SE508045C2 - Adhesion inhibitors, preparations containing the same and process for their preparation - Google Patents

Abstract

Products for inhibiting the adhesion of pathogens to gastrointestinal epithelial mucosa in animals including humans are described. The products contain high molecular weight carbohydrates produced by Lactobacillus strains of enteric origin preferably having a composition of N-acetylglucose-amine:galactose:glucose in a ratio of 1:3:2. There is also described preparations for oral administration comprising as active ingredient the products above, when purified, partially purified, or modified chemically while retaining the biological activity, as well as subunits and complexes thereof which retain the biological activity, or Lactobacillus strains producing the products, or culture supernatant resulting from culturing Lactobacillus strains producing the products. Further, there is described a method of preparation of the products by culturing Lactobacillus strains producing the products.

Description

10 15 20 25 30 508 045 L 2 TABLE 1 Strains used to produce products which inhibits the adhesion of E. coli strains expressing fimbriae Stem Origin Lactobacillus fermentum 104r pig, intestine Lactobacillus murinus C39 pig, intestine Lactobacillus fermentum KLD human, intestine Lactobacillus sp. HBL8 Lactobacillus sp. LMN9 Lactobacillus fermentum 104s Lactobacillus sp. LAB32 human, intestine human, intestine pig, intestine pig, intestine Target organisms are E. coli strains that express ñmbrier. These Umbrians enables the bacteria to adhere to the brush edge of small intestinal epithelial cells and overlying mucus (25). Examples of target organisms are given in Table 2 below.

TABLE 2 E. coli strains expressing various fimbriae and used as target organisms Escherichia coli strain origin K12 PMK 002 (K88 ') pig K12 K88ab (1) pig K12 K88ab (2) pig K12 K88ac pig K12 PMK 005 (K88ac) pig 1107 (K88ac) (wild type) pig K12 K88ad pig K88ad (wild type) pig K99 (wild type) pig 987 P (wild type) pig F41 (wild type) cattle CFA / I human CFA / II human CFA / IV human HB101 (pAZZ 50) (S) human 10 15 20 25 30 35 508 045 3 The products of the invention may be administered to animals, including human, in different ways. Thus, preparations for oral dosing may include it viable Lactobacillus strain as such. However, the preparations can also include the culture supernatant derived from the culture of Lactobacillus the strain or the purified, or partially purified, high molecular weight carbohydrate products produced by the Lactobacillus strain, as well as chemically modified products which still retains the activity of the purified products.

The carbohydrate products can be prepared by growing a strain of Lactobacillus of intestinal origin in complex medium during semi-anaerobic growth conditions. The optimal production takes place in the late log phase and early stationary phase of growth. The products are extracted from the growth medium in which they are released during cultivation.

For use in preparations for oral dosing, the carbohydrate products are mixed preferably with pharmaceutically acceptable ingredients.

The inhibitory activity has been detected in late log phase and early stationary phase. It turned out that more of the active products were present after the death of the cell, which may indicate that the active products are derived from lysed cells. This is also supported by the observation that cells did not die in it medium in which acetate was omitted and no adhesion inhibiting activity could not be observed. Despite the fact that cell wall fragments did not turn out to be inhibitory, removed treatment of retentate fractions with lysozyme den adhesion inhibiting activity. From this it could be concluded that they active components are soluble cell wall fragments from lysed cells.

The inhibitory activity was found to be highest at 37 ° C. Although activity decreases sharply at 0 ° C, it is unlikely that this is caused by a inactivation of the enzyme activity because diluted spent culture fluid does not could increase the inhibitory activity upon extended incubation.

Fractionation by gel filtration shows that a form of the active compound has an Mr value of approximately 1700 kDa. After pronase treatment, Mr to about 1100 kDa, indicating that the active component may be associated with protein. It is not clear whether this is a 600 kDa protein or a smaller protein, which binds several subunits together or gives it active component a conñrmation with a larger Stoke radius. The additional fractions ions with adhesion inhibitory activity found after pronase treatment ling probably contained pronas because this is heat sensitive and of approx same size. 10 15 20 25 30 35 508 045 4 From the above it appears that the molecular weight of the products according to the invention should not be limited to 1700 kDa without products with both higher and lower molecular weights, such as subunits and complexes thereof, are included below provided that they have the same activity.

