junction protein formation) ++ strong light (high level of tight junction protein formation) +++ very strong light (very high level of tight junction protein formation)

Conclusions • Both the Lactobacillus rhamnosus KL53A and Bifidobacteńum breve PB04 strains, as well as their mixture, have a special ability to stimulate Caco-2 and HT-29 tissue cells to produce occludin.

• In the case of close junction proteins from the zonulin group (ZO-1), a significant stimulation of the activity of forming these proteins was observed on the cells of the Caco-2 line after the application of Lactobacillus rhamnosus KL 53A.

• In the case of using the enteropathogenic strain of Escherichia coli (EPEC), a very strong effect was observed, preventing the formation of tight junction proteins between cells of both the Caco-2 and HT-29 lines.

• The tested mixture of Lactobacillus rhamnosus KL53A and Bifidobactenum breve PB 04 strains strongly stimulated Caco-2 cells to produce occludin and ZO-1 zonulin with a population size of about 10 ⁵ cfu / ml.

• When examining the mixtures of Lactobacillus rhamnosus KL 53A and Bifidobacteńum breve PB 04 (weight ratio 1: 1), it was observed that increasing the total number of probiotic bacteria population from 10 ⁵ to 10 ⁸ CFU / ml significantly reduced the ability of intestinal epithelial cells to produce proteins tight connections, i.e. occludin and zonulin ZO-1.

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III. Adherence studies to HT-29 ΜΤΧ and Caco-2 human tissue lines.

The study was carried out according to the methodology given in the work: Surface properties of Lactobacillus strains. II. Adherence to the surface of tissue culture cells. Struś M., Kukla G., Rurańska-Smutnicka D., Przondo-Mordarska A., Heczko P. Med. Exp. Microbiol. 2001; 53 (3): 253-8.

Single probiotic strains of Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 and their mixture in a weight ratio of 1: 1 were selected for the study. The following strains were used as controls: Lactobacillus rhamnosus GG and Bifidobacterium breve DSM 20213.

The preparation was assessed under a light microscope under magnification of 1000x. The cells of the tested bacteria of the genus Lactobacillus and Bifidobacterium, which adhered to the tissue line, were counted in 20 fields of view of the preparation. The mean score was calculated and assigned the appropriate degree of adherence according to the range given below.

The degree of adherence Average number of bacterial cells in the field of view +++ Over 80 ++ Range 60-80 + Range 40-60 - Below 40

Table 7. Adherence of the mixture of bacteria of the genus Lactobacillus and Bifidobacterium to CaCo-2 tissue.

Tested mixture of probiotic strains 1st experiment (results from 20 fields of view of the preparation) II experiment (results from 20 fields of view of the preparation) Average result a mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB 04 strains 110 (+++) 70 (++) 90 (+++) Control 1 Lactobacillus rhamnosus GG 100 (+++) 100 (+++) 100 (+++) Control 2 Bifidobacterium breve DSM 20213 58 (+) 54 (+) 56 (+)

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Table 8. Adherence of Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 strains to CaCo-2 tissue.

Test probiotic strain 1st experiment (results from 20 fields of view of the preparation) II experiment (results from 20 fields of view of the preparation) Average result Lactobacillus rhamnosus KL53A 100 (+++) 100 (+++) 100 (+++) Bifidobacterium breve PB 04 54 (+) 38 (-) 46 (+) Control 1 Lactobacillus rhamnosus GG 95 (+++) 100 (+++) 97 (+++) Control 2 Bifidobacterium longum 68 (++) 75 (++) 71 (++) DSM 20219 Control 3 Bifidobacterium breve DSM 20213 65 (++) 38 (-) 51 (+)

Table 9. Adherence of mixtures of Lactobacillus and Bifidobacterium bacteria to tkanki-29ΜΤΧ tissue.

Tested mixture of probiotic strains 1st experiment (results from 20 fields of view of the preparation) II experiment (results from 20 fields of view of the preparation) SCORE averaged a mixture of strains Lactobacillus rhamnosus KL53A i Bifidobacterium breve PB 04 90 (+++) 80 (++) 85 (+++) Control 1 Lactobacillus rhamnosus GG 100 (+++) 100 (+++) 100 (+++) Control 2 Bifidobacterium breve DSM 20213 49 (+) 51 (+) 50 (+)

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Table 10. Adherence of Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 strains to tkanki-29ΜΤΧ tissue.

