WO2008143527A1 - Probiotic bacterium - Google Patents

Abstract

A composition to help or improve gastrointestinal health with includes L. plantarum AGR 1526 which is publicly available from DSM under accession number DSM 2648.

Description

PROBIOTIC BACTERIUM

TECHNICAL FIELD

The present invention relates to a probiotic bacterium. In particular the present invention broadly relates to an isolated strain of a bacterium which is particularly beneficial to the gastro-intestinal barrier of host animals.

BACKGROUND ART

The gastrointestinal barrier (GIB) is the tergest interface between man and his external environment. It acts as a "biological bouncer" that protects the host from the entry of bacteria and antigens, and hence, it is critical in maintaining health and wellness. The GIB is comprised of physical,¹' ² chemical,³ immunological^4"6 and microbiologic barriers.⁷ In humans, GIB integrity is compromised in conditions such as Inflammatory Bowel Diseases (Crohn's Disease and Ulcerative Colitis)⁸' ⁹, Irritable Bowel Syndrome¹⁰ and some kinds of food-borne - infections¹¹' ¹². GIB integrity also deteriorates with aging¹³' ¹⁴ and can be temporarily impaired during times of stress¹⁵. In animals, impaired GIB integrity is often associated with acute or chronic inflammation and diarrhoea, such as calf scours, which leads to, at best, impaired growth and reduced productivity.

It is well accepted that "bad" bacteria, such as Salmonella,™ present in the intestinal lumen can negatively affect GIB integrity; however, it is less well known that "good" bacteria, often referred to as "probiotics", can have a positive effect on gastrointestinal health. There are numerous products on the market that claim to contain probiotic bacteria but few of these are backed up with robust scientific evidence. After extensive searching we found seven probiotic products containing strains of bacteria that had data regarding their efficacy published in international peer-reviewed scientific journals. These published studies are summarised in Table 1.

Table 1 - Scientific evidence for the efficacy of commercial probiotic products.

We also found a probiotic mixture, VSL#3, which contains eight strains of bacteria - Lactobacillus easel, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbruekii subspecies bulgarius, Bifidobacterium Iongum, Bifidobacterium breve, Bifidobacterium infantis, and Streptococcus salvaήus subspecies thermophilus - that is backed up by scientific research. The data on the efficacy of the VSL#3 probiotic mixture is summarised in Table 2.

Table 2 - Scientific evidence for the efficacy of VSL#3.

As indicated by the variation in data presented in Tables 1 and 2, the efficacy of probiotic strains can be measured using numerous different in vitro bioassays and in vivo animal models.

The inventors devised to ascertain whether they could possibly identify any probiotic bacterial strains that had a greater positive effect on GIB integrity than the commercial probiotic strains identified. To do this, we firstly compared the commercial probiotic strains to determine which had the greatest positive effect, then we used this best commercial probiotic strain as a benchmark to compare numerous potential probiotic strains.

For the initial screening for beneficial effects we used the trans-epithelial electrical resistance (TEER) assay⁵⁹' ⁶⁰. We choose this assay because it measures the integrity of the tight junctions between the intestinal epithelial cells (physical barrier) which we believe is a primary determinant of gastrointestinal health. Further details on the TEER assay are given in the materials and methods section. Surprisingly, the inventors found a probiotic strain of Lactobacillus plantarum which not only performs as well as existing probiotic bacteria but out performed such strains in terms of enhancing the integrity of the GIB as measured by the TEER assay.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve_, the right to challenge the accuracy and pertinence of the cited documents. It wilh be clearly understood that, although a number of prior art publications are referred to herein; this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

In general the diseases, ailments, or disorders of the GIB to which the present invention has application are those associated with deficiencies in the integrity of the GIB. The use of probiotic bacteria to assist with gastrointestinal health and the GIB is well known. The inventors have however surprisingly discovered that a particular known strain of Lactobacillus plantarum, namely Lactobacillus plantarum AGR 1526 has a significant positive effect on the integrity of the GIB. In particular, selection of Lactobacillus plantarum AGR 1526 has been shown to positively effect the integrity of the GIB and perform better in this regard than known scientifically proven probiotics tested in this assay, and to exhibit a positive effect for a longer period of time that these well known probiotics, such as Lactobacillus rhamnosus HN 001

According to one aspect of the present invention there is provided a composition to help or improve gastrointestinal health with includes L. plantarum AGR 1526 which is publicly available from DSM under accession number DSM 2648.

According to a second aspect of the present invention there is provided a use of L. plantarum AGR 1526 which is publicly available from DSM under accession number DSM 2648 to manufacture a composition to improve or help maintain gastrointestinal health.

According to a third aspect of the present invention there is provided a use of L plantarum AGR 1526 as publicly available from DSM under accession number DSM 2648 to manufacture a composition to improve or help maintain the integrity of the gastrointestinal barrier.