The active component is probably not proteinaceous, as the activity could still be detected after pronase treatment and after heat treatment at 121 ° C for 20 minutes.

Because lipase treatment interfered with the adhesion assay, it was not possible to exclude the fact that the inhibitory component contained lipid, using the results of the lipase study. After solvent extraction however, the activity was in the aqueous phase and no activity could be observed. be in the chloroform phase. This indicates that the active component is not hydrophobic.

Chemical analysis showed the presence of glucose, N-acetylglucosamine and galactose in a ratio of 1: 3: 2.

In order to investigate how the active component interacts with mucus, E. coli 1107 was treated with retentate and washed. This treatment did not affect the adhesion. If E. coli 1 107 was allowed to adhere to immobilized mucus before retentate treatment, the cells were not removed. This indicates that the component either acts at a site other than the K88 receptor or has one lower affinity for the receptor than KSS fimbriae. The latter possibility is unlikely because concomitant exposure of immobilized mucus to both retentate and E. coli 1107 provides the same adhesion inhibition as pretreatment of mucus with retentat. Without being bound by any theory, it can be assumed that the active component nents have a higher affinity for its place of action than the KSS fimbrias for theirs receptor site. It can therefore be postulated that E. coli 1107 adhesion is inhibited to due to steric obstruction of the active component.

Adhesion to mucus fractions, which contain detectable proteins, can inhibited by treatment with spent culture retentate. Because the adhesion to the neutral lipids from mucus could not be inhibited by retentate, it may be so that the active component only affects the protein receptor. Alternatively can component require non-lipid substances to affect their adhesion neutral lipids and the absence of inhibition of retentate to neutral lipids could be attributed to the method of analysis used.

The invention is described in more detail below in the form of the following non- restrictive embodiments. 10 15 20 25 30 35 508 045 Materials and methods Bacteria and culture conditions L. fermentum strain 104r was isolated from single-layered squamous epithelium from pig stomach (Henriksson et al., 1991). Primary cultures (from storage cultures stored at -80 ° C in 40% glycerol) was allowed to grow overnight in broth (Mann, Rogosa and Sharpe; MRS Oxoid) using a 1% inoculum, 37 ° C and a "candle jar". Cultures were then allowed to grow in a "lactobacillus defined medium". (LDM), produced according to Kotarski & Savage (1979), with the exception that "Tween 80 "was omitted, using a 1% inoculum from the primary culture. Cultures were also allowed to grow in LDM where both "Tween 80" and sodium acetate had been omitted. Consumed culture fluids were recovered by centrifugation of the 24-hour cultures at 10,000 x g for 20 minutes and dialysis (molecular weight limit 12 to 14 kDa), at least 3 times at 4 ° C for about 6 hours, against 5 liters of "Milli-Q" water, purified with "Milli-Q plus" (Millipore Corp.). In order to determine the relationship between the adhesion of spent culture fluid and L. fermentum 104r plant, 10 ml samples were taken from a 250 ml culture at different time intervals and were centrifuged and dialyzed as described above. For- cultured fluids and retentates were stored at -20 ° C before use. As control, the inoculated medium was dialyzed in the same manner as the spent medium the culture fluid.

Primary cultures of E. coli K88ac strain 1107 (hereinafter referred to as E. coli 1107) was grown overnight in tryptone soy broth CTSB; Oxoid) at 37 ° C using a 1% inoculum from stock cultures stored at -80 ° C in 40% glycerol. E. coli 1107 was radiolabeled by inoculation of 5 ml of TSB, containing 1 .mu.Ci.ml / l of methyl-1,2-3H-thymidine. International), with 1% of the primary culture and allowed to grow at 37 ° C to an absorbance (600 nm) of 0.5 in 0.01. Cells were harvested by centrifugation (ca. 3000 x g), washed in phosphate buffered saline (PBS; 10 mM phosphate; pH 7.2) and was resuspended in PBS, and the absorbance at 600 nm was set at 0.5. The effect of the presence of retentate on the adhesion and the effect of retentate on E. coli 1107 was studied by resuspending the cells in retentate (60 min, 37 ° C) and subsequent use of this suspension directly in the adhesion assay or by washing and resuspension in PBS.