Test probiotic strain 1st experiment (results from 20 fields of view of the preparation) II experiment (results from 20 fields of view of the preparation) Average result Lactobacillus rhamnosus KL53 A 100 (+++) 100 (+++) 100 (+++) Bifidobacterium breve PB 04 40 (+) 50 (+) 45 (+) Lactobacillus rhamnosus GG control 100 (+++) 100 (+++) 100 (+++) Control 2 Bifidobacterium longum DSM 20219 78 (++) 72 (++) 75 (++) Control 3 Bifidobacterium breve DSM 20213 45 (+) 51 (+) 48 (+)

Conclusions • The mixture of probiotic strains Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 shows strong adherence properties to both CaCo-2 and ΗΤ-29ΜΤΧ tissue.

• The mixture of probiotic strains has not lost its adherence properties in relation to the adherence of individual probiotic strains.

IV. Study of antagonistic properties against the most common pathogens of the gastrointestinal tract using the quantitative method.

Materials and methods.

The study was carried out in accordance with the methodology given in the work: The scope of antagonistic effect of Lactobacillus strains on the etiological factors of bacterial vaginosis.

Struś M., Malinowska M. Med. Exp. Microbiol. 1999; 51 (1-2): 47-57

Single probiotic strains of Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 and their mixture in a weight ratio of 1: 1 were selected for the study; (abbreviated: KL53A + PB04 mixture). The Lactobacillus rhamnosus GG strain was used as a control. The following bacterial etiological factors (indicator strains) were used in the study:

• Escherichia coli 25922B / 1 (in short: E. coli), • Streptococcus agalactiae B / 3 (in short: S. agalactiae), • Staphylococcus epidermidis B / 2 (in short: S. epidermidis), • Staphylococcus aureus B / 5 (abbreviated: S. aureus), • Enterococcus faecalis (abbreviated: E. faecalis), • Clostridium difficile ATCC 9689 (abbreviated: C. difficile).

The results of the study are presented in the tables below.

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Table 11. Antagonistic effect of the mixture of Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04 (weight ratio 1: 1) against selected indicator bacteria, using the quantitative method.

Probiotic mixture / indicator strain 0 hours after 8 hours after 24 hours mixture KL53A + PB04 4.5 xl0 ⁸ 2.0xl0 ⁸ 2.0xl0 ⁸ E.coli Ι.ΟχΙΟ ⁸ 0 0 mixture KL53A + PB04 5.2xl0 ⁸ 4.5xl0 ⁸ Ι.ΟχΙΟ ⁸ S.agalactiae 2.0xl0 ⁷ 0 0 mixture KL53A + PB04 4.4xl0 ⁸ 2.5Χ10 ⁸ Ι.ΟχΙΟ ⁸ S.epidermidis Ι.ΟχΙΟ ⁸ 0 0 mixture KL53A + PB04 2.0x10® 2.0Χ10 ⁸ Ι.ΟχΙΟ ⁸ S. aureus Ι.ΟχΙΟ ⁸ 0 0 mixture KL53 A + PB04 2.2xl0 ⁸ Ι.ΟχΙΟ ⁸ Ι.ΟχΙΟ ⁸ E.faecalis 2.5xl0 ⁸ 7.0xl0 ⁶ Ι.ΟχΙΟ ² mixture KL53 A + PB04 3.5xl0 ⁸ 4.0xl0 ⁸ Ι.ΟχΙΟ ⁸ C.difficile 2.0xl0 ⁵ 0 0 Control Mixture control KL53A + PB04 6.2xl0 ⁸ 3.5xl0 ⁸ 1.2χ10 ⁸ E. coli control 6.0x10 * 2.0x10 * 1.2x10 * S.agalactiae control 3.0x10 * 3.2x10 * 2.0x10 * S.epidermidis control 5.0x10 * 5.2x10 * 4.0x10 * S. aureus control 8.0x10 * 1.1x10 * 2.0x10 * Control of E.faecalis 3.3x10 * 5.0x10 * 1.0x10 * C.difficile control 1.2x10 * 1x10 * 5x10 *

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Table 12. Antagonistic effect of Lactobacillus rhamnosus GG against selected indicator bacteria using the quantitative method.