According to a fourth aspect of the present invention there is provided a method of preventing, alleviating and/or treating a disease, ailment, or disorder associated with a gastrointestinal disease comprising:

- administering an effective amount of Lactobacillus plantarum AGR 1526 to a person or animal.

According to a fifth aspect of the present invention there is provided a method of preventing, alleviating and/or treating a disease or ailment associated with a gastrointestinal disease comprising:

- administering a composition which includes an effective amount of Lactobacillus plantarum AGR 1526 to a person or animal.

According to a sixth aspect of the present invention there is provided the use of Lactobacillus plantarum AGR 1526 as a food or drink additive.

According to a seventh aspect of the present invention there is provided the use of Lactobacillus plantarum AGR 1526 as an animal feed or drink additive.

According to an eighth aspect of the present invention there is provided a use of Lactobacillus plantarum AGR 1526 to manufacture a medicament for the prevention, alleviation and/or treatment of a disease, ailment, or disorder associated with the gastrointestinal barrier.

According to a ninth aspect of the present invention there is provided a use of Lactobacillus plantarum AGR 1526 as a probiotic.

According to a tenth aspect of the present invention there is a composition which includes Lactobacillus plantarum AGR 1526 to improve or help maintain the integrity of the gastrointestinal barrier.

According to an eleventh aspect of the present invention there is provided a composition which includes an effective amount of Lactobacillus plantarum AGR 1526 to prevent, alleviate and/or treat a disease, ailment, or disorder associated with the gastrointestinal barrier.

According to a twelfth aspect there is provided a use of Lactobacillus plantarum AGR 1526 to reduce serum amyloid A levels in a person or animal.

According to a thirteenth aspect there is provided a use of Lactobacillus plantarum AGR 1526 in the manufacture of a composition to reduce serum amyloid A levels in a person or animal.

Preferably, compositions including the present invention will be formulated for oral administration.

As used herein the term "isolated" means removed from the natural environment in which the bacteria naturally occurs and is separated from some or all of the coexisting materials in the natural system from which the bacteria has been obtained.

As used herein the term "disorder" refers to any abnormality in normal function.

Compositions of the present invention may be formulated in a variety of different ways without departing from the scope of the present invention. In general the type of formulation chosen will be dependent on the end application.

The "effective amount" of Lactobacillus plantarum AGR 1526 to be delivered may vary, but can be easily determined by those skilled in the art, via routine trial and experimentation and/or by reference to existing probiotic products or literature thereon. The present invention may preferably include formulations suitable for direct application to grass or animal feed e.g. spray form.

Other suitable formulations will be known to those skilled in the art.

Thus the selection of L. plantarum AGR 1526 as a probiotic bacterium provides an enhanced effect on GIB integrity over known probiotics, as well as providing the public with a useful choice.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 Schematic diagram of TEER assay setup. TJ = tight junctions;

Figure 2 Photograph of TEER assay setup;

Figure 3 Schematic diagram of the Endohm12 used to measure the resistance across the epithelial cell monolayer;

Figure 4 Images of pulse-field gel electrophoresis of L plantarum strains.

Figure 5 16s rRNA sequence of AGR 1526;

Figure 6 Images of pulse-field gel electrophoresis of AGR 1526 compared to commercial L. plantarum strains.

Figure 7 Change in trans-epithelial electric resistance (TEER) (± SEM, n=4) across Caco-2 monolayers over time in the presence of commercial probiotic bacterial strains

Figure 8 Change in trans-epithelial electric resistance (TEER) (± SEM, n=4) across Caco-2 monolayers over time in the presence of the best commercial strain (L. rhamnosus HN001) and the probiotic bacterium AGR 1526.

Figure 9 Graphs showing the viable cell counts of the bacterial strains in acidic conditions over time. Values are means (n=3) ± SEM. Figure 10 Graphs showing the viable cell counts of the bacterial strains in exposed to bile over time. Values are means (n=3) ± SEM.

Figure 11 Graphs showing the viable cell counts of the bacterial strains in exposed to pepsin over time. Values are means (n=3) ± SEM.

Figure 12 Graphs showing the viable cell counts of the bacterial strains adhered to undifferentiated and differentiated Caco-2 cells over time. Values are means (n=3) ± SEM.

Figure 13 Graphs showing the viable cell counts of the bacterial strains adhered to HEp-2 cells over time. Values are means (n=3) ± SEM.

Figure 14 The treatment group (n=30) was given a composition including AGR1526 in phosphate buffered saline (PBS, pH 7.3), twice daily. Control calves (n=30) received PBS only. SAA was determined from plasma using an ELISA assay as described previously.

BEST MODES FOR CARRYING OUT THE INVENTION

Potential probiotic strains

Previous worked showed that Lactobacillus plantarum had a positive effect on GIB integrity so we choose to test numerous strains of L. plantarum to determine if all strains of this species had a similar effect or if the effects were strain specific. The strains of L. plantarum were obtained from the German culture collection Deutsche Sammlung von Mikroorganismen (DSM).