In an uitro-adhesion assay The adhesion of radiolabeled E. coli 1107 to the ileum mucus from a 35 10 15 20 25 30 508 045 6 days old pig was studied using a modification of it method specified by Laux m. fl. (1984), as described by Conway et al. (1990). In short, this meant that mucus was recovered from the ileum from a 35 days old piglet in Hepes Hanks buffer (HH; pH 7.4) and stored at -20 ° C or -80 ° C. It was immobilized (0.5 mg protein / ml) on polystyrene microtiter wells (Nunc; polysorp) by overnight incubation at 4 ° C. The excess of mucus was removed by washing twice with 0.25 ml HH. Retentat or fractions from dialysis of spent culture fluid (0.10 ml per well) were added the immobilized mucus and microtiter plates were then incubated at 37 ° C under 1 hour. Wells, which had been similarly treated with unvaccinated dialyzed medium, served as controls. Mucus-coated cells were washed then twice with HH (0.25 ml) and the adhesion of the radiolabeled the cells were studied as previously described (Conway et al., 1990). Inhibitions of the adhesion was evaluated as the percentage of radioactivity at the test in relation to the control, in which mucus was treated with oympat medium. All analyzes were performed in quadruplicate.

Kinetics of the adhesion inhibiting component In order to determine the kinetics of the inhibitory component, were performed adhesion analysis on ice (0 ° C) and at 37 ° C and incubation was performed with spent culture fluid during different time periods. To determine whether enzymatic activity was involved, the spent culture fluid was diluted 10-fold "Milli-Q" water and was also incubated at various time intervals. The rest of the experiment was performed as described above.

Fractionation of spent culture fluid retentates Consumed culture fluid from L. fermentum 104r was fractionated, after growth in LDM medium, by gel filtration of culture supernatant retentates using preparation of a preparative grade of "Superose 6" (Pharmacia). Aliquots of spent culture broth (100 ml) was dialyzed against "Milli-Q" water and concentrated was then filtered approximately 25 times by ultrafiltration using a membrane with a molecular limit of 10 kDA (Millipore). This concentrate lyophilized and resuspended in 2 ml of Milli-Q, thus concentrating the material 50 times. A 1 ml sample of this concentrate was applied to an XK 16/70 "column, filled with" Superose 6 "of preparative quality (Pharmacia) and eluted with PBS at a flow rate of 13.4 ml.cm'2.h'1. The fractionation 10 15 20 25 30 35 7 508 045 was performed at 4 ° C and the eluate was collected as 2 ml fractions. Absorbance (280 nm) was recorded and the carbohydrate content was determined with glucose as standard (Dubois et al., 1956), immediately after fractionation. Concentrated supernatant was also treated with pronase (Calbiochem), 0.1 mg.ml'1, and incubated for 30 minutes at 37 ° C and fractionated as described above.

On a "Con A-Sepharose 4B" (Pharmacia) column, bed volume 18.5 ml, 10 ml retentate was applied, d fate rate 13 ml.cm'2.h'1. The column was washed then with 20 mL of buffer (Tris-HCl, 0.02 M; NaCl 0.5 M, pH 7.4). Finally the column was eluted with buffer (as mentioned above) containing 0.5 M α-D methyl mannose, or with a borate buffer (0.1 M, pH 6.5). The fractionation was performed at 4 ° C and the eluate was collected as 2 ml fractions.

Characterization of the adhesion inhibiting component Prior to the addition of retentate of spent culture broth or medium to the immobilized mucus, in order to detect the presence of adhesion inhibitory activity, the retentate was pretreated in various ways. (i) Retentat and medium were autoclaved at 121 ° C for 20 minutes before being tested at the adhesion assay. (ii) Treatment with lipase (Calbiochem) was performed during use of 4 mg / ml and incubation for 1 hour at 37 ° C, thereafter the sample was boiled for 10 minutes and centrifuged at about 13,000 x g for 5 minutes minutes. (iii) A lipid extraction was performed according to Bligh & Deyer (1959). A 5 ml samples of dialyzed spent culture broth were added dropwise to a mixture of 5 ml of chloroform and 10 ml of methanol. The mixture was stirred for 30 minutes.