Probiotic strain / indicator strain 0 hours ρο 8 hours ρο 24 hours Lactobacillus rhamnosus GG 2.5xl0 ⁸ Ι.ΟχΙΟ ⁸ 1.2χ10 ⁸ E.coli Ι.ΟχΙΟ ⁸ 0 0 Lactobacillus rhamnosus GG 2.5xl0 ⁸ 3.5χ10 ⁸ 1.5χ10 ⁸ S.agalactiae 1χ10 ⁸ 0 0 Lactobacillus rhamnosus GG 2.5x10 ⁸ 3.5χ10 ⁸ 3.0x10 * S.epidermidis 4.1Χ10 ⁸ 0 0 Lactobacillus rhamnosus GG 5.0χ10 ⁸ 3.5χ10 ⁸ 2.8χ10 ⁸ S. aureus ό.ΟχΙΟ ⁸ 2.0x1ο ¹ 0 Lactobacillus rhamnosus GG 2.ΟΧ1Ο ⁸ 2.5χ10 ⁸ 1.5χ10 ⁸ E.faecalis 4.2χ10 ⁸ 8.0Χ10 ⁴ 0 Lactobacillus rhamnosus GG 5.0χ10 ⁸ 4.0χ10 ⁸ 2.5χ10 ⁸ C.difficile 3.0χ10 ⁵ 0 0 Control Control Lactobacillus rhamnosus GG 8.5.Χ1Ο ⁸ Ι.ΟχΙΟ ⁹ Ι.ΟχΙΟ ⁹ E. coli control 6.0x10 * 2.0x10 * 1.2x10 * S.agalactiae control 3.0x10 * 3.2x10 * 2.0x10 * S.epidermidis control 5.0x10 * 5.2x10 * 4.0x10 * S. aureus control 8.Οχ 10 ^κ 1.1x10 * 2.0x10 * Control of E.faecalis 3.3x10 * 5.0x10 * Ι.ΟχΙΟ ⁸ C.difficile control 1.2χ10 ⁵ 1x10 ⁵ 5χ10 ⁵

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Table 13. Antagonistic activity of Lactobacillus rhamnosus KL53A against selected indicator bacteria, using the quantitative method.

Probiotic strain / indicator strain 0 hours CFU / ml after 8 hours CFU / ml after 24 hours, CFU / ml L. rhamnosus KL53A 2.5x10 ^y 1,2x10 ^y 1.3xl0 ^y E.coli 1.1x10 * 0 0 L.rhamnosus KL53A 3.4x10 ^y 1.3xl0 ^y 1.5x10 ⁹ S.agalactiae 8, lxl0 ^b 0 0 L. rhamnosus KL53A 2.5x10 ^y 9.6x10 ⁸ 1.3xl0 ^y S.epidermidis 2.0x10 ' 0 0 L. rhamnosus KL53A 3x10 ⁸ 2.5xl0 ⁸ 1.8xl0 ⁸ S. aureus Ι, ΙχΙΟ ⁸ 2xl0 ⁴ 0 L. rhamnosus KL53A ΙχΙΟ ⁹ 1.5x10 ^y Ι, ΙχΙΟ ⁹ / lxl0 ^y E.faecalis 1x10 ' 3x10 * 0/0 * L. rhamnosus KL53A 2.9x10 ⁹ l, 2xl0 ⁹ 1,2x10 ^y CLdifficile Ι, ΟχΙΟ ⁴ 0 0 Control Control L.rhamnosus KL53A 1.8xl0 ⁹ 5.0x10 ⁸ Ι, ΙχΙΟ ⁹ E. coli control 4.0x10 ' 4.0x10 ' 3.0x10 * S.agalactiae control 4.0x10 ' 3.2x10 ⁸ 1.6x10 ⁸ S.epidermidis control 5.0x10 ' 1.7xl0 ⁸ 4.0x10 ⁸ S. aureus control 3.0x10 ⁸ 1.1Χ10 ⁸ 2.0x10 ⁸ Ent.faecalis control 3.0x10 * 4x10 * 6xl0 ⁷ CLdifficile control 4.0x10 ⁵ lxl0 ^b 5x10 °

LEGEND:

* additional incubation time of 72 hours

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Table 14. Antagonistic activity of Lactobacillus rhamnosus GG against selected indicator bacteria using the quantitative method.

Probiotic strain / indicator strain 0 hours CFU / ml after 8 hours CFU / ml after 24 hours CFU / ml Lactobacillus rhamnosus GG l. 4xl0 ⁸ 8.6x10 ⁹ 7.4x10 * E.coli 1x10 * 0 0 Lactobacillus rhamnosus GG l, 2xl0 ^y 9.3x10 * 7x10 * S.agalactiae 1x10 ' 0 0 Lactobacillus rhamnosus GG 5.2x10 * l. 4xl0 ^y 7x10 * S.epidermidis 1x10 ' 0 0 Lactobacillus rhamnosus GG 5.OxlO ⁸ 3.5x10 ⁸ 2.8x10 ^p S. aureus 6.0xl0 ⁸ 0 0 Lactobacillus rhamnosus GG 7x10 * lx10 ^y 8x10 ⁸ E.faecalis 2x10 ' 2xl0 ³ 0 Lactobacillus rhamnosus GG l, 2xl0 ⁸ 3.5xl0 ⁸ 5x10 * Cl.difficile ΙχΙΟ ³ 0 0 Control Control Lactobacillus rhamnosus GG Ι, όχΙΟ ⁹ 2.4x10 ⁹ lxl0 ⁹ E. coli control 2x10 * 1x10 * 1x10 * S.agalactiae control 2.1x10 ' 5.7x10 * 1.2x10 * S.epidermidis control 1x10 * 3x10 * 1.7x10 ⁸ S. aureus control 8.0xl0 ⁸ 1.1x10 ⁹ 2.0xl0 ⁹ Control of E.faecalis 3x10 * 4x10 * 6x10 ' Cl.difficile control 4x10 ' lx 10 ⁶ 5x10 ^b