Pulse-field gel electrophoresis

A 1% (v/v) inoculum of a stationary phase culture was added to 10 ml of fresh MRS broth and allowed to grow overnight. Cells from 1.5 ml samples of the culture were harvested by centrifugation, washed once in 1M NaCI, 10 mM Tris.CI (pH 7.6), and resuspended in 300 μl of the same solution. This was mixed with an equal volume of 2% low melting point agarose (Bio-Rad Laboratories, Richmond, CA.) in 0.125 M EDTA (pH 7.6), and left to solidify in moulds (Bio-Rad). Agarose blocks were removed from the moulds and the cells were lysed by incubating the agarose blocks for 18 hours at 37° C in EC buffer (1 M NaCI, 6 mM Tris.CI, 100 mM EDTA, 1% (w/v) sarkosyl, pH 7.6) containing 5 mg ml^'1 lysozyme. The agarose blocks were then incubated with proteinase K (1 mg ml^"1 in 0.5 M EDTA, 1% sarkosyl, pH 8.0) for 24 hours at 37° C, and with 1 mM phenylmethylsulfonyl fluoride (PMSF) in TE 10/1 (10 mM Tris.CI, 1 mM EDTA, pH 8.0) for 2 hours at 37° C. After treatment the agarose blocks were stored in TE 10/100 (10 mM Tris.CI, 100 mM EDTA, pH 8.0) at 4° C until required.

Slices (1-2 mm) were cut from the agarose blocks with a sterile coverslip, and washed twice with gentle shaking in TE 10/0.1 (10 mM Tris.CI, 0.1 mM EDTA, pH 8.0) at room temperature. Slices were_, transferred to microcentrifuge tubes, washed once at 4° C with restriction enzyme buffer, and incubated for 16-18 hours with the appropriate restriction enzyme in a total volume of 100 μl. The enzymes used to differentiate strains of L plantarum were Asc\ and I-Ceul (New England Biolabs, Beverly, MA).

Following digestion, the slices were washed once with TE 10/1 for one hour at 4° C and loaded into the wells of a 1% agarose gel (Bio-Rad pulsed field certified agarose prepared in 0.5X Tris-borate buffer). The wells were sealed with agarose and the gel run in 0.5X Tris-borate buffer using a CHEF DR III pulsed-field gel electrophoresis apparatus and model 1000 mini chiller (Bio-Rad). Gels were run at 200V for 20 hours at 14° C, and with the pulse time ramped from 1 to 30 seconds for Asc\ digests, and from 10 to 100 seconds for I-Ceul digests. Multimers of bacteriophage lambda prepared for use as pulsed-field gel molecular size standards (New England Biolabs) were used to give an indication of fragment sizes. Gels were stained with Gelstar nucleic acid gel stain (Cambrex Bio Science, Rockland, ME), destained with water, and photographed using a Kodak Gel Logic 200 Imaging System (Eastman Kodak, Rochester, NY).

16s rRN A sequencing

The bacterial strains were identified based on their 16s rRNA sequences. From the isolated genomic DNA the 16s rRNA was amplified using with FD1 (5'- AGAGTTTGATCCTGGCTCAG-S¹) and RD1 (5'-AAGGAGGTGATCCAGCC-S') primers and PCR supermix (Invitrogen, Auckland) using a Thermo Hybaid PX2 thermocycler (Thermo Electron Corporation) and purified using a QIAquick PCR purification kit (QIAGEN). The florescent labelled DNA was prepared using PCR with Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and purified using ethanol/EDTA precipitation. The DNA sequencing was carried out by the Allan Wilson Centre Genome Service at Massey University, Palmerston North, New Zealand. The DNA sequence contigs were aligned using the ContigExpress Vector NTI software (Invitrogen). The determined sequences were compared to known bacterial sequences using the NCBI Blast database.

In vitro bioassay

To test whether the bacterial strains had a positive effect on the integrity of the gastrointestinal barrier the in vitro trans-epithelial electrical resistance (TEER) assay was used. Figures 1 and 2 illustrate the assay setup. The TEER is a function of the strength of the tight junctions between the epithelial cells of the monolayer. When the tight junctions are well formed it is more difficult for the current to pass through the monolayer of cells so the TEER increases. Conversely, when the tight junctions are compromised the current can pass more easily through the monolayer so the TEER decreases.

Caco-2 cells (a human intestinal epithelial adenocarcinoma cell line) were grown on semi-permeable membranes for 5 days until they reached confluence and formed tight junctions between adjacent cells. The Caco-2 cells were grown in M199 with 10% foetal bovine serum, 1% non-essential amino acids and 1% penicillin- streptomycin solution at 37° C in 5% CO₂. The monolayers were prepared the day before the TEER assay by removing the medium, washing three times with phosphate buffered saline and adding M199 with 1% non-essential amino acids, without foetal bovine serum and penicillin-streptomycin to ensure growth of the bacterial strains.