After this period, 5 ml of chloroform and 5 ml of "Milli-Q" water and the mixture was stirred for 5 minutes. The mixture was centrifuged for 5 hours minutes at about 3000 x g. After separation, the chloroform was evaporated off under a nitrogen stream and the aqueous phase was lyophilized. Both fractions were redissolved in 5 ml of Q "water. The lipid fraction was sonicated to enhance the solution. (Iv) Lysozyme (Serva) treatment was performed with 2 mg / ml and incubation for 1 hour at 37 ° C, then the samples were boiled and centrifuged as described above for lipase. (v) Cell wall fragments were obtained using L. fermentum cells, which has been allowed to grow in LDM. The cells were harvested in mid log phase by centrifugation (20 minutes at 4 ° C, 10,000 x g) and washed twice with HH. The cells are homo- was generated for 1 hour at 2 ° C in a "Vibrogen-Zellmühle". The homogenate was centrifuged at 25,000 x g, 20 minutes at 4 ° C. The pellet was washed twice and resuspended in HH to one-eighth of the original culture volume 10 15 20 25 30 35 508 045 8 and then frozen (-20 ° C) until use. (vi) For carbohydrate analysis concentrated retentate of spent culture broth 100x and 900 .mu.l were applied on a "Sepharose CL 4B" column after elution with PBS, and active fractions were brought together. Monosaccharide analysis was performed on pooled fractions, such as described by Hardy m. fl. (1988). (vii) Glucose oxidase (Boehringer Mannheim) - treatment was performed with 5.0 mg / ml and incubation for 1 hour at 37 ° C, then the samples were treated with lipase. (viii) Endo-β-galactosidase excretion Mannheim) treatment was performed with 0.05 mg / ml and incubation for 1 hour at 37 ° C, then the samples were treated as described for lipase.

Characterization of the inhibition mechanism Ellipsometry measurements were performed in a "Rudo1ph Research model 436" with vertical test orientation, in order to detect changes in mucus through treatment with retentate. This technique measures changes in the polarization of light then this reflected on a surface. These changes are greatly affected by the presence of barrel films that have been adsorbed to the surface. The principle of ellipsometry is described by Welin (1992). Led by the elliptical angles, polarizer and analyzer, the thickness of the adsorbed film is calculated. Mucus was immobilized on a hydrophobic silicone surface and treated with retentate or medium as in the adhesion assay. Retentates were also radiolabeled by reductive methylation. using the method specified by Jentoff & Dearborn (1979). An aliquot of retentate (6 ml) was mixed with 1 .mu.l of 3H-formaldehyde (37%). NaCNBH 3 (7.6 mg) and Hepes (15.6 mg), pH 7.5, and incubated overnight at 22 ° C. The reaction was stopped by dialysis against "Milli-Q" water. It marked the retentate was tested for adhesion inhibitory activity. Real Estate serous mucus was incubated with the labeled retentate. After 1 hour at 37 ° C the retentate was transferred to scintillation vials and the activity was determined.

The wells were washed twice with 250 μl HH and the activity in the washing liquids determined by scintillation. The wells were then incubated with 250 μl of 1% SDS at 60 ° C. After 1 hour, the contents were transferred to scintillation vials and activity was determined. Complementary contact angles in immobilized mucus was determined with an N RL contact angle goniometer. Mucus was immobilized as in the adhesion assay and the support surface was immersed in HH with the mucosal laid surface down. Air bubbles were released below this surface and their complementary contact angles were determined. Adhesion and inhibition of E. coli 1107 adhesion to neutral lipids were studied in order to determine which receptor is affected by 10 15 20 25 30 9 508 045 retentatet. Neutral lipids (0.5 mg.ml'1 in methanol) from mucus, prepared as described by Blomberg et al. (1993b), was immobilized by evaporation of the solvent. After evaporation, BSA (0.5 mg / ml) and the remainder were added the experiment was performed as described above for mucus.