Conclusions • By examining the antagonistic effect of the mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 (weight ratio 1: 1), its activity against the selected indicator bacteria was demonstrated.

By analyzing the results of the antagonistic activity of individual probiotic strains, it can be assumed that the reduction of the indicator bacterial population was mainly due to the action of Lactobacillus rhamnosus KL53A metabolites, and to a slightly lesser extent Bifidobacterium breve PB04.

V. Evaluation of antagonistic activity against bacteria of the same kind.

The antagonistic effect of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 strains and their mixtures against bacteria of the same type (Lactobacillus and Bifidobacterium) was determined using the semi-quantitative bar method (Strus M., Pakosz K., Przondo-Mordarska A., Rożynek E., Pituch H ., Meisel-Mikołajczyk F., Heczko PB 2001. Med. Exper. Microbiol. 53: 133-142).

Conclusions

The study showed that:

• The Bifidobacterium breve PB04 strain does not show antagonistic activity against any of the tested indicator strains of the genus Lactobacillus and Bifidobacterium.

• Lactobacillus rhamnosus KL53A has a weak antagonistic effect against Bifidobacterium strains, and no such effect against Lactobacillus rhamnosus strains.

• Based on the above results, it can be concluded that probiotic preparations based on the mixture of the tested strains (Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04) do not exert a mutual, antagonistic effect leading to the reduction of the population of these bacteria.

VI. Study of resistance to artificial gastric juice and bile salts.

In order to determine the degree of resistance of Lactobacillus rhamnosus K153A and Bifidobacterium breve PB04 to gastric pH, the modified Clark method was used (Clark PA, Cotton LN, Martin JH 1993. Manuscript J-8381 of the Mississippi Agriculture and Forestry Experiment Station .; 11-14 ).

The tested strains of Lactobacillus rhamnosus K153A and Bifidobacterium breve PB04, as well as a mixture of these two strains, showed high resistance to low pH and the effect of pepsin in artificial gastric juice. After 20 minutes, these strains reduced their population by only 1-2 logs, which may indicate their perfect adaptation to the unfavorable conditions in the human gastrointestinal tract.

The complete decomposition of bile salts at a concentration of 1.2, 5, 10, 20 g / l was demonstrated by the Lactobacillus rhamnosus strain KL53A and the mixture of Lactobacillus rhamnosus K153A and Bifidobacterium breve PB04. In the case of the Bifidobacterium breve PB04 strain, the bile salt decomposition was typical for the genus Bifidobacterium and the species Bifidobacterium breve, i.e. the strain showed complete bile salt decomposition for a concentration of 1 g / l (+++), and for the remaining concentrations, i.e. 2, 5, 10.20 g / L, a poor bile salt degradation was observed in the semi-quantitative method defined as (+).

VII. The invention will be described in an example concerning the study of the effect of an orally administered mixture of probiotic strains on the composition of the intestinal microflora and on the function of the intestine of gnotobiotic (germ-free) mice.

The experimental model consisting in the oral administration of the test bacteria to gnotobiotic mice was chosen because it is considered the closest to the situation in a newborn with low birth weight and an undeveloped immune system and / or born via caesarean section, whose gastrointestinal tract is not colonized with bacteria from the mother.

The research work included, among others translocation test, i.e. determination of the presence of lactic acid bacteria in the mesenteric lymph nodes, blood and selected internal organs (liver, spleen), as well as the assessment of gastrointestinal colonization by probiotic strains using the breeding method and the fluorescence method of in situ hybridization. Germ-free mice are the best model to verify the safety of probiotic bacteria.

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In a study on germ-free mice, the safety of using a mixture of probiotic strains Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 was proven. In the experimental qualitative and quantitative assessment, it was proved that the tested probiotic strains do not have the ability to exceed / weaken the intestinal barrier and penetrate into the host's organs. The presence of Bifidobacterium breve PB04 was not detected in any of the examined organs.