Before the TEER assay, the bacterial strains were grown overnight in MRS broth at 37 ⁰C in 5% CO₂ (except the E. coli K12 negative control which was grown in LB broth aerobically). The bacterial cells were collected by centrifugation and suspended in tissue culture media (M199 with 1% non-essential amino acids) to the required optical density at 600 nm. At this wavelength the turbidity of the solution is proportional to the bacterial cell concentration.

The bacterial solutions were added to the top to the monolayer of Caco-2 and the TEER was measured every 2 hours for 12 hours. For each experiment three bacterial strains were tested and the following controls were included; tissue culture media without bacteria added, positive bacterial strain (L. plantarum VSL#3 for commercial strain testing and the best commercial strain for the experimental strain testing) and negative bacterial strain (E. coli K12). Each bacterial strain was tested in quadruplicate.

To measure the resistance across the monolayers, each insert was lifted into an electrode chamber with electrodes at the top and bottom (ENDOHM-12 tissue culture chamber, World Precision Instruments, Florida, USA) using sterile tweezers and the resistance was measured using a voltohmmeter (EVOM Epithelial Tissue Voltohmmeter, World Precision Instruments, Florida, USA). A schematic diagram of the EndOhm chamber is given in Figure 3. The resistance was measured every 2 hours for 12 hours.

The TEER was calculated from the resistance using the formula: TEER (Ω cm²) = resistance (Ω) x membrane area (cm²), where the membrane area was 1.54 cm².

The change in TEER for each insert was calculated using the following formula: change in TEER (%) = TEER (Ω cm²) / initial TEER (Ω.cm²) - 100 (%). The mean change in TEER was plotted against time, with the error bars showing the standard error of the mean. A Student's t-Test was used to compare treatments. Statistical differences between treatments were declared at a probability less than 0.05 whilst a probability below 0.1 but above 0.05 was considered to represent a trend.

To compare the seven commercial probiotic strains the mean change in TEER over 12 hours and standard error of the means (SEM) were calculated. Student's t-Test was used to compare each bacterial strain to the controls. Statistical differences between treatments were declared at a probability less than 0.05, and a positive trend at a probability less than 0.10.

Results

Pulse-field gel electrophoresis

Pulse field gel electrophoresis was used to compare the L. plantarum strains to confirm that they were all different. Figure 4 shows the resulting images. AGR 1526 has a unique profile compared to the other L. plantarum strains that were tested.

16s rRNA sequence

To confirm the species of AGR 1526 16s rRNA sequencing was used. The 16s rRNA sequence is given in Figure 5. This was compared to other known sequences in the NCBI Blast database and it confirmed that AGR 1526 is a strain of Lactobacillus plantarum (99% match). The pulse-field gel electrophoresis profile of AGR 1526 in Figure 6 shows this strain is different to the three commercial L plantarum strains tested.

In vitro bioassay

The commercial probiotic strains were tested using the in vitro bioassay to determine which strain had the greatest positive effect on the TEER. For these experiments L. plantarum isolated from the commercial product VSL#3 was used as the positive control to normalise between assays because this strain has shown a consistent positive effect of TEER in previous experiments. The results of the assays are summarised in Figure 7 and Table 3.

In the Table 1 the values given are the mean change in TEER compared to the control media with L. plantarum VSL#3 normalised to 100%. Therefore, negative values mean that the strain had a negative effect on TEER compared to the control media and positive values mean that the strain had a positive effect on TEER compared to the control media. Values greater than 100% indicate that the strain performed better that L plantarum VSL#3 and values lower than 100% indicate the strain performed worse than L plantarum VSL#3.

Table 3 - Summary of the results of the in vitro assays testing the effect of commercial probiotic strains on trans-epithelial electrical resistance (TEER).

Mean change in TEER (± SEM) % compared to Commercial Probiotic Strain control media (n=4) expressed relative to

Lactobacillus plantarum VSL-3 strains (100%)

Lactobacillus rhamnosus HN001 222 (19)

Bifidobacterium lactis Bb12 148 (31 )

Lactobacillus plantarum 299 158 (33)

Lactobacillus plantarum VSL#3 100 (33)

Lactobacillus plantarum 299v 66 (32)

Lactobacillus casei Shirota 14 (57)

Lactobacillus rhamnosus GG -11 (79) Bifidobacterium lactis HN019 -115 (34)

The three best performing commercial probiotic strains in the TEER assays were L plantarum 299, L rhamnosus HN001 and B. lactis Bb12. All three strains had a positive TEER effect (compared to control media); however, only L rhamnosus HN001 produced a statistically greater TEER _va|ue compared to L plantarum VSL#3 over a 10 hour period from 2 to 12 hours after the start of the assay. Therefore, it was decided that L. rhamnosus HN001 was the best commercial strain to which all experimental strains should be compared.