A 2 ml sample (4.5 mg / ml protein) of freshly prepared porcine ileum mucus fractionated using an "XK 16/70" column, filled with Sepharose CL 4B "(Pharmacia) and eluted with HH at a leaching rate of 15 ml.cm'2.h'1. The eluate was collected as fractions (2 ml each) and monitored by measuring the absorbance (280 nm). Fractions were immobilized as described above for the in vitro adhesion assay, in order to block any uncovered sites, and the wells were incubated for 1 hour at 37 ° C with 0.5 mg.m1'1 BSA, after removal of the fractions. The rest of the adhesion assay was performed as described above.

Results Relationship between growth phase of L. fermentum 104r and inhibition of E. coli 1107 adhesion The adhesion inhibitory effect is only detectable late log phase and early stationary phase of L. fermentum 104r. When a sample, taken after 24 hours of growth, diluted 10-fold, no adhesion inhibitory effect could be observed (94.5% SD 15.21, n = 4); for a sample taken after 86 hours of growth and diluted 10 times however, an adhesion of 54.2% SD 2.3 (n = 4) was observed. When L. fermentum 104r was allowed to grow in LDM without "Tween 80" and sodium acetate, could any sion inhibitory activity is not detected in spent culture fluid nor died cells as fast as in LDM where only "Tween 80" had been omitted, results is not displayed.

Kinetics of the adhesion inhibitory activity The maximum inhibitory activity was obtained after about 15 minutes, when the analysis was performed on ice or at 37 ° C; the adhesion of E. coli 1107 was reduced however, to about 70% and 15% at 0 ° and 37 ° C incubation, respectively. When used up culture fluid was diluted 10 times, the adhesion was reduced to about 90% and remained at this level during prolonged incubation.

Fractionation of culture sugarynate antententate When concentrated retentates of cultures grown in LDM broth, 10 15 20 25 30 sus 045 10 fractionated by gel filtration, the adhesion inhibiting activity was found the fractions corresponding to a relative molecular weight (Mr) of about 1700 kDa.

When the retentate was treated with pronas, the activity of the fractions was detected as corresponded to a Mr of about 1100 kDa. These fractions contained practically no some protein but significant amounts of carbohydrate. After pronas treatment additional fractions with adhesion inhibitory activity could be observed.

This activity was sensitive to heat treatment and has a Mr equivalent it went pronas.

In affinity chromatography with "Concanavaline-A" some activity was possible not recovered from the column, neither by elution with 0.5 M α-D-methylmannose or when eluting with 0.1 M borate buffer.

Characterization of the adhesion inhibiting component Autoclaving of the retentate of spent culture fluid did not affect it adhesion inhibitory effect (autoclaved adhesion 6.78% SD 0.04 compared to untreated adhesion 7.23% SD 1.46, n = 3). Results from lipase treatments could not be interpreted as these enzyme treatments interfered with adhesion the analysis when both treated medium and retentate caused a 1.5- to 3-fold increase increase in adhesion compared to wells that had been exposed to untreated medium. After lipid extraction, very little inhibitory activity was found in the chloroform phase (94.50% adhesion SD 10.35, n = 3) with most of the activity present in the water-methanol phase (34.7 6% adhesion SD 24.02, n = 3). Treatment with lysozyme, the activity was totally removed (100.46% SD 1.42, n = 3). Contrary in addition, cell wall fragments had a stimulating effect on the adhesion in concentrates. higher than the original culture concentration and no effect when diluted to the initial concentration. Carbohydrate analysis of retentate fractions of spent culture revealed the presence of N-acetylglucosamine, galactose and glucose in a ratio of 1: 3: 2 in the most active fractions.

Based on these results, retentates were treated with glucose oxidase and endo-β galactosidase. Glucose oxidase removed most of the activity (81.90% SD 9.37, n = 4), while endo-β-galactosidase did not affect the adhesion inhibitory activity. teten (27.51% SD 9.38, n = 3).