The effectiveness of colonization was also proven in a study on germ-free mice. The detection of the presence of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 strains in the faeces of mice showed the ability of these bacteria to effectively colonize the gastrointestinal tract. On the first day after colonization, Lactobacillus rhamnosus KL53A was the dominant strain in the intestines and its concentration reached the value of 2.7x109 CFU / g of faeces. The next day of the experiment the number of number of lactobacilli in faeces slightly decreased, while the concentration of Bifidobacterium breve PB04 reached 1,27x10 ¹⁰ CFU / g of feces, and this strain remained predominant in the gut colonized mice. The degree of colonization with probiotic bacteria in various parts of the intestine (ileum, cecum and colon) was also assessed. The results obtained are shown in Fig 1.

Fig. 1 shows the degree of colonization with probiotic bacteria in various parts of the intestine (ileum, cecum and colon).

The animal section showed significant differences in the gut structure of germ-free mice and mice colonized with probiotic bacteria as shown in Fig. 2, where (A) shows the intestines of germfree mice with an extremely distended cecum, while (B) shows the intestines of Lactobacillus rhamnosus KL53A colonized mice and Bifidobacterium breve PB04 with reduced cecum.

The method of fluorescence in situ hybridization (FISH) was based on the hybridization of short-chain nucleotide probes to the complementary fragment of the 16S rRNA ribosomal. These probes are designed for a 16S rRNA fragment that is typical of the respective type of bacteria. At the 5 'end, these probes are labeled with an appropriate fluorescent dye. The FISH method enables the detection of particular types and even species of bacteria directly in the crushed material. The slides were viewed under a fluorescence microscope at 1000x magnification.

Based on the applied fluorescent in situ hybridization method, the presence of Lactobacillus and Bifidobacterium bacteria in the intestinal contents was confirmed in all gnotobiotic (germ-free) mice colonized with probiotic bacteria, and the presence of probiotic bacteria in the intestinal wall was confirmed in four out of five mice receiving probiotics. The number of probiotic bacteria per gram of tissue or faeces was approximately from 1x10 ⁷ CFU to 1x108 CFU. In the control group (germ-free mice that were not administered probiotic bacteria) all materials collected for microbiological analysis gave negative results.

The obtained results confirm the effectiveness of colonization of the intestine of gnotobiotic (germ-free) mice after oral administration of a mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04.

The influence of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 colonization on the expression of tight junction proteins in the intestine was also tested. The expression of occludin and zonulin (ZO-1) was slightly elevated in the group receiving probiotics compared to the group of germ-free mice (but the differences were not statistically significant).

Summarizing the results of the study on germ-free mice, it should be stated that the strains included in the test product are safe and do not show the risk of translocation. The obtained results of microbiological tests also indicate the effectiveness of colonization, and thus the potential benefits of using the product in the group of newborns with low birth weight.

VIII. Studies on the influence of orally administered probiotic bacteria on the intestinal barrier functions of stressed neonatal rats.

The experimental model based on the oral administration of the test bacteria to neonatal rats was chosen because it is considered the closest to the situation in a low birth weight newborn with an undeveloped immune system, whose gastrointestinal tract is not colonized with mother's bacteria and who is stressed, e.g. related to hospitalization in the neonatal intensive care unit.

The study was carried out on 30 newborn Wistar rats. The research procedures started on the second day of life of the young and were carried out in three research groups:

• Control group H - Handled (Control)

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Newborn rats fed on their mother's milk. They were not supplemented with probiotics and were not discontinued. The female did not receive the probiotic.

• SH research group - Stress, Handled (Stress)

Neonatal rats subjected to weaning stress for 180 minutes a day for 13 days. At that time, they were placed in an incubator with a water jacket at 32 ° C (+/- 0.5 ° C). Apart from the natural food of the female, the newborns received sterile water administered once a day in the amount of 100 μΙ. The female did not receive the probiotic.

• SP research group - Stress, Probiotic (Probiotic)

Neonatal rats subjected to weaning stress for 180 minutes a day for 13 days. At that time, they were placed in an incubator with a water jacket at 32 ° C (+/- 0.5 ° C). Apart from the natural food of the female, the newborns were supplemented with an oral probiotic, administered once a day, dissolved in 100 μΙ of water. The female also received a probiotic.

At 15 days of age, newborns from each group were sacrificed by decapitation after anesthesia. Then, the intestines, liver and spleen were dissected under sterile conditions, and the collected materials were subjected to microbiological, immunohistochemical and histopathological analysis.

1. Microbiological examination of the intestinal wall and contents.

These studies were carried out by the breeding method. A qualitative and quantitative microbiological analysis of aerobic and anaerobic bacteria was performed from all biological samples, with particular emphasis on Lactobacillus and Bifidobacterium bacteria.