The effect of AGR 1526 on TEER compared to L. rhamnosus HN001 and control media (no bacteria) is illustrated in Figure 8. AGR 1526 and L. rhamnosus HN001 both had a significantly greater effect (P<0.05) on TEER compare to control media from 2 to 8 hours. AGR 1526 caused a larger average increase in TEER than L rhamnosus HN001 but this was only significantly different (P<0.05) at 2 hours.

Discussion

The probiotic bacterium AGR 1526 (a strain of L. plantarum) performed better than the best commercial probiotic strain L. rhamnosus HN001 in the TEER assay.

Both strains caused a significant increase in TEER compared to the untreated controls from 2 to 8 hours; however, AGR 1526 caused a greater increase in TEER compared to the untreated controls than L. rhamnosus HN001 (only significant at 2 hours). This means that AGR 1526 was able to increase the strength of the tight junctions between the intestinal epithelial cells, and therefore, indicates that it would be able to enhance the integrity of the GIB.

Further in vitro experiments will be carried out to investigate other potential probiotic properties of AGR 1526 compared to L. rhamnosus HN001 such as:

Resistance to acid, bile salts and pepsin to determine if it can survive passage through the gastrointestinal tract (if not it could be encapsulated for protection)

Ability to adhere to intestinal epithelial cells

Ability to modulate immune responses (e.g. decrease pro-inflammatory cytokines, increase anti-inflammatory cytokines).

A mouse trial will also be carried out to determine the efficacy of AGR 1526 in vivo. For this trial, interleukin-10 knockout (IL10^"'^") mice will be used as a model for human Inflammatory Bowel Diseases. The effect of AGR 1526 on key performance indices including growth rate, general health score, gut histology, immune profile and bacteria composition will be monitored.

INTRODUCTION - IN VIVO MOUSE TRIAL

To further test Lactobacillus plantarum AGR1526 and its ability to promote intestinal health and wellness in vivo, IL10^"'^" mice were used as a surrogate model. When raised in conventional conditions these mice spontaneously develop chronic colonic inflammation with symptoms similar to those observed in human patients with Inflammatory Bowel Diseases (IBD).

MATERIALS AND METHODS

Animals

The trial was done in two blocks due to limitations on the numbers of IL10^"'^" mice available at any one time. At 5 weeks of age, mice were randomly assigned to the following treatments according to a complete block design (15 IL10^"'^" mice (C57BL/6J background) and 8 C57BL/6J (C57) mice per group):

1) daily oral placebo (vehicle fluid only) dosing

3) daily oral strain AGR1526 dosing

The mice were orally dosed daily via pipette with the probiotic bacterial strain (approximately 10⁹ colony forming units (CFU)) prepared in the vehicle fluid (20% glucose in skim milk). A week after the start of the probiotic dosing, the mice were inoculated with endogenous intestinal bacteria Enterococcus species (EF) (10⁶ CFU) and complex intestinal flora (100 ml) by oral gavage to ensure consistent and more rapid development of intestinal inflammation in IL10 ^Λ mice (Roy et al. 2007).

A commercial mouse diet (AIN-76A) was offered and food consumption was recorded daily. All mice were weighed and a faecal sample collected thrice weekly. Each day, mice were checked for the presence of loose stools or blood in faeces (an indication of intestinal inflammation) and the General Health Score (GHS; commonly used criteria for rating mouse wellness in a range from 1 (very ill) to 5 (healthy)) was established. At 12 weeks of age the mice were euthanized by CO₂ asphyxiation and cervical dislocation. Blood and tissue samples will be immediately collected post-mortem and rapidly frozen in liquid nitrogen or fixed in formalin and stored at room temperature until analysis.

Plasma serum amyloid A concentrations

Inflammation was determined by analysis of serum amyloid A levels in plasma using a murine specific Tridelta Phase™ range serum amyloid A kit and an Enzyme Linked lmmuno Sorbent Assay (ELISA) according to manufacturer (Tridelta Development Limited, Maynooth, . County Kildare, Ireland). A volume of 50 μL of anti-serum amyloid A/streptavidin-horse radish peroxidase conjugate and 50 μL of diluted plasma sample or standard were added to each well that is coated with a monoclonal antibody specific for serum amyloid A. After 1 hour incubation (37°C) the plates were washed four times with 400 μL diluted wash buffer to remove all of the unbound material. Then 100 μL of tetramethylbenzidine substrate solution was added to induce the colour change in the reaction and incubated at room temperature for 5 to 10 minutes before adding 100 μl of stop solution. The absorbance of each well was read at 450 nm with 630 nm as reference using a plate reader (VERSAmax™, Molecular Devices Corporation, Sunnyvale, California). The intensity of the colour produced was proportional to the concentration of SAA present in the original specimen.