Characterization of the inhibition mechanism Using ellipsometry, no change could be detected in -2 thickness of the absorbed mucus membrane (4.3 ng.mm immobilized material). 10 15 20 508 045 11 Retentate was labeled radioactively for the purpose of determining whether material in the retentate remained on mucus after washing. Of the total radioactivity in the retentate which had been added to mucus, 92.61% SD 0.38 (n = 3) remained in the retentate when recovered from the wells and 4.90% SD 0.37 (n = 3) of the activity was in the washing liquids. Only 2.40% SD 0.15 (n = 3) of the applied radioactivity was directly associated with the mucus layer and was recovered after solubilization with 1% SDS. As a control, it was established that the labeling of the retentate did not affect it adhesion inhibitory activity (labeled retentate allowed 54.26% SD 4.35 adhesion compared to 45.37% SD 5.98 for unlabeled retentate, n = 2). It did not exist any detectable difference in the contact angle of mucus, treated with retentate, in relation to the untreated control. Immobilized mucus had one complementary contact angle of 20.22 ° SD 5.71 (n = 68), while mucus treated with retentate had a complementary contact angle of 15.83 ° SD 5.52 (n = 52). E. coli 1107 cells, which adhered to mucus not treated with retentate, were removed not by subsequent addition of retentate (adhesion 99, 15% SD 0.78, n = 2).

However, the adhesion was inhibited when E. coli 1107 cells were resuspended in retented and applied to the immobilized mucus (15.00% SD 2.22, n = 2).

This inhibition was removed by washing the retentate from the cells before the adhesion assay (85.01% SD 8.51, n = 2). Adhesion to neutral mucus lipids could not be inhibited by retentate (99.1% SD 6.1, n = 2). The number of cells that adhered neutral lipids were about 58% of those who adhered mucus. E. coli 1107 cells adheres to most of the mucus fractions. 10 15 20 25 30 508 045 12 Summary of properties of an example of the adhesion inhibitor Size Thermal stability Chemical composition peljodat resistance to enzymatic activity: lipas pronas lysozyme endo-β-galactosidase glucose oxidase Concanavalin A Anion exchanger absorption on activated carbon absorption to 104r at low pH solubilization with detergents Precipitation: ammonium sulfate PEG-GOOO PEG-4OO cold acetone Lipid extraction Production medium ca 1 700 000 Da (gel filtration) 20 minutes at 121 ° C N-acetylglucosamine: galactose glucose (1: 3: 2) No inhibition of 104r cell walls incomplete results unknown, interferes with adhesion analysis yes No yes reduced activity, mucus destroys activity no activity to recycle no activity to recycle call recycled activity No detergents (SDS, Na-deoxycholate, Brij 58) interferes with adhesion analysis no precipitated activity no precipitated activity no precipitated activity no precipitated activity activity in the aqueous phase late log / early stationary phase several LAB of intestinal origin high acetate concentration is required no production when growing in mucus plant production at several C sources (glucose, arabinos, galactose) depending on biomass formation Mechanism 5 10 15 Activity 508 045 13 no inhibition of adhesion to neutral mucus lipids (lipid receptor) no binding of radiolabeled supernatant natant no binding of HRF-labeled mucus to immobilized supernatant no reduction in HPR activity from the HPR marked mucus no change in immobilized layer thickness (ellipsometry) affects only specific protein peaks in the fraction tioned mucus active at 37 ° C, not 0 ° C no release of adherent E. coli 1107 cells adhesion inhibition with both immobilized and soluble mucus against KSSab / ac / ad and S ñmbrier. 10 15 20 25 30 85 508 045 14 References: Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and puriñcation, Canadian Journal of Biochemistxy and Physiology, 37: 911-917.

Blomberg, L., Henriksson, A. & Conway, P. L. (1993a) Inhibition of adhesion of Escherichia coli K88 to piglet ileal mucus by Lactobacillus spp., Applied and Environmental Microbiology, 59: 34-39.