The following results were obtained:

• Microbiological tests of materials collected from liver and spleen fragments of laboratory animals gave negative results in the SP group (stress + probiotic) and in the SH group (stress). This indicates that there was no translocation of these bacteria from the intestinal lumen with their extensive oral supplementation, even with the simultaneous effect of a strong stress factor.

• When analyzing the mean population numbers of Lactobacillus and Bifidobacterium bacteria per 1 gram of intestinal contents, no differences between the groups were observed. They amounted to about 10 ⁹ CFU / gram of feces for all treatment groups.

• On the other hand, the average number of probiotic bacteria per 1 gram of colon tissue in the SP group (stress + probiotic) increased by 3 logarithms in relation to the other two groups of rats. Such a large increase in the average number of bacteria of the genus Lactobacillus and Bifidobacterium in the SP group per 1 gram of colon tissue may indicate a high adherence of the probiotic strains used to the surface of the colon epithelium.

Table 15. The average number of Lactobacillus and Bifidobacterium bacteria in individual groups of animals (c / gram; CFU / gram) calculated on the basis of the breeding method.

Study groups of rats Average number of Lactobacillus and Bifidobacterium bacteria in individual groups [CFU / gram] lC3ł Large intestine H- no stress (control) 1.2x10 ⁹ 1.5xl0 ⁵ SH- stress 2.9x10 ' 1.6x10 ⁶ SP - stress + probiotic 4.0x10 ' 8.2x10 ⁸

2. Research using fluorescence in situ hybridization (FISH).

The method of fluorescence in situ hybridization (FISH) was based on the hybridization of short-chain nucleotide probes to the complementary fragment of the 16S rRNA ribosomal. These probes are designed for a 16S rRNA fragment that is typical of the respective type of bacteria. On

PL 235 912 '1' 5 'end, these probes are labeled with a suitable fluorescent dye. The FISH method enables the detection of particular types and even species of bacteria directly in the crushed material. The slides were viewed under a fluorescence microscope at 1000x magnification.

Conclusions • Fluorescence in situ hybridization (FISH technique) used to quantify the bacterial flora of the genus Lactobacillus and Bifidobacterium contained in the intestinal contents confirmed similar populations of these bacteria in all three groups of tested neonatal rats.

• In the case of colon tissues, a similar tendency was observed as in the breeding method, i.e. the highest mean value of the Lactobacillus and Bifidobacterium populations was observed in the SP group (stress + probiotic), and the lowest mean value of the Lactobacillus and Bifidobacterium populations in the control group H.

Table 16. Total number of bacteria and total number of Lactobacillus and Bifidobacterium bacteria in individual test groups, calculated by fluorescent in situ hybridization (FISH).

Study group of rats / number of consecutive rat Total Bacterial Count [CFU] Total number of Lactobacillus and Bifidobacterium [CFU] Average result for Group H Intestine content -lxl0 ⁷ Intestine content - 1χ10 ^ϋ Wall j elite-1 χ 10 ⁴ Intestinal wall - 1x10 ^L Average result for the SP Group The content of the intestine -lx10 ^z Intestine content- ΙχΙΟ ⁶ Intestinal wall-1x10 ⁴ f 'J Intestinal wall - 1x10 Average result for the SH Group Intestine content -lx10 ^B Intestine content - 1x10 ⁶ Intestinal wall-1x10 ⁴ Intestinal wall - 1x10

3. Examination of the integrity of the large intestine.

In order to examine the integrity of the large intestine, the presence of tight junction proteins (occludin and zonulin ZO-1) was detected by the fluorescence method. The analysis was performed on paraffin sections made of cut fragments of the large intestine. The intensity of illumination of the connections was observed in the OLYMPUS fluorescence microscope (ΒΧ51) at the wavelength: wzb. 494 nm - emi 520 nm, at 400x magnification, and the intensity of the light was assessed on the semi-quantitative scale.

Luminous intensity according to the half-quantity scale:

- no light (no tight junction proteins forming between cells) + low light (low tight junction protein formation) ++ strong light (high level of tight junction protein formation) +++ very strong light (very high level of tight junction protein formation)

It was found that:

• The strongest signal (indicating the presence of tight junction proteins) was observed on sections collected from neonatal rats from the control group H. In the SP group (stress + probiotic), the intensity of light was slightly weaker.

• In the case of the SH (stress) group, no signal of occludin and zonulin ZO-1 was observed in the tissue fragments taken from the colon epithelium. The lack of a signal in this group may indicate a strong influence of the applied stress factor on limiting the formation of tight junction proteins between intestinal epithelial cells.