Histopathology

The histological analysis of colon samples was carried out at Gribbles Veterinary Pathology in Hamilton. The colon tissue samples were fixed in formalin and embedded in paraffin. The embedded tissue samples were sliced to obtain 5 μm thick sections which were stained with haematoxylin and eosin for light microscopy examination. The stained sections were assessed for inflammation based on a combination of inflammatory cell infiltration (monocytes, neutrophils, eosinophils, plasmocytes, fibrin exudation and lymphangiectasis), tissue destruction (enterocyte loss, ballooning and degeneration, oedema and mucosal atrophy) and tissue repair (hyperplasia, angiogenesis, granuloma and fibrosis). A rating score of between 0 (no change from normal tissue) and 3 (lesions involved in most areas and all layers of the intestinal section including mucosa, muscle and omental fat) was applied to each colonic section for each aspect of inflammation (e.g. monocytes, oedema, hyperplasia etc). The sum of the score for inflammatory cell infiltration, tissue destruction and tissue repair was used to represent the total histological injury score for each colonic section. The sum of the inflammatory cell infiltration was multiplied by 2 to give more weight to this value, as this represented the main characteristic of the observed inflammation.

Calculations and statistical analysis

For each mouse the mean daily live weight gain was calculated using the following equation: daily live weight gain over the experimental period = (final live weight - initial live weight)/number of days. Additionally mean daily food intake was calculated as the total food intake/number of days. These two variables were analysed using Analysis of Variance (ANOVA). Repeated measures analysis was also performed for daily feed intake and daily body weight to compare the time profiles of the different treatment groups. Overall effect on histology injury score and plasma concentration of serum amyloid A were analysed using the Residual Maximum Likelihood (REML) procedure. All analyses examined mouse strain and AGR strain differences, and their interaction. One-sided and two-sided tests were used to compare the plasma levels of serum amyloid A between strains. Analyses were performed using Genstat (version 9).

RESULTS

Feed intake

The mean daily feed intakes for each treatment group for the whole experimental period are summarised in Table 1. There were no significant differences in mean daily feed intakes between mouse strains or between AGR strain-inoculated mice treatment groups over the experimental period. Additionally, there were no mouse strain effects, AGR strain treatment effects or interaction on feed intake at most times.

Table 1. Mean daily feed intake (g/d) of C57 and ILIO⁺ mice between 5 and 12 weeks of age inoculated with and without AGR 1526.

Treatment Mouse strain C57 ILIO⁺

Control 3.38 ± 0.06 3.45 ± 0.04

L plantarum AGR1526 3.43 ± 0.06 3.43 ± 0.04

Data represent mean ± standard error for C57 (n=8) and ILIO⁺ (n=15) mice. Body weight gain

There was no significant difference of the mean daily body weight gains between AGR 1526-inoculated C57 or IL10^"'^" mice treatment groups (P>0.1) indicating that the AGR1526 strain had no toxic effect on the C57 or ILIO^''^' mice that were manifested as a decrease in body weight gain. However, there were significant differences in daily body weight gains between mouse strains (P<0.05) and as expected, the C57 mice gained more weight than the IL10^"'^" micei 526-inoculated IL10^"'^' mice treatment groups.

Table 2. Mean daily body weight gain (g/d) of C57 and IL10^''^" mice between 5 and 12 weeks of age inoculated with and without AGR 1526.

Treatment Mouse strain

C57 IL10*

Control 0.19 ± 0.01 0.18 ± 0.01

L plantarum AGR1526 0.20 ± 0.01 0.17 ± 0.01

Immune biomarkers and histopathology

The general health score was not affected by the mouse strain or the inoculation with AGR strains which means there were no noticeable indications of physical deterioration (e.g. loose stools, blood in stools, loss of hair etc). The histology injury score was greater in IL10^"'^" control mice (inoculated with vehicle only) compared to C57 control mice or C57 mice inoculated with the AGR strains (Table 3). The plasma concentration of serum amyloid A was higher in IL10^"'^" mice inoculated with the AGR strains or the vehicle only (191 μg/ml) compared to the C57 mice (as expected, not detectable).

The concentration of serum amyloid A in the plasma of IL10^"'^" mice inoculated with AGR 1526 was lower than IL10^"Λ inoculated with the vehicle only (Table 3; P=O.19).

DISCUSSION

As expected, there was a lower growth rate in the IL10^"Λ mice compared to the C57 mice over the duration of the trial and at most time points during the experimental period. There is evidence that AGR1526 reduced the concentration of an important biomarker of inflammation, plasma serum amyloid A, in the IL10^"'^" mice compared to IL10^Λ mice inoculated with the vehicle only.

Table 3. Colonic histological injury score and plasma serum amyloid A of IL^"'^'1O and C57 mice at 12 weeks of age inoculated with and without AGR 1526.

Mouse strain

Treatment

C57 ILK)-⁷-

Serum amyloid A (μg/ml)

Control (vehicle only) ND 181.45 ± 54.09

L plantarum AGR1526 133.89 ± 39.91

ND

Data represent mean ± standard error of the means for C57 (n=8) and IL10^"Λ (n=15) mice.