Blomberg, L., Krivan, H. C., Cohen, P. S. & Conway, P. L. (1993b) Piglet ileal mucus contains protein and glycolipid (galactosylceramide) receptors specific for Escherichia coli K88 fi mbriae, Infection and Immunity, 612526-2531.

Conway, P. L., Welin, A. & Cohen, P. S. (1990) Presence of K88 specific receptors in porcine ileal mucus is age dependent, Infection and Immunity, 583178-3182.

Dubois, M., Gilles, K. A., Hamilton, J.K., Rebers, P. A. & Smith, F. (1956) Colorimetric method for determination of sugars and related substances, Analy- tical Chemistry, 281350-3556.

Gaastra, W. & de Graaf, F. W. (1982) Host-specific b mbrial adhesins of noninva- sive enterotoxigenic Escherichia coli strains, Microbiological Reviews, 46: 129-161.

Henriksson, A., Szewzyk, R. & Conway, P. L. (1991) Characteristics of the adhesive determinants of Lactobacillus fermentum 104, Applied and Environ- mental Microbiology, 57: 499-502.

J entoft, N. & Dearborn, D. G. (1979) Labeling of proteins by reductive methyl- tion using sodium cyanoborohydride, Journal of Biological Chemistry, 11: 4359- 4365.

Jonsson, E. 8: Conway, P. L. (1992) Development of probiotics for pigs, p. 260- 316. In R. Fuller (ed.), Probiotics. Chapman & Hall, Ltd., London.

Kotarski, S. F. & Savage D. C. (1979) Models for study of the specificity by which indigenous lactobacilli adhere to murine gastric epithelia, Infection and Immu- nity, 26: 966-975.

Laux, D.C., McSwegean, E. F., Williams, T. J., Wadolkowski, E. A. & Cohen, P. S. (1986) Identification and characterization of mouse small intestine mucosal receptors for Escherichia coli K-12 (K88), Infection and Immunity, 52: 18-25.

Lindgren, S. E. & Dobrogosz, W. J. (1990) Antagonistic activities of lactic acid bacteria in food and feed fermentations, FEMS Microbiology Reviews, 87: 149-164.

Welin Klíntström, S. (1992) PhD. thesis. Linköping University, Linköping, Sweden.

Claims (7)

10 15 20 25 30 508 045 1 5 PATENT CLAIMS Products for inhibiting the adhesion of pathogens to gastrointestinal epithelial mucosa in animals, including humans, characterized in that they are high molecular weight carbohydrates produced by Lactobacillus strains of intestinal origin and derivatives thereof. Products according to claim 1, characterized in that they have been produced from Lactobacillus strains derived from the gastrointestinal tract of the host to which the products are to be administered. Products according to claim 1 or 2, characterized in that they have a molecular weight of about 200-1700 kDa and a composition of N-acetylglycosamine: galactose glucose in a ratio of 1: 3: 2. Preparation for inhibiting the adhesion of pathogens to gastrointestinal epithelial mucosa in animals, including humans, characterized in that the active ingredient is selected from the group consisting of viable Lactobacillus strains of intestinal origin, producing high molecular weight carbohydrate products, wherein culture supernatant of Lactobacillus strains of intestinal origin, which produce high molecular weight carbohydrate products, and purified, partially purified or chemically modified forms of, or subunits or complexes with retained biological activity of high molecular weight carbohydrate products, produced from Lactobacillus strains of intestinal origin. A composition according to claim 4, characterized in that the Lactobacillus strains are derived from the gastrointestinal tract of the host to which the composition is to be administered. Preparation according to Claim 4 or 5, characterized in that the high-molecular-weight carbohydrate products have a molecular weight of approximately 200 to 1700 kDa and comprise N-acetylglucosamine / galactose glucose in a ratio of 11322. Methods of preparing products for inhibiting the adhesion of pathogens to gastrointestinal epithelial mucosa in animals, including humans, which products are high molecular weight carbohydrates, characterized in that a strain of Lactobacillus is grown in complex medium under semi-anaerobic growth conditions, wherein sos 045 The optimal production of the products takes place in the late log phase and early stationary phase of the plant, and the desired products are extracted from the growth medium in which they are released during cultivation.