• Comparing the SP (stress + probiotic) and SH (stress) groups, it was found that the intensity of tight junction proteins formation may be modified to some extent by bacterial supplementation

PL235 912B1 was probiotic, because in the group which was administered probiotics, many more newborn rats showed the presence of occludin and zonulin ZO-1 in the collected sections of the large intestine.

The obtained results confirm the effectiveness of colonization of the intestines of newborn rats after oral administration of a mixture of probiotic bacteria of the genus Lactobacillus and Bifidobacteńum, ie Lactobacillus rhamnosus KL53A and Bifidobacteńum breve PB04.

4. Histopathological analysis.

Measurements were taken:

• length of intestinal villi, • height of intestinal epithelial cells, • height of mucus droplets in goblet cells.

Statistical analysis was performed using the Statistica 10 program (StatSoft corp., USA). The Kruskal-Willis ANOVA test along with tests for multiple comparisons were used to compare the differences between the groups. A significance level of 0.05 was adopted.

a) Mucus drop height

Tab. 17 Mucus drop height - ANOVA test

Kruskal-Wallis rank ANOVA; mucus drop height Independent (grouping) variable: Kruskal-Wallis test group: H (2, N = 363) = 36.57480 p = .0000 Code N - important Sum - Rank Average - Rank control 1 108 25,080.50 232.2269 stress 2 75 12,942.00 172.5600 probiotic 3 180 28 043.50 155.7972

Tab. 18 Mucus drop height - p values

P-value for multiple (two-tailed) comparisons; mucus drop height Independent (grouping) variable: Kruskal-Wallis test group: H (2, N = 363) = 36.57480 p = .0000 control - R: 232.23 stress-R: 172.56 probiotic - R: 155.80 control 0.000465 0.000000 stress 0.000465 0.735304 probiotic 0.000000 0.735304

The size of the mucus droplets is greatest in the unstressed group. In the case of the stressed group, less variability in results is noticeable. The administration of a probiotic does not normalize the mean values, but increases the spread. The differences between the groups are statistically significant (Tab. 1); in the post-hoc test (Tab. 2) it turns out that the difference between the control group and the stressed group and the group in which the probiotic was used is statistically significant, while the difference between the "stress" group and the "probiotic" group is not statistically significant. The comparison of the results is illustrated in Fig. 3.

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b) The length of the intestinal villi

Table 19 Villus length - ANOVA test

Kruskal-Wallis rank ANOVA; villi length Independent (grouping) variable: Kruskal-Wallis test group: H (2, N = 331) = 46.12594 p = .0000 Code N - important Sum - Rank Average - Rank control 1 114 14,100.50 123.6886 stress 2 80 17427.00 217.8375 probiotic 3 137 23,418.50 170.9380

Table 20 Villus length - p values

P-value for multiple (two-tailed) comparisons; villi length Independent variable (grouping): Kruskal-Wallis test group: H (2, N = 331) = 46.12594 p = .0000 R-control: 123.69 stress - R: 217.84 probiotic - R: 170.94 control 0.000000 0.000295 stress 0.000000 0.001488 probiotic 0.000295 0.001488

The villi length in the stressed group increases; administration of a probiotic leads to a partial normalization of this parameter. The differences between the groups are statistically significant (Tab. 3); in the post-hoc test (Tab. 4) it turns out that the differences between all three groups are statistically significant. The results are shown in Fig. 4.

c) Height of epithelial cells

Table 21 Height of epithelial cells - ANOVA test

Kruskal-Wallis rank ANOVA; epithelial cell height Independent variable (grouping): Kruskal-Wallis test group: H (2, N = 390) = 9.741693 p = .0077 Code N - important Sum - Rank Average - Rank control 1 119 20,814.00 174, 9076 stress 2 80 18,062.00 225.7750 probiotic 3 191 37,369.00 195.6492

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Table 22 Height of epithelial cells - p values

P-value for multiple (two-tailed) comparisons; epithelial cell height Independent variable (grouping): Kruskal-Wallis test group: H (2, N = 390) = 9.741693 p = .0077 R-control: 174.91 stress - R: 225.78 probiotic -R: 195.65 control 0.005406 0.345413 stress 0.005406 0.134341 probiotic 0.345413 0.134341

The height of the epithelial cells in the stressed group increases; administration of a probiotic leads to a partial normalization of this parameter. The differences between the groups are statistically significant (Tab. 5); in the post-hoc test (Tab. 6) it turns out that only the difference between the control group and the "stress" group is statistically significant. The results are shown in Fig. 5.

5. Conclusions.

In the summary of the histopathological analysis, it can be stated that stress causes an increase in the size of intestinal epithelial cells, an increase in the length of the villi and a decrease in the size of mucus droplets. The administration of a probiotic leads to the normalization of these parameters, the normalization being most clearly visible in the case of the size of epithelial cells, while it is small in the case of the size of the mucus droplets.