The C57 and IL10^"'^" mice were significantly different for all treatments (P<0.05).

ND: not detectable (as expected).

INTRODUCTION - IN VITRO COMPARISON OF L. PLANTARUM AGR1526 TO L. RHAMNOSUS HN001.

In the bioassays described AGR 1526 were compared to the commercial probiotic L. rhamnosus H N 001.

The bioassays chosen for this milestone were a cell adherence assay and an immune challenge assay. The cell adherence assay tested the ability of the bacterial strains to adhere to intestinal cells. This property is important because it is a crucial and a prerequisite step for colonisation of the intestinal tract. We also tested the tolerance of the bacterial strains to acid, bile and pepsin. These properties are important to ensure the bacterial strains are able to survive passage through the upper gastrointestinal system to colonise the intestinal tract of the host.

MATERIALS AND METHODS

Acid, bile and pepsin tolerance

The bacterial cultures (L. rhamnosus HN001 , AGR1526) were grown overnight in 4OmL of MRS broth at 37°C with 5% CO2. The cultures were mixed and then aliquoted into lots of 1OmL in sterile tubes. The tubes were centrifuged (20 minutes at maximum speed) to collect the bacterial cell pellets and the supernatant was removed. The pellets were resuspend to an approximate cell concentration of 10⁸ CFU/mL in the following test solutions:

1) MRS broth control

2) MRS broth adjusted to pH 2

3) MRS broth adjusted to pH 4

4) 0.5% bile 5) 1% bile

6) 0.3% pepsin

7) 1% pepsin

The number of viable bacterial cells was determined after 2 and 4 hours in triplicate using standard enumeration techniques. These time points were chosen to represent the amount of time it would take the bacterial strains to pass through the upper gastrointestinal system to the intestinal tract. The concentrations of pepsin and bile and the pH values were chosen to represent the range of these variables (high and low concentrations) found in the human gastrointestinal system.

Cell adherence

The cell adherence assay was carried out with two different epithelial cell lines, HEp-2 and Caco-2 cells. HEp-2s are a human epithelial larynx cell line commonly used in adherence assays because they rapidly grow to confluence (complete layer of cells; 48 hours). Caco-2 cells are a human colon epithelial cell line that better represents the intestinal barrier but take longer to grow (5 to 7 days to grow to confluence and 15 to 20 days to differentiate). The epithelial cells were grown in the presence of foetal calf serum and antibiotics in 24-well tissue culture plates at 37°C with 5% CO₂. The antibiotics were removed from the epithelial cells by washing with pre-warmed phosphate buffered saline (PBS) to allow the survival and growth of the test bacterial strains, and the cells were bathed in either DMEM with 1% non-essential amino acids for HEp-2 cells or M199 with 1% non-essential for Caco-2 cells. Bacterial strains to be assessed were grown overnight in MRS broth and approximately 10⁷ (10μl) were added to each well with each strain being assessed in triplicate. After 3 hours incubation at 37°C (5% CO₂) bacteria were removed from one 24-well tissue culture plate and the epithelial cells were washed gently (x5) with pre-warmed PBS. Bacterial separation from epithelial cells was mediated by adding 1ml of a 1% solution of Triton X-100 to each well and stirring for 10 minutes. Bacteria were quantified by using standard enumeration techniques. To assess adherence over 6 hours, non-adherent bacteria were removed after 3 hours and the epithelial cells were washed gently (x5) with PBS as described previously. Fresh media was then added to each well and the tissue culture plate incubated for a further 3 hours prior to enumeration.

Calculations and statistics

For all the experiments the mean and standard error of the mean (SEM) were calculated using Excel. Student's t-Test was used to compare each bacterial strain to the controls. Statistical differences between treatments were declared at a probability less than 0.05, and a positive trend at a probability less than 0.10.

RESULTS

Acid, bile and pepsin tolerance

The effects of acid, bile and pepsin on the viability of the bacterial strains are summarized in Figures 1 , 2 and 3 respectively. Of the bacterial strains, AGR1526 tolerated the gastrointestinal tract mimicking conditions the best. All of the bacterial strains were able to tolerate conditions of pH 4 for 4 hours without loss of cell viability; however, they all had significantly reduced viability in conditions of pH 2 (decrease in 7 log units). The viability of L rhamnosus HN001 was decreased by 0.5% bile (2-3 log units) and by 1% bile (5-7 log units); whereas the viability of AGR1526 was only reduced at the highest bile concentration after 4 hours (decrease of 2 log units). None of the bacterial strains were affected by pepsin.