The experiments showed that none of the strains mentioned that were fed to the mice migrated into their blood, lymph nodes, spleen and liver. On the other hand, the administered bacteria multiplied in high numbers in the small and large intestines (numbers above 10 ⁹ cfu [CFU] / gram for the Lactobacillus rhamnosus KL 53A strain and above 1O ¹⁰ cfu [CFU] / gram for the Bifidobacterium breve PB 04 strain), which is indicated by for effective colonization of the digestive tract. Moreover, the mixture of the tested strains stimulated the synthesis of tight junction proteins in the intestinal epithelium of the tested animals, which indicates their protective properties, consisting in sealing the intestine and preventing bacterial translocation leading to systemic infection.

The conducted studies showed the lack of invasiveness in vivo, and therefore the safety of the tested mixture of strains, as well as their protective properties, expressed by their influence on the tightness of the intestinal wall. Studies on the ability of the assessed bacterial strains to colonize (colonize) the intestines of gnotobiotic mice, carried out by classical breeding methods and, moreover, by the molecular method of fluorescent in situ hybridization (FISH), showed that both strains multiplied on the intestinal wall in high numbers, therefore they effectively colonized the gastrointestinal tract.

The results of the conducted in vitro tests prove that the strains contained in the composition survive the passage through the gastrointestinal tract and colonize it.

Studies carried out on appropriately selected animal models of rats show that the method of preventing disease by administering the composition can be used in inflammation of the gastrointestinal tract, especially in low birth weight newborns.

An embodiment of a pharmaceutical product.

Lactobacillus rhamnosus KL 53A and Bifidobacterium breve PB 04) strains are stored in a freeze-dried form in vacuum ampoules as a master seed lot. Next production pools are drawn up from this pool (working seed lot). This system allows for a certain number of passages for each product series.

The production process is based on the multiplication and lyophilization of properly prepared bacterial suspensions. The cultivation of probiotic bacteria is carried out using a stationary method on a liquid medium (Lactobacillus rhamnosus KL 53A on an acetate medium, Bifidobacterium breve PB 04 on a cistern medium. During centrifugation, the biomass is separated and washed. The obtained bacterial cell pellet is suspended in a medium composed of: maltodextrin, ascorbic acid. and subjected to a freeze-drying process The freeze-drying cycle is conducted in such a way as to maintain all functions

Physiological bacteria characteristic of probiotic strains. The lyophilisate is ground to a specific grain size.

The lyophilisates of the suspensions of both strains are weighed, then combined and mixed until a homogeneous mixture is obtained. The requirements for the mixture are not less than 1 x 10 ⁸ CFU in 100 mg. 1 portion of the product contains: 1 billion lactic acid bacteria in the following proportions: 50% Bifidobacterium breve PB 04, 50% Lactobacillus rhamnosus KL 53 A and auxiliary substances: maltodextrin filler, ascorbic acid - antioxidant.

The mixture of lyophilisates and the excipient - maltodextrin contained in the product are weighed according to the recipe, combined and mixed to obtain a homogeneous powder.

Then the product is dispensed into capsules / vials / sachets.

Product requirements are not less than 1.0 x 106 CFU / dose.

Dosage

The pharmaceutical preparation is administered in the amount of 1 to 2 doses per day throughout the stay in the hospital and after leaving the hospital for 2 to 6 weeks.

Claims (3)

Patent claims 1. Probiotic composition containing lactic acid bacteria, characterized in that it contains a mixture of the Bifidobacterium breve PB 04 strains, deposit No. B / 00028, and Lactobacillus rhamnosus KL 53A, deposit No. B / 00011, deposited in the Polish Microbiological Collection in Wrocław, in the proportion 1: 1 to 1: 2 in an amount ranging from 1x10 ⁵ to 1x10 ⁸ CFU. 2. Probiotic composition containing a mixture of Bifidobacterium breve PB 04 strains and Lactobacillus rhamnosus KL 53A in a ratio of 1: 1 to 1: 2 in an amount ranging from 1x10 ⁵ to 1x10 ⁸ CFU, for the use of the preparation of a pharmaceutical product for the treatment of related diseases with disturbances of the intestinal bacterial flora. 3. Pharmaceutical product containing a composition of lactic acid bacteria, characterized in that the dose of the pharmaceutical product contains a mixture of Bifidobacterium breve PB 04 and Lactobacillus rhamnosus KL 53A strains in a ratio of 1: 1 to 1: 2 in an amount of 1x10 ⁵ to 1x10 ⁸ CFU and pharmaceutically an acceptable carrier containing maltodextrin and ascorbic acid.