Cell adherence

The ability of the bacterial strains to adhere to epithelial cells was determined using undifferentiated and differentiated Caco-2 cells (Figure 4) and HEp-2 cells (Figure 5). For both the undifferentiated and differentiated Caco-2 cells, AGR1526 was the better strain at adhering (higher number of viable cells attached) and remained attached (lower drop in viable cells attached between 3 and 6 hours) to the epithelial cells. For the HEp-2 cells, AGR1526, and L rhamnosus HN001 adhered to the epithelial cells to a similar extent. Although the HEp-2 model is often used due to its ease of growth, the Caco-2 model is more reliable as it is a human colon epithelial cell line.

DISCUSSION

Of the bacterial strains, AGR1526 was the best at tolerating the conditions that mimicked the gastrointestinal tract and at adhering and remaining attached to intestinal epithelial cells.

These results show that both AGR1526 has properties that are considered very important for probiotic bacteria (ability to tolerant gastrointestinal conditions and adhere to intestinal epithelial cells).

IMPACT of AGR1526 PROBIOTIC ON CALVES

The present study was undertaken to assess the impact of probiotic supplementation on the pre and post-weaning health and growth of calves under artificial rearing conditions. Purebred Friesian bull calves (n = 120, 5 ± 1 days of age, approximately 40kg bodyweight (BW)) were reared indoors over a period of 70 days. The rearing process comprised a 48 day pre-weaning period of twice daily calf milk replacer feeding following by a 22-day post-weaning period. Throughout the trial the calves were allowed ad libitum access to clean water, barley straw and concentrate meal. Post-weaning, Lucerne pellets were also offered twice daily ad libitum. The calves were allocated to a treatment group which received a composition including AGR1526 in phosphate buffered saline (PBS, pH7.3). The untreated control group received the diluent solution (phosphate buffered saline, as placebo). The animals were weaned off calf milk replacer with concurrent removal of the treatments, from day 42 to day 48 of the trial.

The plasma concentration of serum amyloid A was measured using an ELISA assay substantially the same as that used in the mouse trial described above.

RESULTS AND DISCUSSION

The plasma concentration of serum amyloid A measured in the treatment and control groups are shown in Figure 14. As can be seen, the serum amyloid A concentrations in the calves treated with the AGR1526 containing composition were lower (P<0.05) than that of the control group.

Potential uses for AGR 1526 include, but are not limited to:

■ Addition to food and drink products or encapsulation in pill form for humans for the purpose of:

Enhancing general wellness in healthy individuals

Limiting GIB deterioration during aging and times of stress'

Increasing GIB integrity during illness (e.g. Inflammatory Bowel Diseases, Irritable Bowel Syndrome) or infection (e.g. food poisoning).

Replacing gut bacteria after taking antibiotics.

^■ Inclusion in feed or as a feed additive for livestock (e.g. cattle, pigs, sheep) for the purpose of:

Decreasing diarrhoea during weaning (e.g. scours in calves)

Promoting growth and increasing live weight gain

Increasing general wellness.

^■ Inclusion in feed or as a feed additive for companion animals (e.g. cats, dogs) for the purpose of:

Decreasing diarrhoea during weaning

Promoting growth

Increasing general wellness.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the appended claims. References

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Claims

WHAT WE CLAIM IS:

1. A composition to help or improve gastrointestinal health with includes L plantarum AGR 1526 which is publicly available from DSM under accession number DSM 2648.

2. A use of L. plantarum AGR 1526 which is publicly available from DSM under accession number DSM 2648 to manufacture a composition to improve or help maintain gastrointestinal health.

3. A use of L plantarum AGR 1526 as publicly available from DSM under accession number DSM 2648 to manufacture a composition to improve or help maintain the integrity of the gastrointestinal barrier.

4. A method of preventing, alleviating and/or treating a disease, ailment, or disorder associated with a gastrointestinal disease comprising:

- administering an effective amount of Lactobacillus plantarum AGR 1526 to a person or animal.

5. A method of preventing, alleviating and/or treating a disease or ailment associated with a gastrointestinal disease comprising:

administering a composition which includes an effective amount of Lactobacillus plantarum AGR 1526 to a person or animal.

6. A use of Lactobacillus plantarum AGR 1526 as a food or drink additive.

7. A use of Lactobacillus plantarum AGR 1526 as an animal feed or drink additive.

8. A use of Lactobacillus plantarum AGR 1526 to manufacture a medicament for the prevention, alleviation and/or treatment of a disease, ailment, or disorder associated with the gastrointestinal barrier.

9. A use of Lactobacillus plantarum AGR 1526 as a probiotic.

10. A composition which includes Lactobacillus plantarum AGR 1526 to improve or help maintain the integrity of the gastrointestinal barrier.

11. A composition which includes an effective amount of Lactobacillus plantarum AGR 1526 to prevent, alleviate and/or treat a disease, ailment, or disorder associated with the gastrointestinal barrier.

12; A use of Lactobacillus plantarum AGR 1526 to reduce serum amyloid A levels in a person or animal.

13. A use of Lactobacillus plantarum AGR 1526 in the manufacture of a composition to reduce serum amyloid A levels in a person or animal.